UV-Visible Spectroscopy

Dr.M.Jagadeeswaran
Spectroscopy

Electromagnetic
radiation

Interaction

Atoms (or)
Molecule
Laws of Photometry

Io = Ia+It+Ir
Types of Spectroscopy
 Absorption spectroscopy
• UV-Visible Spectroscopy

• Infrared Spectroscopy

• Nuclear Magnetic Resonance Spectroscopy

• Atomic Absorption Spectroscopy

 Emission Spectroscopy
• Fluorimetry

• Flame Emission Spectroscopy

Principle
Types of electrons

• Sigma electrons (σ)

• Pi electrons (π)
 Types electronic transitions

σ σ* > n σ* > π π* > n π*

 σ σ* transitions
• An electron in a bonding σ orbital is excited to the
corresponding orbital
• High energy process because σ bonds are very
strong
• Ex: Methane can only undergo σ σ* transitions
Absorption - 150 nm
 n σ* transitions
• Saturated compounds containing atoms with
lone pairs are capable of n σ* transitions
• Need less energy than σ σ* transitions
• Ex: Methanol - 174 nm, Water - 167 nm, Methyl
chloride - 169 nm
• Amines absorb higher wavelength than alcohol
coz, extinction co-efficient of amine will be larger
 π π* Transitions
• Occurs in the unsaturated compounds
• Excitation of π electron requires smaller energy,
so transition occurs at longer wavelength
• Ex: Alkenes, alkynes, carbonyl compounds, azo…
• Conjugated alkenes (170-190 nm)
 n π* Transitions
• An electron of unshared pair on hetero atom gets
excited to π* antibonding orbital
• It requires least amount of energy than others
• Occurs at longer wavelength
• Ex: Saturated aldehydes
λmax
Types of shifts
Bathochromic shift
Hypsochromic shift
 Chromophore
• “Any isolated covalently bonded group that
shows a characteristic absorption in the UV or
Visible region”
 Auxochrome
• Any group which does not act as a chromophore but
whose presence brings about a shift of the
absorption band towards longer wavelength

• Color enhancing group

• Ex: -OH, -OR, -NH2 ,-NR2, ,-NHR, -SH etc..

• Effect of auxochrome due to its ability to extend the

conjugation of a chromophore by the sharing of
non-bonding electrons
Auxochrome
Beer & Lambert law
A α bc
A = abc
A = log Io/It
 Beer’s law
• The Beer-Lambert law (or Beer's law) is the
linear relationship between absorbance and
concentration of an absorbing species
• A is the measured absorbance
• a is a wavelength-dependent absorptivity
coefficient
• b is the pathlength
• c is the analyte concentration
 Lambert’s law
Mathematically, the Lambert’s law may be expressed as follows.
- dI / dt α I
-dI / dt = K I . . . . . . . . . .(1)
Where I = intensity of incident light
t = thickness of the medium
K= proportionality constant
By integration of equation (1), and putting I=I0 when t=0,
I0/ It = kt or It= I0 e-kt
Where, I0 = intensity of incident light
It = intensity of transmitted light
k = constant which depends upon wavelength and absorbing medium
used.
By changing the above equation from natural log, we get,
It = I0 e-Kt . . . . . . . . . . (2)
Where K = k/ 2.303
So, It = I0 e-0.4343 kt
It = I010-Kt . . . . . . . . . . (3)
 Beer’s law
The above sentence is very similar to Lambert’s law. So,
It = I0 e-k' c
It = I0 10-0.4343 k' c
It = I0 10 K' c . . . . . . . . . . (4)
Where k' and K'= proportionality constants, c = concentration
By combining equation (3) and (4), we get,
It = I0 10 -act
I0 / It = 10 act
Where, K and K' = a or ε
c = concentration
t or b = thickness of the medium
log I0 / It = εbc . . . . . . . . . . (5)
A = log I0 / It . . . . . . . . . (6)
A = εbc . . . . . . . . . (7)
A = abc
 Deviations of Beer’s law
• 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.
 Real Deviations
Concentration
At high concentrations, solute molecules can cause
different charge distribution on their neighboring
species in the solution.
Dilute solutions can be used
For e.g. methylene blue absorptivity at 436 nm fails to
observe Beer Lambert law even at concentrations as
low as 10μM.
High analyte concentrations can also possibly alter the
refractive index (η) of the solution which in turn could
affect the absorbance obtained
 Chemical deviation
• Chemical deviations occur due to chemical
phenomenon involving the analyte molecules
due to association, dissociation and interaction
with the solvent to produce a product with
different absorption characteristics.

• For example, phenol red undergoes a resonance

transformation when moving from the acidic
form (yellow) to the basic form (red).
 Instrumental Deviations

• Due to Polychromatic Radiation

• Due to Presence of Stray Radiation

• Due to Mismatched Cells or Cuvettes

 Solvent Effect
• The position and intensity of an absorption band
may differ when changing the solvents
• Conjugated dienes and aromatic hydrocarbons
have very little solvent shift
• α,β-unsaturated carbonyl compounds show two
different shifts
(a) The π π* band moves to longer wavelength
(b) The n π* band moves to shorter wavelength
Solvent Effect
 Solvent Effect
• The stabilization of nonbonding orbital is
particularly pronounced with hydrogen
bonding solvents
• π* orbitals are more stabilized by solvation
than are π orbitals, because π* orbitals are
more polar
• Net result is the energy of transition π π*
becomes less with solvation, while the energy
of transition π π* becomes greater.
 Instrumentation
• Light source

• Wavelength selector (Monochromator)

• Sample cell

• Detector
 Light source
• Tungsten filament lamp – VISIBLE (350-2000 nm)
It contains tungsten filament in a glass envelope
Life of the lamp is limited by the evaporation of
tungsten
Tungsten-halogen lamps are used in more expensive
instruments
• Deuterium discharge lamp – UV (185-380 nm)
It consists of two electrodes contained in a deuterium
filled silica envelope
When we pass a high voltage across the electrode
causes emission of deuterium
Five times greater light intensity than hydrogen lamps
 Tungsten Deuterium
filament lamp lamp
 Monochromator
• Filters
Glass filters are pieces of colored glass which
transmit limited wavelength.
The color is produced by incorporating oxides of
such metals as vanadium, manganese, iron,
nickel and copper in the glass.
Thus, a filter absorbing yellow light
(575-625 nm) appears blue.
The range of wavelengths transmitted is very
wide and may exceed 150 nm
Types of Filter
 Monochromator
• It is called as wavelength selector
• Polychromatic light is converted into
monochromatic light

Types
1. Prism
2. Gratings
 Prism Monochromator

❑ When a beam of monochromatic light passes through a

prism, it is bend or reflected
❑ The amount of deviation is dependent of wavelength, blue
light being more refracted more than red
❑ Prisms are made of quartz for use in UV region, since glass
absorbs wavelengths shorter than about 330 nm.
❑ Glass prisms are used in visible region
Echellette Grating
Grating monochromator
❑ It consists of a very large
number of equispaced lines
(50-6000 grooves/mm) ruled
on a glass blank coated with a
thin film of aluminium
❑ Transmission gratings or
reflection gratings are used
❑ d sinθ = nλ (n=0,1,2,3)
❑ Plane gratings are most
frequently used in
monochromators
❑ To eliminate the effect of
overlapping orders , filters
have sometimes to be used
 Sample cell

UV Visible
 Sample cell
• Cells are constructed with optically flat fused
glass may be used
• Measurements below the UV cutoff of glass at
about 320 nm require the use of more
expensive fused silica cells which are
transparent to below 180 nm
• Standard pathlength of cells for meausring the
absorption or fluorescence in the UV-Visible
range is 10 mm
 Detectors
• Photo tube
• Photomultiplier tube
• Photo voltaic cell
• Silicon Photodiode
Photo tube
 Photo tube – Working Principle
• Photocathode-anode combinations contained in
evacuated envelope
• It operates on the principle that electrons are
emitted from certain materials in direct
proportion to the number of phtons that strike
their surface
• Elements of high atomic volume e.g. K or Ce are
commonly used and in order to increase the
sensitivity, composite coatings such as
Ce/CeO/SiO
• Lack of linear response, In accuracy
Photomultiplier tube
 Photomultiplier tube - Working Principle
• Incident radiation ejects photoelectrons from the cathode
• The emitted photoelectrons are focused by an electrostatic
field and accelerated toward a curved electrode, the first
dynode coated with BeO, GaP or CsSb that ejects several
electrons
• Several anodes at a gradually increasing potential are
contained in one bulb
• The final photocurrent 106-108 times greater than the
primary current
• 90-120 voltage should maintain in the tube
• The current never falls to zero
• A small residual current called dark current is produced, due
to spontaneous discharge at the high voltage of the dynodes
Photo voltaic cell
 Photo voltaic cell – Working Principle
• It requires no power supply but gives a current which
under suitable conditions α light intensity
• It consists of a metallic plate, usually copper or iron,
upon which is deposited a layer of selenium
• Selenium and metallic oxides and sulphides have
extremely small electrical conductivities
• The current flow as shown by the deflection of the
galvanometer needle when the two electrodes are
connected
• The current output also depends upon the
wavelength of the incident light
• Lack of sensitivity
Silicon Photodiode
Silicon Photodiode – Working Principle
• The process starts with a very high resistivity
intrinsic silicon material
• Very shallow p and n diffusions are made in the
top and bottom surfaces, respectively
• Top surface covered with a protective SiO2 layer
• Metal contacts formed on the top and bottom
surfaces provide electrical connections
• A photon must reach the active area to produce
current flow in the external circuit
• Diode responsivity is typically 250-500 mA W-1
Single Beam Spectrophotometer
Double Beam Spectrophotometer
 Qualitative analysis
• Functional group analysis
• Impurity studies
• Quantitative analysis
• Chemical kinetics
• Differentiate the conjugated polyenes and
eneynes
• Charge-transfer transitions
 Applications
• Quantitative spectrophotomertic assay
Use of a standard absorptivity value
• Use of a calibration graph
 Single or double point standardization

Ctest = Atest x Cstd

Astd

Ctest = (Atest-Astd1)(Cstd1-Cstd2)+Cstd1(Astd1-Astd2)
Astd1 – Astd2
Std1 and std2 – more concentrated standard
and less concentrated standard respectively
Simultaneous equation method
 Woodward – Feiser Rules

• Woodward gave certain rules for correlating

λmax with molecular structure
• This rule can be used to calculate the position
of λmax and degree of substitution of
chromophore
• Mostly it used to calculate the conjugated
dienes, trienes and polyenes
Homoannular diene

Heteroannular diene
Total = 274 nm