This presentation covers UV region, principle, absorption law, chromophore & auxochrome, instrumentation & application.

1. Presentation
on
UV-Visible Spectroscopy
Dr. A. B. Roge
Department of Pharmaceutical Chemistry
Nanded Pharmacy College,
Nanded
05-03-2021
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2. Introduction
It is molecular absorption spectroscopy based on measurement UV–Visible radiation absorbed by molecules of the
substance under investigation.
UV region: 10 nm – 400 nm
Far UV region (vacuum region): 10 nm – 200 nm
Near UV region : 200 nm – 400 nm
Visible region : 400 nm – 800 nm
Principle: UV spectroscopy is an important tool in analytical chemistry. UV spectroscopy is type of absorption
spectroscopy in which light of ultra-violet region (200-400 nm.) is absorbed by the molecule. Absorption of the ultra-violet
radiations results in the excitation of the electrons from the ground state to higher energy state. The energy of the ultra-
violet radiation that are absorbed is equal to the energy difference between the ground state and higher energy states (ΔE
= hf). Molecules containing bonding and non-bonding electrons (n-electrons) can absorb energy in the form of ultraviolet
or visible light to excite these electrons to higher anti-bonding molecular orbitals. The more easily excited the electrons
(i.e. lower energy gap between the HOMO and the LUMO), the longer the wavelength of light it can absorb. There are
four possible types of transitions (π–π*, n–π*, σ–σ*, and n–σ*), and they can be ordered as follows :σ–σ* > n–σ* > π–π* >
n–π*.
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3. Absorption Law
The expression of Beer-Lambert law is-UV spectroscopy obeys the Beer-Lambert law, which states that: when a beam of
monochromatic light is passed through a solution of an absorbing substance, the rate of decrease of intensity of radiation
with thickness of the absorbing solution is proportional to the incident radiation as well as the concentration of the solution.
A = log (I0/I) = ECL
Where, A = absorbance
I0 = intensity of light incident upon sample cell
I = intensity of light leaving sample cell
C = molar concentration of solute
L = length of sample cell (cm.)
E = molar absorptivity
From the Beer-Lambert law it is clear that greater the number of molecules capable of absorbing light of
a given wavelength, the greater the extent of light absorption. This is the basic principle of UV
spectroscopy.
Lamberts law: Absorption of solution is directly proportional to thickness of medium. A = K x L
Beers law: Absorption of solution is directly proportional to concentration of solution A = K x C
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4. Deviations from Beers law:
As per beers law, Absorption of solution proportional to concentration of solution. However, sometimes
absorbance does not change proportionally with respect to concentration due to known causes refer as
deviations.
Concentration Deviation: Linear relation between Absorbance and concentration holds only in dilute solution. In
concentrated solution, molecules are very close & incident light is unequally distributed among molecules as
result some molecules failed to participate in absorption phenomenon. Absorption does not proportionally
increase even after concentration is increased.
Chemical Deviation: Absorption of substance at particular wavelength is characteristic to its chemical nature. If
chemical substance changes absorption also changes. Substances susceptible to hydrolysis / photo degradation
undergo chemical deviation during measurement causes deviations in absorbance.
Instrumental Deviation: As absorption coefficient is a function of wavelength, Beer-Lambert law holds good
only for monochromatic light. Change in wavelength will result in deviation for linearity between absorbance and
concentration.
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5. Chromophore and Auxochrome
Chromophore- Chromophore is color imparting group. Chromophore is defined as any isolated covalently
bonded group that shows a characteristic absorption in the ultraviolet or visible region (200-800 nm).
Chromophore can be divided into two groups-
a) Chromophore which contain pie (π) electrons and which undergo pie to pie* (π – π*) transitions.
Ethylene and acetylenes are the example of such chromophore.
b) Chromophore which contain both pie (π)and nonbonding (n) electrons. They undergo two types of transitions,
pie to pie* (π – π*) and nonbonding to pie* (n – π*). Carbonyl, nitriles, azo compounds, nitro compounds etc. are
the example of such chromophores.
Auxochrome- An auxochrome is color enhancing group. An auxochrome can be defined as any group which
does not itself act as a chromophore but whose presence brings about a shift of the absorption band towards the
longer wavelength of the spectrum. Auxochrome causes bathochromic shift due to its ability to extend conjugation
by sharing lone pair of electron present over heteroatom. –OH,-OR,-NH2,-NHR, -SH etc. are the examples of
auxochrome groups.
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6. Absorption and intensity shifts in the UV spectroscopy
There are four types of shifts observed in the UV spectroscopy-
Bathochromic effect- This type of shift is also known as red shift.
Bathochromic shift is an effect by virtue of which the absorption maximum is
shifted towards the longer wavelength due to the presence of an auxochrome
or change in solvents. The nonbonding to pie* transition of carbonyl compounds
observes bathochromic or red shift .
Hypsochromic shift- This effect is also known as blue shift. Hypsochromic
shift is an effect by virtue of which absorption maximum is shifted towards the
shorter wavelength. Generally it is caused due to the removal of conjugation or
by changing the polarity of the solvents.
Hyperchromic effect- Hyperchromic shift is an effect by virtue of which
absorption intensity increases. The introduction of an auxochrome in the
compound generally results in the hyperchromic effect.
Hypochromic effect- Hypochromic effect is defined as the effect by virtue of
intensity of absorption decreases. Hyperchromic effect occurs due to the
distortion of the geometry of the molecule / removal of auxochrome. 05-03-2021
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7. Instrumentation
Most of the modern UV spectrometers consist of the following parts-
Radiation Source- It is one of the important component of photometer. Deuterium lamp and Tungsten lamp are
commonly used as a source of UV radiations and Visible radiation respectively as they meets requirements of ideal
source i.e., Continuous emission, rugged, constant intensity radiation, wide range of wavelength.
Optical system: components of optical system and their function is as below
Mirror: Collect radiations emitted by light source and reflect to Lens.
Lens: Lens condensed scattered radiations and converted to single beam of light.
Entrance Slit: Beam of light passes through entrance slit where width beam of light is controlled and passes
to monochromator.
Monochromator / Filter: From beam of light , radiation of particular wavelength is isolated by absorption
transmission mechanism (filter) Or Dispersion mechanism (monochromator).
Exit slit: It allows isolated radiation to pass through sample tube. It is kept close to sample tube to avoid stray
radiation reaching sample.
Sample cells- They are used to keep solution in light path. Sample tubes are made glass (Visible region) , quartz
fused silica (UV region). They are available in different dimension rectangular. Sample tube with width 1 cm is
commonly used. Sample tube with lid are used to handle volatile sample. They have rough and transparent
surface. They are always hold on rough surface. 05-03-2021
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8.  Detector- Detectors are used to measure intensity of transmitted radiation with respect to incident intensity. Photo
detectors are used in UV spectroscopy. They convert light energy to electrical energy. They consist pair of electrode
enclosed in evacuated tube made of glass. Cathode is coated with photosensitive material e.g. cesium oxide in photo
tube, Selenium layer in photovoltaic cell. As soon as radiation strikes cathode it causes excitation of photosensitive
material and release electrons and generate current proportional to intensity of radiation reaches detector. e.g. Barrier
layer cell , Phototube & Photomultiplier tube (number of dynode between cathode & anode)
 Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled
to a small servomotor. Generally current generated in the photocells is of very low intensity, the main purpose of
amplifier is to amplify the signals many times so we can get clear and recordable signals.
 Recording devices- Most of the time amplifier is coupled to a pen recorder which is connected to the computer.
Computer stores all the data generated and produces the spectrum of the desired compound.
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9. Applications
1.Detection of functional groups- UV spectroscopy is used to detect the presence or absence of
chromophore in the compound. This is technique is not useful for the detection of chromophore in complex
compounds. The absence of a band at a particular band can be seen as an evidence for the absence of a
particular group. If the spectrum of a compound comes out to be transparent above 200 nm than it confirms
the absence of –a) Conjugation b) A carbonyl group c) Benzene or aromatic compound d) Bromo or iodo
atoms.
2.Detection of extent of conjugation- The extent of conjugation in the polyenes can be detected with the
help of UV spectroscopy. With the increase in double bonds the absorption shifts towards the longer
wavelength. If the double bond is increased by 8 in the polyenes then that polyene appears visible to the
human eye as the absorption comes in the visible region.
3.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.
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10. 4. Determination of configurations of geometrical isomers- It is observed that cis-alkenes absorb at
different wavelength than the trans-alkenes. The two isomers can be distinguished with each other when
one of the isomers has non-coplanar structure due to steric hindrances. The cis-isomer suffers distortion
and absorbs at lower wavelength as compared to trans-isomer .
5. Determination of the purity of a substance- Purity of a substance can also be determined with the
help of UV spectroscopy. The absorption of the sample solution is compared with the absorption of the
reference solution. The intensity of the absorption can be used for the relative calculation of the purity of
the sample substance.
6. Chemical kinetics: study of chemical reaction is carried out by UV spectroscopy. UV spectrum of
reaction mixture is recorded at regular interval of time. Initially spectrum containing bands of only
reactant. Over the period of time , spectra consist of bands of reactant and product. At end of reaction,
spectra contains band of only product.
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11. Thank you…..
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