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UV-VISIBLE
spectroscopy
T.Y.B.Pharm
Spectroscopy is the study of the interaction
between matter and electromagnetic
radiation.
 spectroscopy originated through the study
of visible light dispersed according to
its wavelength, by a prism.
Later the concept was expanded greatly to
include any interaction with irradiative
energy as a function of its wavelength or
frequency.
The word spectroscopy implies that we will use the
electromagnetic spectrum to gain information about
organic molecules.
The other name of UV (Ultra-Violet) spectroscopy is
Electronic spectroscopy as it involves the promotion of the
electrons from the ground state to the higher energy or
excited state.
ultraviolet means that the information will come from a
specific region of the electromagnetic spectrum called the
ultraviolet region. The electromagnetic spectrum includes
all radiation that travels at the speed of light c (3 x 1010
cm/sec). The electromagnetic spectrum includes radio
waves, which have long wavelengths, x-rays, which have
short wavelengths, and visible light, which has
wavelengths between those of radio waves and x-rays.
x-rays are most energetic, visible light next,
and radio waves least energetic.
Thus, the shorter the wavelength, the
greater the energy of an electromagnetic
wave.
The heat excites some ground-state
electrons to higher energy levels, then when
the electrons “fall” back to the ground state,
they “emit” energy that corresponds to the
energy difference between the energy states
(orbitals) where the electrons are found
The UV-Vis Spectrometer
The basic idea behind UV-Vis Spectroscopy is to shine light
of varying wavelengths through a sample and to measure
the absorbance at each wavelength. Only the wavelengths
corresponding to the ΔE for an electronic transition will be
strongly absorbed.
A UV-Vis spectrum plots absorbance (or its inverse,
transmittance) of the sample versus wavelength
Here’s the spectrum for ethene. [In this case the wavelength is plotted
versus transmittance, the inverse of absorbance (high absorbance = low transmittance,
and vice versa). ]
Note that the wavelength of maximum transmittance is at 174 nm. We call
this λmax , pronounced “lambda max”. Very little light passes through the sample at this
wavelength, because the wavelength corresponds very closely to ΔE for the π to π*
transition.
For example, knowing that the λmax for ethene is at
174 nm allows us to calculate the energy gap ΔE ,
which turns out to be about 164 kcal/mol.
As the number of conjugated pi bonds increases, the λmax increases as well!
Because longer frequency = smaller energy, this means that the energy
gap ΔE between the highest-occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO)decreases as the number of conjugated pi
bonds increases
The ultraviolet region falls in the range
between 190-380 nm, the visible region fall
between 380-750 nm.
The following electronic transitions are
possible:
π- π* (pi to pi star transition)
n - π* (n to pi star transition)
σ - σ * (sigma to sigma star transition)
n - σ * (n to sigma star transition)
and are shown in the below hypothetical
energy diagram
The σ to σ* transition requires an absorption of a photon with a wavelength which
does not fall in the UV-vis range (see table 2 below). Thus, only π to π* and n to π*
transitions occur in the UV-vis region are observed.
σ to σ* > n to σ* > π to π* > n to π*
highest energy lowest energy
(lowest wavelength) (lowest wavelength)
σ to σ*= c-c(alkanes)
π to π*=c=c ,or triple bond(alkenes, alkynes)
n to π*=c=o(carbonyl compounds)
How Does λmax Relate To The Color We
Perceive?
How does the wavelength of maximum absorbance (λmax)
relate to the actual color?
First, a refresher from the last post. We see the
complementary colour of the major color that is
absorbed. A molecule that absorbs in the blue will
appear orange, because we perceive the colors that
are reflected, and orange is the complementary color of
blue.
For example, this molecule, Rhodamine B [note 2] absorbs
at about 560 nm (green) and appears red , the
complimentary color of green.
Solvent
lower limit
(nm)
Acetonitrile 190
Chloroform 240
Cyclohexane 205
95% Ethanol 205
n-Hexane 195
Methanol 205
Water 190
1. Solvents used in UV-Vis spectroscopy (near UV)
Compound λ(nm) Intensity/ε
transition with
lowest energy
CH4 122 intense σ-σ*(C-H)
CH3CH3 130 intense σ-σ* (C-C)
CH3OH 183 200 n-σ* (C-O)
CH3SH 235 180 n-σ* (C-S)
CH3NH2 210 800 n-σ* (C-N)
CH3Cl 173 200 n-σ* (C-Cl)
CH3I 258 380 n-σ* (C-I)
CH2=CH2 165 16000 π-π* (C=C)
CH3COCH3 187 950 π-π* (C=O)
273 14 n-π* (C=O)
CH3CSCH3 460 weak n-π* (C=S)
CH3N=NCH3 347 15 n-π* (N=N)
a. Common functional groups
Introduction to UV spectroscopy-
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 (deltaE =
hf).
Generally, the most favored transition is from the
highest occupied molecular orbital (HOMO) to lowest
unoccupied molecular orbital (LUMO).
For most of the molecules, the lowest energy occupied
molecular orbitals are s orbital, which correspond to
sigma bonds.
 The p orbitals are at somewhat higher energy levels,
the orbitals (nonbonding orbitals) with unshared paired
of electrons lie at higher energy levels.
The unoccupied or antibonding orbitals (pie*and
sigma*) are the highest energy occupied orbitals.
Some of the important transitions with increasing
energies are: nonbonding to pie*, nonbonding to sigma*,
pie to pie*, sigma to pie* and sigma to sigma*.
Principle of UV spectroscopy
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.
The expression of Beer-Lambert law is-
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.
Instrumentation and working of UV
Light Source- Tungsten filament lamps and Hydrogen-Deuterium
lamps are most widely used and suitable light source as they cover the
whole UV region. Tungsten filament lamps are rich in red radiations;
more specifically they emit the radiations of 375 nm, while the
intensity of Hydrogen-Deuterium lamps falls below 375 nm.
Monochromator- Monochromators generally composed of prisms
and slits. The most of the spectrophotometers are double beam
spectrophotometers. The radiation emitted from the primary source
is dispersed with the help of rotating prisms. The various wavelengths
of the light source which are separated by the prism are then selected
by the slits such the rotation of the prism results in a series of
continuously increasing wavelength to pass through the slits for
recording purpose. The beam selected by the slit is monochromatic
and further divided into two beams with the help of another prism.
Sample and reference cells- One of the two divided
beams is passed through the sample solution and second
beam is passé through the reference solution. Both
sample and reference solution are contained in the cells.
These cells are made of either silica or quartz. Glass can't
be used for the cells as it also absorbs light in the UV
region.
Detector- Generally two photocells serve the purpose of
detector in UV spectroscopy. One of the photocell
receives the beam from sample cell and second detector
receives the beam from the reference. The intensity of the
radiation from the reference cell is stronger than the
beam of sample cell. This results in the generation of
pulsating or alternating currents in the photocells.
Amplifier- The alternating current generated in the
photocells is transferred to the amplifier. The
amplifier is coupled to a small servometer. 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
Concept of Chromophore and Auxochrome in the UV
Chromophore- Chromophore is defined as any isolated covalently bonded group that
shows a characteristic absorption in the ultraviolet or visible region (200-800 nm).
Chromophores can be divided into two groups-
a) Chromophores which contain p electrons and which undergo pie to
pie* transitions. Ethylenes and acetylenes are the example of such chromophores.
b) Chromophores which contain both p and nonbonding electrons. They undergo two
types of transitions; pie to pie* and nonbonding to pie*. Carbonyl, nitriles, azo
compounds, nitro compounds etc. are the example of such chromophores.
Auxochromes- 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. –OH,-OR,-NH2,-NHR, -SH etc. are the
examples of auxochromic groups.
Absorption and intensity shifts in the UV spectroscopy
a) 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.
b) 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.
c) Hyperchromic effect- Hyperchromic shift is an effect by virtue of which absorption
maximum increases. The introduction of an auxochrome in the compound generally results
in the hyperchromic effect.
d) Hypochromic effect- Hyperchromic effect is defined as the effect by virtue of intensity of
absorption maximum decreases. Hyperchromic effect occurs due to the distortion of the
geometry of the molecule with an introduction of new group.
Chromophores-
unsaturated groups like c=o,c=c
Auxochromes-
saturated groups contains non bonding
electrons to chromophore
Solvent Effect
Solvents play an important role in UV
spectra. Compound peak could be obscured
by the solvent peak. So a most suitable
solvent is one that does not itself get
absorbed in the region under investigation.
A solvent should be transparent in a
particular region. A dilute solution of sample
is always prepared for analysis. Most
commonly used solvents are as follows.
Solvent λ of absorption
Water 191 nm
Ether 215 nm
Methanol 203 nm
Ethanol 204 nm
Chloroform 237 nm
Carbon
tetrachloride
265 nm
Benzene 280 nm
Tetrahydrofuran 220 nm
Applications of UV spectroscopy
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.
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.
Uv spectroscopy
Uv spectroscopy
Uv spectroscopy

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Uv spectroscopy

  • 2. Spectroscopy is the study of the interaction between matter and electromagnetic radiation.  spectroscopy originated through the study of visible light dispersed according to its wavelength, by a prism. Later the concept was expanded greatly to include any interaction with irradiative energy as a function of its wavelength or frequency.
  • 3. The word spectroscopy implies that we will use the electromagnetic spectrum to gain information about organic molecules. The other name of UV (Ultra-Violet) spectroscopy is Electronic spectroscopy as it involves the promotion of the electrons from the ground state to the higher energy or excited state. ultraviolet means that the information will come from a specific region of the electromagnetic spectrum called the ultraviolet region. The electromagnetic spectrum includes all radiation that travels at the speed of light c (3 x 1010 cm/sec). The electromagnetic spectrum includes radio waves, which have long wavelengths, x-rays, which have short wavelengths, and visible light, which has wavelengths between those of radio waves and x-rays.
  • 4. x-rays are most energetic, visible light next, and radio waves least energetic. Thus, the shorter the wavelength, the greater the energy of an electromagnetic wave. The heat excites some ground-state electrons to higher energy levels, then when the electrons “fall” back to the ground state, they “emit” energy that corresponds to the energy difference between the energy states (orbitals) where the electrons are found
  • 5.
  • 6.
  • 7.
  • 8. The UV-Vis Spectrometer The basic idea behind UV-Vis Spectroscopy is to shine light of varying wavelengths through a sample and to measure the absorbance at each wavelength. Only the wavelengths corresponding to the ΔE for an electronic transition will be strongly absorbed. A UV-Vis spectrum plots absorbance (or its inverse, transmittance) of the sample versus wavelength
  • 9. Here’s the spectrum for ethene. [In this case the wavelength is plotted versus transmittance, the inverse of absorbance (high absorbance = low transmittance, and vice versa). ] Note that the wavelength of maximum transmittance is at 174 nm. We call this λmax , pronounced “lambda max”. Very little light passes through the sample at this wavelength, because the wavelength corresponds very closely to ΔE for the π to π* transition.
  • 10. For example, knowing that the λmax for ethene is at 174 nm allows us to calculate the energy gap ΔE , which turns out to be about 164 kcal/mol.
  • 11. As the number of conjugated pi bonds increases, the λmax increases as well! Because longer frequency = smaller energy, this means that the energy gap ΔE between the highest-occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO)decreases as the number of conjugated pi bonds increases
  • 12. The ultraviolet region falls in the range between 190-380 nm, the visible region fall between 380-750 nm. The following electronic transitions are possible: π- π* (pi to pi star transition) n - π* (n to pi star transition) σ - σ * (sigma to sigma star transition) n - σ * (n to sigma star transition) and are shown in the below hypothetical energy diagram
  • 13. The σ to σ* transition requires an absorption of a photon with a wavelength which does not fall in the UV-vis range (see table 2 below). Thus, only π to π* and n to π* transitions occur in the UV-vis region are observed.
  • 14. σ to σ* > n to σ* > π to π* > n to π* highest energy lowest energy (lowest wavelength) (lowest wavelength) σ to σ*= c-c(alkanes) π to π*=c=c ,or triple bond(alkenes, alkynes) n to π*=c=o(carbonyl compounds)
  • 15. How Does λmax Relate To The Color We Perceive? How does the wavelength of maximum absorbance (λmax) relate to the actual color? First, a refresher from the last post. We see the complementary colour of the major color that is absorbed. A molecule that absorbs in the blue will appear orange, because we perceive the colors that are reflected, and orange is the complementary color of blue. For example, this molecule, Rhodamine B [note 2] absorbs at about 560 nm (green) and appears red , the complimentary color of green.
  • 16.
  • 17. Solvent lower limit (nm) Acetonitrile 190 Chloroform 240 Cyclohexane 205 95% Ethanol 205 n-Hexane 195 Methanol 205 Water 190 1. Solvents used in UV-Vis spectroscopy (near UV)
  • 18. Compound λ(nm) Intensity/ε transition with lowest energy CH4 122 intense σ-σ*(C-H) CH3CH3 130 intense σ-σ* (C-C) CH3OH 183 200 n-σ* (C-O) CH3SH 235 180 n-σ* (C-S) CH3NH2 210 800 n-σ* (C-N) CH3Cl 173 200 n-σ* (C-Cl) CH3I 258 380 n-σ* (C-I) CH2=CH2 165 16000 π-π* (C=C) CH3COCH3 187 950 π-π* (C=O) 273 14 n-π* (C=O) CH3CSCH3 460 weak n-π* (C=S) CH3N=NCH3 347 15 n-π* (N=N) a. Common functional groups
  • 19. Introduction to UV spectroscopy- 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 (deltaE = hf).
  • 20. Generally, the most favored transition is from the highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO). For most of the molecules, the lowest energy occupied molecular orbitals are s orbital, which correspond to sigma bonds.  The p orbitals are at somewhat higher energy levels, the orbitals (nonbonding orbitals) with unshared paired of electrons lie at higher energy levels. The unoccupied or antibonding orbitals (pie*and sigma*) are the highest energy occupied orbitals. Some of the important transitions with increasing energies are: nonbonding to pie*, nonbonding to sigma*, pie to pie*, sigma to pie* and sigma to sigma*.
  • 21. Principle of UV spectroscopy 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. The expression of Beer-Lambert law is- 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.
  • 22. Instrumentation and working of UV Light Source- Tungsten filament lamps and Hydrogen-Deuterium lamps are most widely used and suitable light source as they cover the whole UV region. Tungsten filament lamps are rich in red radiations; more specifically they emit the radiations of 375 nm, while the intensity of Hydrogen-Deuterium lamps falls below 375 nm. Monochromator- Monochromators generally composed of prisms and slits. The most of the spectrophotometers are double beam spectrophotometers. The radiation emitted from the primary source is dispersed with the help of rotating prisms. The various wavelengths of the light source which are separated by the prism are then selected by the slits such the rotation of the prism results in a series of continuously increasing wavelength to pass through the slits for recording purpose. The beam selected by the slit is monochromatic and further divided into two beams with the help of another prism.
  • 23. Sample and reference cells- One of the two divided beams is passed through the sample solution and second beam is passé through the reference solution. Both sample and reference solution are contained in the cells. These cells are made of either silica or quartz. Glass can't be used for the cells as it also absorbs light in the UV region. Detector- Generally two photocells serve the purpose of detector in UV spectroscopy. One of the photocell receives the beam from sample cell and second detector receives the beam from the reference. The intensity of the radiation from the reference cell is stronger than the beam of sample cell. This results in the generation of pulsating or alternating currents in the photocells.
  • 24. Amplifier- The alternating current generated in the photocells is transferred to the amplifier. The amplifier is coupled to a small servometer. 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
  • 25.
  • 26.
  • 27. Concept of Chromophore and Auxochrome in the UV Chromophore- Chromophore is defined as any isolated covalently bonded group that shows a characteristic absorption in the ultraviolet or visible region (200-800 nm). Chromophores can be divided into two groups- a) Chromophores which contain p electrons and which undergo pie to pie* transitions. Ethylenes and acetylenes are the example of such chromophores. b) Chromophores which contain both p and nonbonding electrons. They undergo two types of transitions; pie to pie* and nonbonding to pie*. Carbonyl, nitriles, azo compounds, nitro compounds etc. are the example of such chromophores. Auxochromes- 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. –OH,-OR,-NH2,-NHR, -SH etc. are the examples of auxochromic groups.
  • 28. Absorption and intensity shifts in the UV spectroscopy a) 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. b) 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. c) Hyperchromic effect- Hyperchromic shift is an effect by virtue of which absorption maximum increases. The introduction of an auxochrome in the compound generally results in the hyperchromic effect. d) Hypochromic effect- Hyperchromic effect is defined as the effect by virtue of intensity of absorption maximum decreases. Hyperchromic effect occurs due to the distortion of the geometry of the molecule with an introduction of new group.
  • 29.
  • 30. Chromophores- unsaturated groups like c=o,c=c Auxochromes- saturated groups contains non bonding electrons to chromophore
  • 31. Solvent Effect Solvents play an important role in UV spectra. Compound peak could be obscured by the solvent peak. So a most suitable solvent is one that does not itself get absorbed in the region under investigation. A solvent should be transparent in a particular region. A dilute solution of sample is always prepared for analysis. Most commonly used solvents are as follows.
  • 32. Solvent λ of absorption Water 191 nm Ether 215 nm Methanol 203 nm Ethanol 204 nm Chloroform 237 nm Carbon tetrachloride 265 nm Benzene 280 nm Tetrahydrofuran 220 nm
  • 33. Applications of UV spectroscopy 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.
  • 34. 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. 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.