UV-VISIBLE SPECTROSCOPY

PRESENTED BY
RESHMI PAUL
VP22PHAR0300002
DEPARTMENT OF PHARMACEUTICAL CHEMISTRY
GITAM DEEMED TO BE UNIVERSITY
VISHAKHAPATNAM
 CONTENTS

 INTRODUCTION
 THEORY
 LAWS
 INSTRUMENTATION
 CHOICE OF SOLVENTS
 EFFECT OF SOLVENTS
 APPLICATION

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 INTRODUCTION
 UV spectroscopy is concerned with the
study of absorption of UV radiation
which ranges from 200nm to 400nm,
colored compounds absorb the radiation
from 400nm to 800nm(visible region).

• Colorless compounds absorb the radiation

at UV region. In both UV spectroscopy and
visible spectroscopy, the valence electrons
absorb energy and there by molecules Fig- Double beam Uv-vis spectroscopy

undergo transition from ground state to

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excited state.
 THEORY

Principle

 It is the measurement and interpretation of

Electromagnetic radiation absorbed or emitted
when the molecules or atoms or ions of sample
move from one energy state to another.

 Electromagnetic radiation is given by:

E = hʋ

Where, E = energy (in joules)h = Planck's constant ʋ

ʋ=frequency
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ELECTRONIC TRANSITIONS
 The absorption of light by a sample in the ultraviolet or visible region is
accompanied by a change in the electronic state of the molecules in the sample.

 Any molecule has either n, л or combination of these electrons.

 σ-σ⁎ transitions:

 σ electron from orbital is excited to corresponding anti- bonding orbital σ*.

 High energy required is for this transition.

 Example: Methane (CH,) has C-H bond only and can undergo σ-σ* transition

 shows absorbance maxima at 125nm.

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 Cont.…
 л-л*transitions:

 л electron in a bonding orbital is excited to corresponding anti-bonding orbital л⁎

 Compounds containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic
compounds etc undergo л-л*transitions.

 Shows absorbance maxima at 170-205 nm.

 n-σ⁎transitions:

 Saturated compounds containing atoms with lone pair of electrons like O, N, S shows n-
σ⁎transitions.

 Requires less energy than σ-σ⁎ transitions. And shows absorbance maxima at 150-250nm.
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Cont.….

 n-л⁎ transitions:

 An electron from non-bonding orbital is

promoted to anti-bonding л⁎ orbital.

 Compounds containing double bond involving

hetero atoms(C=0, C≡N, N≡O) undergo such
transitions.

 Requires minimum energy and show absorbance

maxima at 300 nm.

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ABSORPTION & INTENSITY SHIFT

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 LAWS OF UV-VIS SPECTROSCOPY
 Lambert’s law: When a beam of monochromatic radiation Lambert’s law- A=log10(I0/I)αl
is passed through the absorbing medium then the decrease Aαl
in the intensity of the radiation will be directly A=
proportional to the thickness or pathlength of the solution.
 Beer’s law: When a beam of monochromatic radiation is Beer’s law - A=log10(I0/I)αc
passed through the absorbing medium then the decrease in Aαc
the intensity of the radiation will be directly proportional A=
to the concentration of the solution.
Lambert’s & Beer’s law -
 Lambert-Beer’s law:When the beam of monochromatic A=log10(I0/I)αl*c
radiation is passed through the absorbing medium then the Aαc
decrease in the intensity of the radiation will be directly A=
proportional to the thickness as well as concentration of Where, l= pathlength, c=
the solution. concentration, = molar absorptivity
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 INSTRUMENTATION
Components of UV- Vis Spectroscopy:
 Light source – there are mainly two types of lamps used in radiation source H 2-D2 lamp,
Tungsten filament lamp.
 Filters and Monochromators –There are various parts of monochromator: Entrance slit,
Collimating lens, Gratings/Prism, Reflecting lens , Exit slit.
 Sample cell
 Detectors- detectors used are- Photomultiplier tube, photodiode array, charged coupl.ed
detectors.
 Amplifiers
 Recording device
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Cont.…

Fig- Instrumentation of UV-Vis spectrophotometer

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 CHOICE OF SOLVENTS

 A solvent is a liquid that dissolves another solid, liquid or gaseous solute resulting in a
solution at specified temperature.

 Solvents can be broadly classified into two categories: polar and non polar.

 A drug may show varied spectrum at particular wavelength in one particular condition
but shall absorb partially at the same wavelength in another conditions.

 The changes in the spectrum are due to: Nature of solvent, Nature of absorption band,
Nature of solute.

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 Common solvents used in UV spectra:

SOLVENT WAVELENGTH(nm)
Water 205
Methanol 210
Ethanol 210
Ether 210
Cyclohexane 210
Dichloromethane 220

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 EFFECT OF SOLVENT

 A solvent exerts a profound influence on the quality and shape of spectrum.

 The absorption spectrum of pharmaceutical substance depends practically

upon the solvent that has to been employed to solubilize the substance.

 A drug may absorb a maximum radiation energy at particular wavelength in

one solvent but shall absorb partially at the same wavelength in another
solvent.

 Example: Acetone in n-hexane λmax at 279nm.

Acetone in water λmax at 264nm.

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 APPLICATIONS

 When the word spectroscopy is used it means that we are going to use electromagnetic
spectrum to get information organic molecules. The range of UV region is from 200-
400nm and that of visible region is 400-800mm.

 Following are the applications of UV-visible spectroscopy:

1)Detection of Impurities:

 UV absorption spectroscopy is one of the best methods to determine the impurities in

organic molecules. Additional peaks can be observed due to impurities in the sample and it
can be compared with that of standard raw material. By also measuring the absorbance at
specific wavelength, the impurities can be detected.
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Cont.…

2) Quantitative analysis:
 UV absorption spectroscopy can be used for the quantitative determination of

compounds that absorb UV radiation. It is determined by using beer's law which is

given as:

A log 10/It=log 1/T=-log T=abc =£bc

Where:£ -is extinction co-efficient, c- is concentration, and b- is the length of the cell

that is used in UV spectrophotometer.

3)Qualitative analysis: UV absorption spectroscopy can characterize those types of

compounds which absorbs UV radiation. Identification is done by comparing the

absorption spectrum with the spectra of known compounds.

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Cont.…

4)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.

5)Detection of functional groups: This technique is used to detect the presence or absence of
functional group in the compound Absence of a band at particular wavelength regarded as an
evidence for absence of particular group.

6)Quantitative analysis of pharmaceutical substances: Many drugs are either in the form of
raw material or in the form of formulation. They can be assayed by making a suitable solution
of the drug in a solvent and measuring the absorbance at specific wavelength.

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 Cont.…

7)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.

8) Molecular weight determination: Molecular weights of compounds can be measured

spectrophotometrically by preparing the suitable derivatives of these compounds. For
example, if we want to determine the molecular weight of amine then it is converted in to
amine picrate. Then known concentration of amine picrate is dissolved in a liter of solution
and its optical density is measured at Amax 380 nm.
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THANK YOU