UV-Visible

spectrophotometry
By Jagdish Singh (b.sc
microbiology
Principle of UV
Spectroscopy:
 Basically, spectroscopy is related to the
interaction of light with matter.
 As light is absorbed by matter, the result is an
increase in the energy content of the atoms or
molecules.
 When ultraviolet radiations are absorbed, this
results in the excitation of the electrons from the
ground state towards a higher energy
 Molecules containing π-electrons or nonbonding
electrons (n-electrons) can absorb energy in the
form of ultraviolet light to excite these electrons
to higher anti-bonding molecular orbital.
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Beer–Lambert law The Beer-Lambert law states that
there is a linear relationship
between the concentration and
the absorbance of the solution,
which enables the concentration
of a solution to be calculated by
measuring its absorbance.
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Conceptual Basis of Beer’s Law:
 Light of a particular wavelength enters the ‘sample’.
 Light scatters from particles in solution reducing light transmission
 Light is absorbed by molecules/particles and remitted at different
wavelengths, reducing light transmission.
 A = ECl
 A = absorbance of the sample
 I0 = intensity of the radiation
entering the sample.
 I = intensity of the radiation
emerging from the sample
 l = length of the light path through
the sample, in centimeter
 c = concentration of the sample, in
moles/liter
 ε = molar absorptivity (liter mol-1
cm-1 or M-1 cm-1 ) where M = mol
L-1
The amount of
absorbed radiation
may be measured in
different ways
Absorption spectrum:
Absorption spectrum represents the
dependence of absorbance from
wavelength.
From the absorption spectrum can be
determined: -
• The position of the absorption
maxima (λ max)
• The intensity of the absorption
maxima (A)
The calibration
curve:
For concentration determinations the
calibration curve (already existing or
determined in the laboratory) can be
used. According to the Beer-Lambert
Law, absorbance is proportional to
concentration.
To build a calibration curve:
• Plotted the absorbance versus
concentration A = f (c) to produce a
calibration ‘curve
• tain equation of the straight line (y =
mx + c) from line of ‘best fit’
Limits of Beer’s Law:
High Concentration and
Chemical Deviations:
The Beer-Lambert Law breaks
down for solutions of higher
concentration
For accurate results:
• Dilute the sample
• Use a narrower cuvette.
Electronic levels and transitions
In U.V spectroscopy molecule
undergo electronic transition
involving σ, π and n electrons .Four
types of electronic transition are
possible
1- σ ⇾ σ* transition
2- n ⇾ σ* transition
3- n ⇾ π* transition
4- π ⇾ π* transition
 Applications of UV Spectroscopy:
It is useful in the structure elucidation of organic molecules, such as in detecting the
presence or absence of unsaturation, the presence of hetero atoms.

UV absorption spectroscopy can be used for the quantitative determination of compounds

that absorb UV radiation.

This technique is used to detect the presence or absence of functional group in the
compound.

Kinetics of reaction can also be studied using UV spectroscopy

It is one of the best methods for determination of impurities in organic molecules