UV-Visible Spectroscopy

Instrumental Methods of Analysis

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 Syllabus:

• 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

2
 Spectroscopy:
It is the branch of science that deals with the study of interaction of matter with light.
OR
It is the branch of science that deals with the study of interaction of electromagnetic
radiation with matter.

 The UV-VIS spectrometry is one of the oldest instrumental

techniques of analysis and is the basis for a number of
ideal methods for the determination of micro and semi-
micro quantities of analytes in a sample.
 These determinations find applications in research,
industry, clinical laboratories and in the chemical analysis
samples from various industries.

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 Electromagnetic Radiation
EMR is type of energy that is transmitted trough space at enormous velocities.
Electromagnetic radiation consist of discrete packets of energy which are called as
photons.
A photon consists of an oscillating electric field (E) & an oscillating magnetic field
(M) which are perpendicular to each other.

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 Electromagnetic Radiation
• Wave theory of duality is applicable to EMR.
• Wave nature: Reflection, Diffraction, Interference
• Corpuscular nature: Interaction between radiation and matter such as absorption or emission of
energy.
• Frequency (ν):
– It is defined as the number of times electrical field radiation oscillates in one second.
– The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second
1Å = 10 cm = 10 m
-8 -10

1nm = 10Å = 10-7 cm = 10-9 m

• Wavelength (λ): 1µm = 103 nm = 10-4 cm = 10-6 m
– It is the distance between two consecutive troughs or crests.
– Expressed in cm, nm, Å
5
 Electromagnetic Radiation

• The relationship between wavelength & frequency can be written as:

c=νλ
• As photon is subjected to energy, so
E=hν=hc/λ
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Electromagnetic Radiation

7
Electromagnetic Radiation

8
9
Interaction of EMR with matter

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 Principles of Spectroscopy:
• The principle is based on the measurement of spectrum of a
sample containing atoms / molecules.
• Spectrum is a graph of intensity of absorbed or emitted radiation
by sample verses frequency (ν) or wavelength (λ).
• Spectrometry is the technique used to
asses the concentration or amount of a
given species.
• Spectrometer is an instrument design
to measure the spectrum of a
compound.

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 Principles of Spectroscopy:

1. Atomic Absorption Spectroscopy :

• An analytical technique which concerns with the measurement of absorption of
electromagnetic radiation.
e.g. UV (185 - 400 nm) / Visible (400 - 800 nm) Spectroscopy, IR
Spectroscopy (0.76 - 15 μm)
2. Atomic emission Spectroscopy:
3. Molecular Absorption Spectroscopy
4. Emission Spectroscopy:
• An analytical technique in which emission (of a particle or radiation) is dispersed
according to some property of the emission & the amount of dispersion is measured.
e.g. Fluorimetry, Flame Photometry

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A line (L) spectrum of an atom

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A band spectrum of a molecule

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 Interaction of EMR with matter:
1. Electronic Energy Levels:
• At room temperature the molecules are in the lowest energy levels
E 0.

• When the molecules absorb UV-visible light from EMR, one of the
outermost bond / lone pair electron is promoted to higher energy
state such as E1, E2, …En, etc is called as electronic transition and
the difference is as:
∆E = h ν = En - E0 where (n = 1, 2, 3, … etc)
∆E = 35 to 71 kcal/mole
15
 Interaction of EMR with matter:
2. Vibrational Energy Levels:
• These are less energy level than electronic energy levels.

• The spacing between energy levels are relatively small i.e. 0.01 to 10
kcal/mole.

• e.g. when IR radiation is absorbed, molecules are excited from one

vibrational level to another or it vibrates with higher amplitude.

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 Interaction of EMR with matter:
3. Rotational Energy Levels:
• These energy levels are quantized & discrete.

• The spacing between energy levels are even smaller than

vibrational energy levels.

∆Erotational < ∆Evibrational < ∆Eelectronic

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•Absorption Spectroscopy is selected when fewer molecules
interact with incident radiation and get excited from ground
state to the excited state.
•Emission spectroscopy is selected when a large no. of molecules
interact with incident radiation and get excited from ground
state to the excited state

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 Origin of Spectrum:
 The absorption of radiation in the UV-VIS region of
the spectrum is dependent on the electronic structure
of the absorbing species like, atoms, molecules, ions
or complexes.

 A given electronic energy level has a number of

vibrational energy levels in it and each of the
vibrational energy level has a number of rotational
energy levels in it.

 When a photon of a given wavelength interacts with

the molecule it may cause a transition amongst the
electronic energy levels if its energy matches with the
difference in the energies of these levels.

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20
 Optical
Instruments
• Spectroscope: An optical instrument used for visual identification of emission lines,
produced as a result of emission of light from the excited sample.
• Spectrograph: A record of emission lines on photographic plate or a film.
• Photometer: An instrument used to measure intensity of a radiation. It is used for
measurement of absorption in UV-VIS regions.
• Fluorimeter: A photometer designed for fluorescence measurement
- A photometer makes use of source, filter, photoelectric detector
• Colorimeter: A photometer used in the visible radiation.
• Spectrometer: An optical instrument used to measure properties of radiations over a
specific portion of EMR, typically used in spectroscopic analysis to identify materials. It
is an instrument which makes the use of a monochromator.
• Spectrophotometer: A photometer that can measure intensity as a function of the
color, or more specifically the wavelength of radiation.
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 Principle:
• The UV radiation region extends from 10 nm to 400 nm and the visible
radiation region extends from 400 nm to 800 nm.
Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
• Far UV spectroscopy is studied under vacuum condition.
• The common solvent used for preparing sample to be analyzed is either
ethyl alcohol or hexane.

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•When a beam of light is passed through a transparent cell containing a
solution of an absorbing substance, reduction in the intensity of the light may
occur.
•This is due to,
a) Reflection at the inner and outer surfaces of the cell
b) Scatter by particles in the solution
c) Absorption of light by molecules in the solution
•Reflections at the surfaces can be compensated by a reference cell containing
the solvent only, and scatter may be eliminated by filtration of solution.
•The intensity of the light absorbed is then given by
I absorbed = I0- IT

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 Common terms used in Absorption
Spectroscopy
•Radiant Power (P) or Intensity (I): The power of radiation is the energy, of
the beam reaching a given area per second, whereas intensity is the
power per unit solid angle.
•Transmittance: Fraction of incident radiation transmitted by the solution.
T=
Absorbance (A) = -log10 T
= -log10
If the attenuation of the beam is more,
transmittance is less and absorbance is more

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Beer- Lambert’s Law

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 Lambert’s
Law
• The intensity of a beam of parallel monochromatic radiation decreases
exponentially as it passes through a medium of homogenous thickness OR
absorbance is proportional to thickness (pathlength) of the solution.

- α I0
• Let I0 be the intensity of incident radiation, b be the thickness of the solution.

- =k I0 --Eq.1

- =kdb --Eq. 2
On rearranging eq. 2 =-kdb --Eq. 3

On integrating eq. 3

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 = -k

--Eq. 4
Taking antilogs
=
I =I0 --Eq. 5

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 Iabs = I0-I
= I0-I0--Eq. 6

By changing to log 10
From eq. 4

2.303
log10= - b
log10= - --Eq. 7 [ where, k’ =- ]
= 10-k’b
= I010-k’b k’ =
extinction/absorption coefficient

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 Physical

From eq. 7
significance of k’
log10= -
On rearranging, k’ = - log
= log ()-1
k’ =
log ()
If, log () =1
k’ =
But, log () = 1 only when =10
I= I0
K’ (absorption coefficient) is thus defined as the reciprocal of thickness of absorbing
medium which is required to reduce the intensity of transmitted light to 1/10th of the
intensity of the incident radiation.

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 Beer’s Law
• The intensity of a beam of the parallel monochromatic radiation decreases
exponentially with the number of absorbing molecules. Or the absorbance is
proportional to the concentration.
- αI --- Eq. 1
- = kI --- Eq. 2
Where I= Intensity of the radiant beam, k= proportionality constant, n= concentration
- --- Eq. 3
Where I0= incident radiation when number of interacting molecules, n =0, I= Intensity of
transmitted radiation when number of interacting molecules , n=N and N=total no. of
molecules in the path of radiation over thickness (b) of the solution.

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 = -k

--Eq. 4
Taking antilogs
=
I =I0 --Eq. 5

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 Iabs = I0-I
= I0-I0--Eq. 6

By changing to log 10
From eq. 4

2.303
log10= - N
log10= - --Eq. 7 [ where, k’’ =- ]
= 10-k’’N
= I010-k’’N k’’ =
extinction/absorption coefficient

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 Beer-Lambert Law
A= abc
Equation
Where a= absorptivity, b= pathlength, c=conc.
If, c is in moles/l, a= Є= molar absorptivity
And eqn can take the form of
A= Є bc

Є = Molar absorptivity at a specified wavelength of a substance in solution is

the absorbance at that wavelength of a 1 mol/l solution in a 1 cm cell.
(Hence, unit mol/cm)

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•A1%1 cm = Specific absorbance is the absorbance of 1% solution in

A= A bc
a cell of 1 cm pathlength.
1%
1 cm

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Conditions that need to be fulfilled in order for Beer’s law to
be valid
These are:
• The absorbers must act independently of each other.

• The absorbing medium must be homogeneous in the interaction volume—dilute solutions are required.

• The absorbing medium must not scatter the radiation – no turbidity. Clear solutions required.

• The incident radiation must consist of parallel rays, each traversing the same length in the absorbing medium

• The incident radiation should preferably be monochromatic, or have at least a width that is narrower than that
of the absorbing transition; and

• If any of these conditions are not fulfilled, there will be deviations from Beer’s law.

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 Limitations of beer lambert law
The limitations of Beer-lambert law are given as:
1.Beer-Lambert law is only valid on monochromatic light
2.This law is applicable under a low concentration range where
interactions between molecules are not considered.
3.This law is also invalid when radiations of very high intensities
are used.

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 Deviations from Beer-Lambert Law
• Under certain conditions Beer-Lambert law fails to maintain a linear relationship between absorbance
and concentration of analyte. These deviations are classified into three categories:

• Real Deviations - These are fundamental deviations due to the limitations of the law itself.

• Chemical Deviations- These are deviations observed due to specific chemical species of the sample
which is being analyzed.

• Instrument Deviations - These are deviations which occur due to how the absorbance measurements
are made.

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 Real Limitations of Beer Lambert’s law
• Applicable to low analyte concentration/ dilute solutions. This is because of the change in the molar
absorptivity (ε) of an analyte.

• High Conc. (> 0.01M) , the extent of solute-solute interactions or Hydrogen bonding can affect the
analyte environment and its absorptivity

• As conc. becomes high Average distances between molecules and ions decrease-Each particle
affects the charge distribution of others

• This, in turn, affects the ability of analyte to absorb the given radiation

• Absorptivity also depends on RI of the medium. Higher Conc. of analyte may change RI of the medium,
causing deviations

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In the absence of hydrogen bonding
Excitation of an amine is brought
about at a longer wavelength
(smaller energy).
In the presence of hydrogen
bonding
Excitation of an amine is brought
about at a shorter wavelength
(larger energy).
This change in λmax with change in
conc. results in a change in, ε,
resulting in deviation from Beer’s
law

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• At a low conc. of an amine as
the absorbing species, the
molecules are far apart, and
solvent molecule (eg.
Ethanol) occupy the space
between the two molecules
of an amine.
This interaction changes the
charge distribution in an
absorber, which results in a
change in λmax and molar
absorptivity of an absorber,
ultimately leading to the
deviation from Beer’s law.
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Chemical deviations
•At times, even electrolyte concentrations (such as those present in
buffers) play an important role in altering the charge distributions and
affecting UV-visible absorbance.

•It is also possible that the concentration is so high, that the molecules
create a screen for other molecules, thereby shadowing them from the
incident light.

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Chemical deviations
• Occur due to chemical phenomenon involving the analyte molecules, due to association, dissociation,
polymerisation, complex formation and interaction with the solvent to produce a product with different
absorption characteristics
• Eg. Benzyl alcohol
4C6H5CH2OH (C6H5CH2OH)4

Monomer Polymer
λmax = 275 nm λmax = 300 nm

• At a less conc. the solution remains in monomeric form and a straight

line (B) is observed at 275 nm.
• As conc. increases, polymerization takes place as a result, a negligible
amount of monomer remains in solution, leading to negative deviation
(D) In conc. vs absorbance plot.
• As conc. of polymer is more, greater absorbance is observed at 300 nm. Leading to positive deviation (E).
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• Another example of apparent chemical deviation is
the deviation from linearity between absorbance and
total concentration of chromium because of a change
in color of unbuffered dichromate solution on
dilution.
• An orange colored dichromate solution is converted (Acidic) (Alkaline)
to yellow chromate on dilution.
• As the conc. of dichromate ions increases in solution
(Concentrated solution), its conversion to chromate is
reduced, since same amount of water is available for
the hydrolysis of increasing dichromate ions.

• If the absorbance of this solution is measured at 550

nm then positive deviation is seen. Same
measurement at 450 nm leads to negative deviation.

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• A dichromate solution appears orange because a radiation corresponding to orange
color is not absorbed but is transmitted out. The transmitted radiation reached to our
eyes and gives the perception of an orange appearance to the dichromate solution.
• The radiation corresponding to its complementary color (blue) is absorbed ,maximum
by an orange color solution, whereas a yellow colored solution of chromate ions
absorbs radiation of its complementary color (violet).

Color Wheel
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For example, phenol red undergoes a resonance
transformation when moving from the acidic
form (yellow) to the basic form (red).

Due to this resonance, the electron distribution

of the bonds of molecule changes with the pH
of the solvent in which it is dissolved.

Since UV-visible spectroscopy is an electron-

related phenomenon, the absorption spectrum
of the sample changes with the change in pH of
the solvent.

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 Instrumental
Deviations
• 1. Due to Polychromatic Radiation (Also the reason why absorbance measurements are taken at the
wavelength of maximum absorbance λmax) :
• Beer-Lambert law is strictly followed when a monochromatic source of radiation exists.
• In practice, however, it is common to use a polychromatic source of radiation with continuous distribution of
wavelengths along with a filter or a grating unit (monochromators) to create a monochromatic beam from
this source.
• Molecules have different molar absorptivity at different λ. Consider a molecule having molar absorptivities ε’
and ε” at wavelengths λ’ and λ”. The absorbance (Am) for such a species can be calculated as:

At λ’, A’ =ε.b.c
Atλ”, A” =ε”.b.c
Therefore, Am =A’+ A”

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• When the molar absorptivities are the same at both wavelengths (i.e. ε’ = ε”) , the relationship between
absorbance and concentration follows Beer-Lambert law to obtain a straight line.

• However, as the difference between ε’ and ε” increases, the deviations from linearity also increases.

• Absence of monochromatic radiation can be due to wide slit width (narrow opening through which light
or other waves pass) also

• If absorptivity of the substance at this extraneous wavelength is less than that at the wavelength of
measurement, the measured absorbance will be lesser than true absorbance: Negative Deviation

• If absorbance at extraneous wavelength is greater than wavelength of measurement, then positive

deviation will be observed.

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48
Instrumental Deviations
B] Due to the Presence of Stray Radiation

• Stray radiation or scattered radiation consists of a mixture of wavelengths which differ

greatly from that of the principle radiation.
• Stray radiation is produced due to reflection from various internal surfaces of the
instrument.
• Multiple reflections bring about loss in energy of radiation and correspondingly a
change in their wavelength.
• They cause deviation from Beer’ law .

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Instrumental Deviations
• C] Due to Mismatched Cells or Cuvettes

• If the cells holding the analyte and the blank solutions are having different path-lengths, or unequal
optical characteristics, it is obvious that there would be a deviation observed in Beer-Lambert law.

• In such cases when a plot of absorbance versus concentration is made, the curve will have an intercept
k and the equation will be defined as:
A = εbc + k

• In today’s instrument this problem is generally not observed, however if it is present, appropriate linear
regression to quantify this deviation must be made.

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 Why absorption measurements are taken at wavelength of maximum absorbance λ max?

• If the band of wavelength selected on the spectrometer is such that the molar absorptivities of the
analyte is essentially constant, deviations from Beer-Lambert law are minimal.

• However, if a band is chosen such that the molar absorptivity of the analyte at these wavelengths changes
a lot, the absorbance of the analyte will not follow Beer-Lambert law.

• It is observed that the deviations in absorbance over wavelengths are minimal when the wavelength
observed is at the λmax.
• Due to this reason, absorption measurements are taken at wavelengths.

51
 Causes of nonlinearity

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors.

• Deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions

between molecules in close proximity ( Real)

• scattering of light due to particulates in the sample ( Real)

• fluorescence or phosphorescence of the sample (Real)

• changes in refractive index at high analyte concentration (Real)

• shifts in chemical equilibrium as a function of concentration (Chemical)

• non-monochromatic radiation ( Instrumental)

• stray light (Instrumental)

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 Electronic Transitions:

s
* Unoccupied
p levels
*

Energ Atomic Atomic

orbital n orbital
y
Occupied levels
p

s
Molecular orbitals

53
 Electronic Transitions:
The possible electronic transitions can graphically shown as:

54
 The possible electronic transitions are

• σ → σ* transition
• π → π* transition
• n → σ* transition
• n → π* transition
• σ → π* transition
• π → σ* transition Different energy levels of electronic transitions

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 • σ → σ* transition
• σ electron from orbital is excited to corresponding anti-bonding
orbital σ*.
• The energy required is large for this transition.
• This energy can be produced when a substance is exposed to
wavelength in the region of 100-200 nm usually < 185 nm.
e.g. Methane (CH4) has C-H bond only and can undergo σ →
σ* transition and shows absorbance maxima at 125 nm.
• Interference due to atmospheric N2 or O2. Vacuum UV: adds
cost, breakdown of bonds.
• Never observed in the readily accessible UV region.
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 • n → σ* transition
• Saturated compounds containing atoms with lone pair of
electrons like O, N, S and halogens, are capable of n → σ*
transition.

• These transitions usually require less energy than σ → σ*

transitions.

• The number of organic functional groups with n → σ* peaks in

UV region is small (150 – 250 nm).

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• In the presence of polar solvents, the non-bonding pair of an analyte interacts with
the electropositive atom of the solvent. Eg. N of amino and ethanol (hydrogen bond)
• When a hydrogen bond is formed system becomes more stable i.e the ground state
energy of non-bonding electron goes down. When an n → σ* transition of one of the
electron occurs, the remaining electron cannot maintain a hydrogen bond.
• Thus, in presence of hydrogen bond the non bonding electron will require higher
energy for their excitation to σ* antibonding orbital
• Hence, in a polar solvent absorption maxima shift towards shorter wavelengths.
(Hypsochromic/Blue shift)
• Shift will be more depending upon the polarity of the solvent.

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 • n → π* transition
• An electron from non-bonding orbital is promoted to anti-bonding
π* orbital.
• Compounds containing double bond involving hetero atoms (C=O,
C≡N, N=O) undergo such transitions.
• n → π* transitions require minimum energy and show absorption at
longer wavelength around 300 nm.
• The commonly used solvents do not absorb any radiation beyond
215-220 nm. The wavelength of radiation, beyond which a solvent
does not show any absorption, is called as cut off wavelength.
• No interference of any atmospheric component in between 220-380
nm. (readily accessible UV region).
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•In case of molecules involving this type of transition, interaction
of non-bonding electrons with polar solvent is observed.
•The extent of interaction depends on the polarity of the
solvent.
•In the presence of non-polar solvent interaction with non-
bonding electron does not occur. With polar solvents non-
bonding electrons form a hydrogen bond, resulting in a
hypsochromic shift. This hypsochromic shift is to the extent of 30
nm depending on the polarity of solvent.

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 • π → π* transition

• π electron in a bonding orbital is excited to corresponding anti-

bonding orbital π*.

• Compounds containing multiple bonds like alkenes, alkynes,

carbonyl, nitriles, aromatic compounds, etc undergo π → π*
transitions.

• e.g. Alkenes generally absorb in the region 170 to 205 nm.

61
•In case of n → π* and π → π* transitions bathochromic shift or red
shift( shift of the absorption maxima towards longer wavelength) is also
observed.
•When an alkene or an unsaturated compound with an electronegative
element (C=O, cyanide) is dissolved in polar solvent, electrons in double/triple
or on the electronegative element are dragged away from them towards
electropositive atom of the solvent.
•Because of dragging of electrons from a pi bond towards polar solvent, bond
becomes weaker and less energy is required to excite a electron, and hence
transition can occur at longer wavelength . This is seen only to the extent of 5
nm.
•In compounds involving of n → π* transitions, hypsochromic shift is more
predominant as compared to bathochromic shift.
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 • σ → π* transition
6
• π → σ* transition
• These electronic transitions are
forbidden
transitions & are only theoretically possible.

• Thus, n → π* & π → π* electronic transitions

show absorption in above
region
200 nm which is accessible to UV-
visible
spectrophotometer.
• The UV spectrum is of only a few broad of
absorption.
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64
65
 Chromophore
The part of a molecule responsible for imparting color, are
called as chromospheres.
OR
The functional groups containing multiple bonds capable of
absorbing radiations (usually above 200 nm due to n → π* & π
→ π* transitions).

e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

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 Chromoph
ore
To interpretate UV – visible spectrum following points
should be noted:

1. Non-conjugated alkenes show an intense absorption

below 200 nm & are therefore inaccessible to UV
spectrophotometer.

2. Non-conjugated carbonyl group compound give a weak

absorption band in the 200 - 300 nm region.

67
 Chromoph
e.g. O ore
Acetone which has λmax = 279 nm O
C
H 3C CH3

and that
cyclohexane
has λmax = 291
nm.
e.g. 1,5 - hexadiene has λmax = 178 nm
2,4
When- hexadiene has λmaxbonds
double = 227 nm are
conjugated in
CH 2 a compound λmax isCHshifted
3
H 2C H C
to longer wavelength.3 68
 Chromoph
ore
3. Conjugation of C=C and carbonyl group shifts
the λmax of both groups to longer wavelength.
e.g. Ethylene has λmax = 171 nm
O
Acetone has λmax = 279 nm
C
H 2C CH H 3C CH
2 Crotonaldehyde has λmax3 = 290 nm
O

C
H2
CH
C 69
3
 Auxochro
me
The functional groups attached to a chromophore which
modifies the ability of the chromophore to absorb light, altering
the wavelength or intensity of absorption.
OR
The functional group with non-bonding electrons that does not
absorb radiation in near UV region but when attached to a
chromophore alters the wavelength & intensity of absorption.

70
 Auxochro
me
e.g. Benzene λmax = 255 nm

O
H

Phenol λmax = 270 nm

NH
2

Aniline λmax = 280 nm

71
 1
Bathochromic Shift (Red Shift)
• When absorption maxima (λmax) of a compound shifts to longer
wavelength, it is known as bathochromic shift or red shift.

• The effect is due to presence of an auxochrome or by the change of

solvent.

• e.g. An auxochrome group like –OH, -OCH3 causes absorption of compound

at longer wavelength.

72
 1
Bathochromic Shift (Red Shift)
• In alkaline medium, p-nitrophenol shows red shift. Because
negatively charged oxygen delocalizes more effectively than the
unshared pair of electron.
O O - -
+ O + O
N N

-
OH

Alkaline

m edium -
O H O

p-nitrophenol
λmax = 255 nm λmax = 265 nm
73
 2
Hypsochromic Shift (Blue Shift)
• When absorption maxima (λmax) of a compound shifts to shorter
wavelength, it is known as hypsochromic shift or blue shift.

• The effect is due to presence of an group causes removal of

conjugation or by the change of solvent.

74
 2
Hypsochromic Shift (Blue Shift)
• Aniline shows blue shift in acidic medium, it loses
conjugation. + -
N H 2 + NH 3 Cl
H

Acidic

m edium

Aniline
λmax = 280 nm λmax = 265 nm

75
 Hyperchromic Effect
• 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.

N N CH 3

Pyridine
2-methyl pyridine
λmax = 257 nm
λmax = 260 nm
76
 Hypochromic Effect
• When absorption intensity (ε) of a compound is decreased, it is
known as hypochromic shift.

CH 3

Naphthalene 2-methyl naphthalene

ε = 19000 ε = 10250

77
 Shifts and Effects
Hyperchromic shift

Blue Red
Absorbance ( A )

shift
shift

Hypochromic shift

λmax
78
Wavelength ( λ )
Find the absorbance of a solution if its concentration is 1
mole/liter, the molar absorption coefficient is 6000 M/cm and
path length is 0.02 m.

79
Find the path length of a solution if its absorbance is 0.45,
the molar absorption coefficient is 3725 mM/cm and
concentration is 50 μM.

80
Find the concentration of a solution if its absorbance is 5.25,
the molar absorption coefficient is 2254 mM/cm and path
length is 300 nm.

81
Self-test

82
• In 1945 Robert Burns Woodward gave certain rules for

correlating λmax with molecular structure.

• In 1959 Louis Frederick Fieser modified these rules with more

experimental data, and the modified rule is known as

Woodward-Feiser Rules.

• It is used to calculate the position and λmax for a given

structure by relating the position and degree of substitution of

chromophore.

83
• Each type of diene or triene system is having a certain

fixed value at which absorption takes place; this

constitutes the Base value or Parent value.

• The contribution made by various alkyl substituents or

ring residue, double bond extending conjugation and

polar groups such as –Cl, -Br etc are added to the basic

value to obtain λmax for a particular compound.

84
 Woodward–Fieser Rules
 for Diene
Woodward (1941) predicted max values only for the
lowest energy transition ( π  π*) from HOMO to
LUMO.
 Base value for an unsubstituted, conjugated,
Base values:
acyclic or heteroannular diene 217 nm
 Base value for an unsubstituted, conjugated, 253 nm
homoannular
Increments for: diene
Each extra double bonds in conjugation + 30
nm
Exocyclic double bond (effect is two fold if the bond is exocyclic to
two rings) +5
nm
Substituent effect:
A. -OCOR or –OCOAr + 0 nm
B. Simple alkyl substituents or ring + 5 nm
residue
C. Halogen (-Cl, -Br) + 5 nm
D. OR (R=Alkyl) + 6 nm
E. SR (R=Alkyl) + 30 nm
F. NR2 (R=Alkyl) + 60 nm
85
 1 2
3

EtO

86
 1
2
3

1
2 3

87
 R

2
3
1
4
5

HOOC

2
5 4 3

AcO

88
 1

2
3
4
5

89
 Rules of Enone & Dienone
Absorption
Base values:
 Acyclic α,β-unsaturated ketones 215 nm
Woodward-Fieser Rule for Enones
 6-membered cyclic α,β-unsaturated 215 nm
ketones
 5-membered cyclic α,β-unsaturated 202 nm
ketones
 α,β-unsaturated aldehydes 210 nm
 α,β-unsaturated carboxylic acid & esters 195 nm
Increments
for:
Double bond extending conjugation (DEC): +30

Exocyclic double bond: +5

Homodiene component: +39

90
 Increments for:
Alkyl group/ring residue: α
+10
β

Woodward-Fieser γ & higher

+12 Rule for Enones
+18
Polar groups: -
OH: α
β +30
+35
δ +50
-OAc: α,β,γ +
-OMe: 6
α +35
β +30
γ +17
-SAlk: δ +31
β
-Cl: α +15
β
+85
-Br: α
+12
+25
β
-NR2: β
+30
+95

91
 O
1'
2
CH3
 
5
O 3
4


OH
HO 
  

92
 O
CH3
 O
OCOCH3
 


O
O
Br

31 93
O O

94
 Aromatic
Parent chromophore: Ar =
Compounds
C 6H 5
Ar-CO-R 246 nm
Ar-CHO 250 nm
Ar-COOH or Ar-COOR 230 nm

Increment for each substituent on

Ar: o, m + 3 nm
Alkyl or ring residue
p + 10 nm
OH, OCH3, OAlk o, m + 7 nm
p + 25 nm
NH2 o, m + 13 nm
p + 58 nm
NHCOCH3 o,m + 20 nm
p + 45 nm
NHMe p + 73 nm
NMe2 o, m + 20 nm
p + 85 nm
Cl o, m + 0 nm
p + 10 nm
Br o, m + 2 nm
p + 15 nm
95
 Aromatic Compounds
+25 +3
MeO

Cl
+0 +3 CO2Et

+7
OH O

OMe
MeO +25
+7
+3

96
 Violations of Woodward Rules
Bipheny
CH3
ls:
CH3

H3C CH3
CH3
H3C CH3
H3C CH3

CH3
250 237
nm 266
nm
nm
Not Substitutions cause loss of co-planarity of
planar orbitals. Loss of overlap, blue shift with
45 0C reduced intensity.
angle

97
 Steric Inhibition of Resonance
(1) In alkenes, trans isomers exhibit absorption at longer
wavelength and high intensity.
(2)Woodward rules are applicable only if there is no strain
around chromophore.

Cross
Ring
conjugation
strain
O

Steric inhibition of resonance

(3)If calculated and experimental values of max do not

match, one can say that the molecule must have some
strain.
(4)Thus UV can be used indirectly to determine if the
molecule has any strain.
98
 Home Work
Predict max values of the following compounds using
Fieser Woodward- R OMe
Me Me
MeO
rules: Me Me

O O
a b c

O
Me Me
CH3

CH3 AcO O
d e f

99
 Home Work

Match three steroid structures shown below to the

for hexane
following
ma : Compound
values A, 275 nm; B, 304 nm, and C,
356 nm.
x

C9H19 C9H19 C8H17

O O

H3C H3C HO
O 1 O 2 3

Partial hydrogenation of the triene shown

below results in two compounds, D and E, both of
molecular
shows a ma formula
hexane =235Cnm
10H14 . Compound
and E, 275 nm.DAssign the
structures.
x

A
100
 Instrumentation
Light sources

Wavelength selector

Sample compartment

Detector

Recorder or signal processor

101
Instrumentation

102
 Instrumentat
ion
Simple schematic that covers most modern UV
spectrometers:

log(I0/I) =
UV-VIS sources I0 I A

sampl
20 70

detecto
0 l, 0
nm

monochromator/

r
referenc
beam splitter I0 I0
optics

103
 Light sources
Various UV radiation sources are as
follows
a.Deuterium lamp
b.Hydrogen lamp
c.Tungsten lamp
d.Xenon discharge lamp
e.Mercury arc lamp
Various Visible radiation sources are as
follow
f. Tungsten lamp
g.Mercury vapour lamp
h.Carbonone lamp
104
• A spectrophotometric radiation source must provide a stable high energy output over a broad range of
wavelengths.

• Sources for UV Region

• For measurements in the UV region, electric discharge sources like hydrogen or a deuterium lamp are
used.
• In these, the excitation of the gaseous molecules is brought about by the passage of electrons through
the gas at low pressures.
• A hydrogen lamp is commonly used in the spectrophotometers and gives light in the wavelength
region of 160-375 nm.
• The radiant power of the hydrogen lamp is low and these are replaced by deuterium lamps but it
increases the cost of the instrument.

105
• Sources for Visible Region
• The modern instruments use a tungsten filament lamp as the radiation source.
• This consists of a thin, coiled tungsten wire that is sealed in an evacuated glass bulb.
• This gives radiations in the range of 350-2200 nm.
• As the output depends on the voltage, the tungsten lamp is energized by a 6 or 12 volt storage battery
or by the output of a constant voltage transformer.
• Now a days some instruments use tungsten-halogen lamps that contain a small amount of iodine in the
quartz bulb housing the tungsten filament. The iodine reacts with gaseous tungsten formed by
sublimation producing volatile WI2. When molecules of WI2 hit the filament , they decompose,
redepositing tungsten back onto the filament. Increase in life and efficiency of tungsten radiation
source
• The presence of iodine extends the output wavelength range of the lamp from 240 -2500 nm.

• In some spectrophotometers, Xenon flash lamps are also used which covers a broad range of
wavelength 180nm- 2000nm

106
Radiation sources: a) deuterium lamp for UV range b) tungsten lamp for
visible range

107
 Monochromat
• In spectrophotometric measurements weors
need to use a narrow band of wavelengths of light. Monochromators
are used for this purpose.

A monochromator is an optical device that transmits a mechanically selectable narrow band of wavelengths of
light or other radiation chosen from a wider range of wavelengths available at the input.

• Wavelength selector (monochromator)

• All monochromators contain the following component parts; An entrance slit ,A collimating lens ‘A dispersing
device (usually a prism or a grating) ,A focusing lens,An exit slit

• Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the
entrance slit. The beam is collimated, and then strikes the dispersing element at an angle. The beam is split into
its component wavelengths by the grating or prism.

• By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the
monochromator through the exit slit.

108
Monochromators

109
Different types of monochromators

1) Filters
2) Prisms
3) Diffraction gratings

110
 Instrumentation
Wavelength Selectors:
Absorption Filters:

• In low cost instruments catering to measurements in the visible range, coloured glass filters are used to cut off
undesirable wavelengths.

• A typical filter is a coloured piece of glass, which absorbs light of certain wavelength and allows that of the other to
pass through.

• When white light falls on an object, a part of it is absorbed and rest is transmitted.

• These transmitted components add up to give the observed colour of the object. The absorbed component and the
observed colour can again add up to give white light.

• These are therefore called as complementary to each other.

• An object of a particular colour looks of that colour because this colour is transmitted and its complementary colour is
absorbed.
111
• The colour of the filter should be the complementary to colour of the solution to be measured.

• If a solution appears orange, this implies that orange light is not being absorbed but that the
complement to orange i.e. blue is being absorbed.

• Therefore, for measuring absorbance of a orange solution you would employ a blue filter.

112
• In order to find the colour of the filter to be used we can take the help of a colour wheel.
• In a colour wheel, the colours, which face one another, are said to be complementary to each
other.
• In order to measure a red coloured solution, its complementary, that is, a green coloured filter
should be used.
• In the colour wheel P, S and T refer to the primary, secondary and tertiary colours, respectively.

113
 Interference filters

•The interference filters cover a wider range than the absorption filters.
•Interference filters are essentially composed of several optical layers
deposited on a glass substrate or transparent quartz.
•Such interference filters are available for ultraviolet, visible and near-
infrared region.
• The performance characteristics of interference filters are significantly
superior to those of absorption (coloured) filters.
•The effective bandwidths of these filters are narrower than absorption
filters.
•It uses the principle of constructive interference to increase the intensity of
desired wavelength and destructive interference to cancel out the
undesired wavelengths.
114
The critical physical phenomenon regulating the construction of interference
filters is the reflection that occurs when light is incident upon a smooth
interface between two transparent media.
Reflection characteristics at such an interface depend on the refractive
indices of the two materials, the angle of incidence, and the polarization
orientation (if any) of the incident light.
When light is incident upon the interface, a fraction of the light enters the
second medium and is refracted, while another portion is reflected at the
interface
The variation in reflectance that is generated by refractive index mismatches
between two adjoining media, combined with the phase changes that occur
upon reflection, provide a mechanism by which optical interference can be
exploited to modulate the transmission and reflection of desired wavelength
regions by an optical element

115
 Monochromato
rs
• Prism and grating monochromators

• A prism disperses sunlight into seven different colours. This occurs due to the refraction of the
light when it passes through the prism.

• The radiations of different colours having different wavelengths are refracted to different
extent due to the difference in the refractive index of glass for different wavelengths.

• Shorter wavelengths are refracted more than longer wavelengths

116
 Prism
monochromator
• If a prism is rotated, different wavelengths of the radiation,
coming out after refracting through it, can be made to pass
through the exit slit.
• In a prism monochromator, shown in Fig., a fine beam of the
light from the source is obtained by passing through an
entrance slit.
• This is then collimated on the prism with the help of a lens.
• The refracted beams are then focused on an exit slit.
• The prism is then rotated in a predetermined way to provide
the desired wavelength from the exit slit.

117
 Grating
Monochromators
• A grating is made by cutting or etching a series of closely spaced parallel grooves
on the smooth reflective surface of a solid material (200-2000 per mm).

• The surface is made reflective by making a thin film of aluminium on it and the
etching is done with the help of a suitably shaped diamond tool.
• In grating monochromator a fine beam of the light from the source falls on a
concave mirror through an entrance slit. This is then reflected on the grating
which disperses it. The dispersed radiation is then directed to an exit slit. The
range of wavelengths isolated by the monochromator is determined by the
extent of dispersion by the grating and the width of the exit slit. Rotation of the
grating in a predetermined way can be used to obtain the desired wavelength
from the exit slit.
• The intensity of radiation reflected by a grating varies with the wavelength, the
wavelength of maximum intensity being dependent on the angle from which the
radiation is reflected from the surface of the line of the grating.

118
Grating
Monochromators

119
Wavelength selector
Prism

Grating

120
 Gratings Prisms
The isolation of wavelength is because of the The isolation of wavelength is because of the
diffraction of wavelength. difference in the velocities of wavelengths inside
the prism.
Gratings are more efficient than prisms because of Prisms are less efficient than gratings because of
the higher degree of dispersion of radiation. the lower degree of dispersion of radiation.
Dispersion is linear. Hence fabrication of the Dispersion is non-linear. Hence fabrication of the
instrument is easy. instrument is difficult.

Manufacturing is tedious, hence cost is high. Manufacturing is simple, hence cost is low

Gratings are used in UV, Visible and IR regions. Prisms are mainly used in visible region.
Gratings produce higher order of spectra. Prisms do not produce higher order of spectra.

121
 Sample compartment

Quartz cell
Glass cell
Plastic cell ex. polystyrene

122
• In order to take the UV spectrum of the analyte it is taken in a cell called a cuvette
which is transparent to the wavelength of light passing through it.
• A variety of quartz cuvettes are available for the spectral determination in the vapour
phase.
• These are of varying path lengths and are equipped for measurements on solutions in
the visible region the cuvettes made of glass can also be used.
• However, since glass absorbs the ultraviolet radiations, these cannot be used in the
UV region.
• Therefore, most of the spectrophotometers employ quartz as these can be used for
both visible and UV region. Usually square cuvettes having internal path length 1.0 cm
are used, though cuvettes of much smaller path lengths say of 0.1 mm or 0.05 mm
are also available.

123
124
 Detector
Photo multiplier tube (PMT)
Phototube
Photodiode array detector
Barrier Layer Cell/ Photovoltaic
cell

125
•Phototube
•A phototube consists of a photoemissive cathode and an anode in an
evacuated tube with a quartz window
•These two electrodes are subjected to high voltage (about 100 V)
difference.
•When a photon enters the tube and strikes the cathode, an electron is
ejected and is attracted to the anode resulting in a flow of current which
can be amplified and measured.
•The response of the photoemissive material is wavelength dependent and
different phototubes are available for different regions of the spectrum.

126
 Photomultiplier (PM) Tube

• A photomultiplier tube consists of a series of electrodes, called dynodes.

• The voltage of successive electrodes is maintained 50 to 90 volt more
positive than the previous one.

• When a radiation falls on the cathode an electron is emitted from it. This is accelerated
towards the next photoemissive electrode by the potential difference between the two.
• Here, it releases many more secondary electrons.
• These, in turn are accelerated to the next electrode where each secondary electron
releases more electrons.
• The process continuous upto about 10 stages of amplification.
• The final output of the photomultiplier tube gives a much larger signal than the
photocell.

127
128
 Photodiode array Detectors
The photodiode array detector passes a wide spectrum of light through the sample and then the light is
separated into individual wavelengths after passing through the sample.
The spectrum of light is directed to an array of photosensitive diodes.
Each diode can measure a different wavelength which allows for the monitoring of many wavelengths
at once.
They can detect as many wavelengths simultaneously as their number of individual diodes

https://www.youtube.c
om/watch?v=IVHPnBeM
NYU

129
 Barrier Layer Cell/
Photovoltaic Cell
• It consist of a semiconductor such as selenium, which is deposited on metallic block of iron or copper.
Another surface of selenium is coated with a thin layer of silver or gold.
• The two metals form two electrodes of the detector.
• The entire assembly is enclosed in a transparent material.
• Both the electrodes are connected to ammeter to record the current in the circuit.
• After interaction with the sample, when transmitted radiation reaches the semiconductor, the covalent
bonds in the latter are broken, as a result of which electrons and the holes are liberated in the
semiconductor.
• Electrons migrate towards gold or silver and holes migrate towards block of iron.
• Electrons do not pass through the semiconducting selenium layer but are free to move through
external circuit into iron box.
• When two electrodes are connected to the ammeter, electrons accumulated on silver layer start
moving through the external circuit and produce electric current.
• The magnitude of the current produced depend upon no. of electrons produced, which in turn
depends upon energy of radiation reaching the detector.

130
Recorder or Signal Processor
Printer
Monitor
Computer

131
 Single Beam
Spectrophotometer
•The first step is to close the shutter and adjust the
scale to 0% T or infinite absorbance to offset the dark
current from the detector to zero
•The Second step is to open the shutter and place the
cell containing only the solvent in the light beam. The
scale is set to read 100% T ( equivalent to 0
absorbance)
•The third step is place the sample cell in the light path
and to measure the intensity of transmitted light (I T)
or the equivalent absorbance.
•Advantages of Single Beam Systems
•Single beam instruments are less expensive
•High energy throughput due to non-splitting of source
beam results in high sensitivity of detection
132
 Double Beam Spectrophotometer
• In a double beam spectrometer, the radiation coming from the monochromator is split into two beams
with the help of a beam splitter.
• These are passed simultaneously through the reference and the sample cell.
• The transmitted radiations are detected by the detectors and the difference in the signal at all the
wavelengths is suitably amplified and sent for the output.

133
•Advantages of Double Beam Systems
• Modern improvements in optics permit high level of automation and offer the same
or even better level of detection as compared to earlier single beam systems.
• Instability factors due to lamp drift, stray light, voltage fluctuations do not affect the
measurement in real-time.
• Little or no lamp warm up time is required. This not only improves throughput of
results but also conserves lamp life

134
 Applications of UV Visible spectroscopy

• Widely used in QC, QA and R&D department of Pharmaceutical industry

Qualitative analysis: The compounds which absorbs in UV/Vis radiation range can be identified by
comparing the resulted spectra with the spectra of known compounds (reference compound).
• Λmax can give an idea about the level of conjugation and unsaturation in the molecule.
Identification of a particular functional group is not possible

Quantitative analysis:
Assay methods for various APIs (which absorb radiations in the UV-Visible region) and their
formulations, single and multicomponent analysis
Certain compounds which may not absorb UV-visible radiations can be derivatised by a
chemical reaction in such a way that the resulting products can absorb UV-visible radiation and
hence quantitative analysis can be performed.

135
• Determination of dissociation constants of acids and bases: The pKa values of any acid or base can
be determined spectrophotometrically by plotting a graph between absorbance and wave length at
different pH values.
• Chemical kinetics study: UV/Vis spectroscopy can be used to study the kinetics of the reaction. To
determine kinetics of reaction the change in concentration of either a reactant or product with time is
measured. As absorbance is directly proportional to concentration, this technique helps to follow the
progress of the reaction.
• Charge-transfer transition: When charge transfer takes place between two chemical species, a
complex is formed. This formed complex absorbs at different wave length than the individual chemical
components.
• Tautomeric equilibrium analysis: UV/Vis spectroscopy can be used to determine the percentage of
various keto and enol forms present in in a tautomeric equilibrium.
e.g. Ethyl acetoacetate – the keto form has λmax 275 nm and ε = 16; whereas the enol form has
λmax 244 nm and ε = 16000.

136
Spectrophotometric Titrations

137
 Spectrophotometric titration

• The process of determining the quantity of a sample by adding measured increments

of a titrant until the end-point, at which essentially all of the sample has reacted, is
reached.

• The titration is followed by measuring the absorbance of radiation in the range

ultraviolet to near-infrared by the sample.

• If at least one component in a titration process absorbs electromagnetic radiation, we

can identify the end point by monitoring the titrand's absorbance at a carefully
selected wavelength

138
 Spectrophotometric titration
• For example, we can identify the end point for a titration of Cu 2+ with EDTA, in the presence of
NH3 by monitoring the titrand's absorbance at a wavelength of 745 nm, where the Cu(NH 3)42+
complex absorbs strongly.

• At the beginning of the titration the absorbance is at a maximum.

• As we add EDTA the concentration of Cu(NH 3)42+, and thus the absorbance, decreases as EDTA

displaces NH3.

• After the equivalence point the absorbance remains essentially unchanged.

Acorr = A × (VEDTA + VCu)/VCu

Note that the titration curve’s y-axis is not the actual absorbance, A, but a corrected absorbance, A corr

139
 Spectrophotometric titration

• Correcting the absorbance for the titrand’s dilution ensures that the
spectrophotometric titration curve consists of linear segments that we can
extrapolate to find the end point.

(a) only the titrant absorbs;

(b) only the titrant absorbs;
(c) only the product of the titration reaction
absorbs;
(d) both the titrand and the titrant absorb;
(e) both the titration reaction’s product and the
titrant absorb;
(f) only the indicator absorbs.
140
 Multi-component Analysis:
• The Spectrophotometric assay of drugs rarely involves the measurement of
absorbance of samples containing only one absorbing component.

• The pharmaceutical analyst frequently encounters the situation where the

concentration of one or more substances is required in samples known to
contain other absorbing substances, which potentially interfere in the assay.

• If the formula of the samples is known, the identity and concentration of the
interferents are known and the extent of interference in the assay may be
determined.

141
 Some of the commonly used Spectrophotometric methods

1. Simultaneous equation method (Vierdott’s method)

2. Derivative Spectrophotometric method
3. Absorbance ratio method ( Q-Absorbance method)
4. Solvent extraction method
5. Dual wavelength method
6. Geometric correction method
7. Orthogonal poly nominal method
8. H-point standard addition method
9. Least square approximation method

142
The basis of all the Spectrophotometric techniques for
multicomponent samples is the property that at all wavelengths:

• The absorbance of a solution is the sum of absorbance of the individual components

Or
• The measured absorbance is the difference between the total absorbance of the
solution in the sample cell and that of the solution in the reference cell.

• And most importantly the excipients present in the formulation are not absorbing at the
wavelength of experiment.

• If all of these conditions are satisfied than we can

apply these methods satisfactorily.

143
144
145
146
It depends on the property that for a substance which obeys Beer’s
law at all wavelengths, the ratio of absorbances at any two
wavelengths is a constant value independent of concentration or
pathlength

Q value (Ratio of absorbance at two wavelengths = A1/A2

147
147
 148
148
 149
149