UV- Visible Spectrophotometry: Introduction, Spectral Shifts, Instrumentation and Applications.pptx

1
UV-VISIBLE SPECTROSCOPY
Presented by- Jaskiran Kaur
Assistant Professor
SCOP, Barabanki

2
CONTENTS
1. Introduction
2. The Nature Of Electronic Excitations
3. Electronic Transitions
4. Chromophores
5. Auxochromes
6. Spectral Shifts
7. Solvent Effects On The Absorption Spectra
8. Beer and Lambert’s Law
9. Instrumentation
10. Applications

3
INTRODUCTION
Spectroscopy is the measurement and interpretation of Electro Magnetic Radiation (EMR)
absorbed or emitted when the molecules or atoms or ions of a sample move from one energy
state to another energy state.
This change may be from Ground State to excited state or excited state to Ground state.
At ground state, the energy of a molecule is the sum total of rotational, Vibrational and
electronic energies.
In other words, spectroscopy measures the changes in rotational, vibrational and /or
electronic energies.

6
THE NATURE OF ELECTRONIC
EXCITATIONS
• When continuous radiation passes through a transparent material, a portion of the radiation may
be absorbed.
• If that occurs, the residual radiation, when it is passed through a prism, yields a spectrum with
gaps in it, called an absorption spectrum.
• As a result of energy absorption, atoms or molecules pass from a state of low energy (the initial,
or ground, state) to a state of higher energy (the excited state).
• The electromagnetic radiation that is absorbed has energy exactly equal to the energy difference
between the excited and ground states.
• In the case of ultraviolet and visible spectroscopy, the transitions that result in the absorption of
electromagnetic radiation in this region of the spectrum are transitions between electronic
energy levels.
• As a molecule absorbs energy, an electron is promoted from an occupied orbital to an
unoccupied orbital of greater potential energy.

8
ELECTRONIC TRANSITIONS
• The electrons that contribute to the absorption characteristics of an organic molecule are:
1. Bonding electrons: These electrons directly participate in bond formation.
2. Non-bonding electrons: Unshared outer electrons that are localized about atoms such as
O, N, Halogens (X), S.
There are different types of electrons contributing electronic transition.
• Sigma (σ) electrons
• Pi (π) electrons
• Non-bonding (n) electrons
• Anti -Bonding (*)

9
• Sigma (σ) electrons: Saturated bonds only contain sigma electrons which are held very
tightly resulting in very high bond strengths. Compounds containing only σ -electrons are
useful as solvents for near UV region. Example : Saturated hydrocarbons, alkanes like n-
hexane are used as solvents in UV region.
• Pi (π ) electrons: Pi electrons are present in unsaturated compounds like alkenes, alkynes
and aromatic compounds. Examples include all alkenes, alkynes and aromatic
compounds.
• Non-bonding (n) electrons: Non-bonding electrons are less fimly held unshared outer
electrons localized on hetero atoms such as oxygen, nitrogen, sulphur or halogens.
• Sigma electron > Pi electron > Non-bonding electron.
• Anti-Bonding electrons: The unoccupied, or antibonding orbitals (π* and *), are the
orbitals of highest energy.

10
Types of Electronic Transitions
1. σ → σ* transition
2. π → π* transition
3. n → σ* transition
4. n → π* transition

11
1. σ → σ* transition
σ electron from orbital is excited to corresponding anti-bonding orbital σ*. The energy required
is large for this transition. e.g. Methane (CH4) has C-H bond only and can undergo σ → σ*
transition and shows absorbance maxima at 125 nm.
2. π → π* transition
π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*. Compounds
containing multiple bonds like alkenes, alkynes, carbonyl, nitriles, aromatic compounds, etc
undergo π → π* transitions. e.g. Alkenes generally absorb in the region 170 to 205 nm.
3. n → σ* transition
Saturated compounds containing atoms with lone pair oaf electrons like O, N, S and halogens
are capable of n → σ* transition.These transitions usually require less energy than σ → σ*
transitions. The number of organic functional groups with n → σ* peaks in UV region is small
(150 – 250 nm).

12
4. n → π* transition
An electron from non-bonding orbital is promoted to anti-bonding π* orbital.
Compounds containing double bond involving hetero atoms (C=O, C≡N, N=O) undergo
such transitions. n → π* transitions require minimum energy and show absorption at
longer wavelength around 300 nm.

13
CHROMOPHORES
Absorption of light in UV-visible region gives rise to absorption spectra. This spectrum
arises due to transitions among electronic energy levels (Eo to E1) of certain groups present
in the molecule.
This group of atoms which is responsible for the absorption in UV-visible region is known
as the chromophore.
Mostly the unsaturated groups, heteroatoms or heteroatoms containing lone pair of electrons
are the possible chromophores.
These chromophores show absorption in certain region of spectrum (Say from 400 to 200
am), however at particular wavelength it shows maximum absorption (say at 254 nm) and
that wavelength is called as “Wavelength of maximum absorption.” It is denoted by max.
The nuclei determine the strength with which the electrons are bound and thus influence the
energy spacing between ground and excited states.

14
Hence, the characteristic energy of a transition and the wavelength of radiation absorbed
are properties of a group of atoms rather than of electrons themselves.
The group of atoms producing such an absorption is called a chromophore.
 As structural changes occur in a chromophore, the exact energy and intensity of the
absorption are expected to change accordingly.

15
AUXOCHROMES
The attachment of substituent groups in place of hydrogen on a basic chromophore
structure changes the position and intensity of an absorption band of the chromophore.
The substituent groups may not give rise to the absorption of the ultraviolet radiation
themselves, but their presence modifies the absorption of the principal chromophore.
Substituents that increase the intensity of the absorption, and possibly the wavelength, are
called auxochromes.
These groups do not absorb the radiations in UV-visible region, however when they are
attached to chromophore they shift the absorption bands cither towards higher
wavelengths or lower wavelengths i.e. they bring out the changes in absorption spectum
of chromophore when attached to it.
These auxochromes generally have nonbonding valence electrons. Eg. –OH, - NH2, -Cl
etc.

16
SPECTRAL SHIFTS
There are four types of shifts observe in the UV – Visible Spectroscopy.
1. Bathochromic shift (Red shift)
2. Hypsochromic shift (Blue shift)
3. Hyperchromic shift
4. Hypochromic shift

17
BATHOCHROMIC SHIFT:
(Red Shift-A shift to lower energy or longer wavelength)
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.
This shift can be caused by an increase in the conjugation of a molecule, a change in the
solvent polarity, or interactions such as hydrogen bonding.
Examples:
 Conjugation: In benzene, the absorption maximum occurs around 254 nm. When
benzene is converted to aniline (with an amino group), the absorption maximum shifts to
around 280 nm due to increased conjugation.
 Solvent Effect: For a compound like 4-nitrophenol, the absorption maximum shifts from
315 nm in water to 400 nm in ethanol due to the solvent polarity effect.

18
HYPSOCHROMIC SHIFT:
(Blue Shift-A shift to higher energy or shorter wavelength)
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.
This shift can be caused by a decrease in conjugation, a change in the solvent polarity, or
steric effects that disrupt the electronic environment.
Examples:
• Conjugation: In the case of benzaldehyde, when it is converted to benzoic acid, the
absorption maximum shifts from 248 nm to 230 nm due to the disruption of conjugation.
• Solvent Effect: For a compound like 4-nitroaniline, the absorption maximum shifts from
380 nm in ethanol to 350 nm in water due to the solvent polarity effect.

19
HYPERCHROMIC SHIFT
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.
This shift can be caused by factors such as an increase in the number of absorbing
species, a change in the molecular environment that enhances the transition probability, or
structural modifications that increase the oscillator strength.
Example:
• Aromatics in Solvents: The addition of a solvent that enhances the solubility of an
aromatic compound can cause a hyperchromic shift due to better solvation and more
efficient light absorption. For instance, dissolving an aromatic compound like naphthalene
in ethanol instead of water can result in a hyperchromic effect

20
HYPOCHROMIC SHIFT
A hypochromic shift is a decrease in the absorbance or intensity of the absorption band.
Hypochromic effect is defined as the effect by virtue of intensity of absorption maximum decreases.
Hypochromic effect occurs due to the distortion of the geometry of the molecule with an introduction
of new group.
This shift can be caused by factors such as a decrease in the number of absorbing species, changes in
the molecular environment that reduce the transition probability, or structural modifications that
decrease the oscillator strength.
Example:
• Complex Formation: When a ligand binds to a metal ion to form a complex, it can result in a
hypochromic shift. For example, the complexation of 2,2'-bipyridine with a metal ion like Fe(II)
often results in a decrease in the intensity of the absorption band due to changes in the electronic
environment of the ligand.

21
SOLVENT EFFECTS ON THE ABSORPTION
SPECTRA
• The choice of the solvent to be used in ultraviolet spectroscopy is quite important, which
can alter the electronic environment of the solute.
• The absorption spectrum of a pharmaceutical substance depends partially upon the
solvent that has been employed to solubilize the substance.
• A drug may absorb a maximum of radiant energy at a particular wavelength in one
solvent but shall absorb practically little at the same wavelength in another solvent.
• These apparent changes in spectrum are exclusively due to various characteristic features,
namely:
(a) Nature of the solvent,
(b) Nature of the absorption band, and
(c) Nature of the solute.

22
Some salient features of 'Solvent Effects' are enumerated below:
1. Absorption bands of many substances are relatively sharper and may also exhibit fine
structure when measured in solvents of low dipole moment
2. Interactions of solvent-solute are found to be much stronger in such substances where
strong dipole forces are involved,
3. Solvent effects do help in reorganizing electronic transitions of the type n-π* that
essentially involve the nonbonding electrons of nitrogen and oxygen,
4. The nonbonding electrons of nitrogen and oxygen usually interact with polar solvents
that ultimately give rise to a characteristic shift to shorter wavelengths.
5. Example: The spectrum of lodine in a nonpolar solvent like CHCl3, is found to be
distinctly different (purple to the naked eye) when the same is compared in a polar
solvent such as C2H5OH (brownish to the naked eye)
6. A spectrum normally shows appreciable changes with varying pH when an ionizable
moiety is present in the molecule and thereby constitutes part of the chromophore
structure.

24
1. Solvent Polarity:
• Bathochromic Shift (Red Shift): In polar solvents, the absorption maximum can shift to
longer wavelengths (lower energy). This occurs because polar solvents can stabilize the excited
state more than the ground state of the solute, lowering the energy gap between the two states.
• Example: The absorption maximum of benzene shifts from around 204 nm in hexane (non-
polar) to around 256 nm in ethanol (polar).
2. Solvent-Solute Interactions:
• Hydrogen Bonding: Solvents that can form hydrogen bonds with the solute can significantly
impact the absorption spectrum. Hydrogen bonding can either stabilize or destabilize different
electronic states, leading to shifts in the absorption band.
• Example: The absorption maximum of phenol shifts to longer wavelengths when it forms
hydrogen bonds with solvents like water or ethanol.

25
3. Solvent Effect on Molar Absorptivity (Intensity):
• Hyperchromic Effect: Some solvents can enhance the intensity (molar absorptivity) of the absorption
band by better solubilizing the solute or by increasing the transition probability.
• Example: Naphthalene in ethanol shows a higher intensity absorption band compared to in hexane.
4. Solvent Polarity and π-π* Transitions:
• Solvent polarity can have a pronounced effect on π-π* transitions, which involve the excitation of an
electron from a bonding π orbital to an antibonding π* orbital. Polar solvents often cause a
bathochromic shift for these transitions.
• Example: The absorption maximum for β-carotene shifts to a longer wavelength in more polar
solvents.
5. Solvent Polarity and n-π Transitions*:
• For n-π* transitions, which involve the excitation of a non-bonding (n) electron to an antibonding π*
orbital, polar solvents generally cause a hypsochromic shift. This is because polar solvents stabilize
the non-bonding electrons more effectively than the antibonding π* orbitals.
• Example: The absorption maximum for acetone shifts to a shorter wavelength in more polar solvents.

26
BEER – LAMBERT’S LAW
The Beer-Lambert law states that:
“For a given material sample path length (L) and concentration (C) of the sample are
directly proportional to the absorbance of the light”
The Beer-Lambert law is expressed as:
A = εLc
where,
• A is Absorbance
• ε is molar extinction coefficient/ molar absorptivity
• L is the distance covered by the light through the solution
• c is the concentration of the absorbing species

27
• Beer’s law was stated by August Beer which states that absorbance and concentration
are directly proportional to each other.
• Johann Heinrich Lambert stated Lambert law. It states that absorbance and path length
are directly proportional.
Monochromatic

28
The Beer Lambert Equation is based on the hypothesis that the intensity (I) of the
monochromatic light is decreased by dI (i.e negative) as it passes through a thickness dx of a
material and is directly proportional to the concentration (C) as well as the intensity (I).
- α C I
- = k C I
= - k C dx
If I represents the incident intensity of the radiation before it passes through a medium of
ₒ
thickness L that has an absorption coefficient k, the transmitted intensity I will be given by the
integrated form of the above equation.
= - k C dx
= -k C [x]
ln I – ln I = - k C (L-0)
ₒ
ln = - k C L
ln = k C L
2.303 log = k C L

29
log =
Assuming as ε (Extinction coefficient/ Molar absorptivity)
log = ε
log = ε C L
 Here ε C L is Absorption/ Absorbance
log = A
Transmittance is the inverse of absorbed intensity
log = A
OR
-log T= A

30
Deviations from Beer Lambert’s Law
 A system is said to obey Beer’s law, when a plot of Concentration Vs absorbance gives a
straight line.
The straight line is obtained by joining the maximum no of points in such a way that
positive and negative errors are balanced or minimised.
When a straight line is not obtained, that is a non-linear curve is obtained in a plot of
concentration Vs absorbance, the system is said to undergo deviation from Beer’s Law.
Such deviation can be positive deviation or negative deviation.
Positive deviation results when a small change in concentration produces a greater change
in absorbance.
Negative deviation results when a large change in concentration produces smaller change
in Absorbance.
It is normally seen that several system obey Beer’s Law only in concentration range,
above which may show deviation (eg) 10 - 50 g/ml, it may obey, but may exhibit
deviation above 50 g/ml.

31
 Several reasons for the observed deviations from Beer’s law are as follows:
1. Instrumental deviations Factors like stray radiation, improper slit width, fluctuations in
single beam and when monochromatic light is not used can influence the deviation.
2. Physicochemical changes in solution Factors like Association, Dissociation, ionisation
(Change in pH), faulty development of colour (incompletion of reaction), refractive
index at high concentrations, can influence such deviations.

32
INSTRUMENTATION
 A typical UV-Visible Spectrophotometer consists of the following components:
1. Radiation source
2. Wavelength selector
3. Sample container
4. Radiation transducer
5. Signal processors
6. Readout device
Schematic diagram of instrumentation of UV
Visible Spectrophotometer

33
Source of Radiation:
They must provide sufficient energy over the wavelength region (400 - 800pm) for easy
detection & measurement.
It must supply continuous radiation of adequate intensity, stable and free from fluctuation
over the entire wavelength region in which it is used.
The output power should be stable for reasonable period.
The following are the example of lamps:
a. Hydrogen Discharge lamp
b. Deuterium lamp
c. Tungsten lamp
d. Xenon discharge lamp

34
a. Hydrogen and Deuterium Discharge lamp:
 A continuum spectrum in the UV region is produced by electrical excitation of hydrogen
or deuterium.
A continuum spectrum is obtained from 160-375nm.
The energy of photon can vary continuously. Only quartz cuvettes are used, because glass
absorb radiation of wavelength less than 350nm.
Advantages:
 Radiation is stable.
 Intensity of radiation is emitted 3-5 times the intensity of hydrogen lamp.
 Disadvantages:
 Expensive

35
b. Tungsten Lamp
Most common light source used in spectrophotometer.
The lamp consist of a tungsten filament in a vacuum bulb it offers sufficient intensity.
 It has long life.
Produces wavelength region 350-2500nm.
 In the visible region the energy output varies with power of operating voltage. The voltage
control is essential for stable radiation.
Tungsten halogen lamps contain small quantity of iodine within a quartz envelop that houses the
filament.
 Quartz is required due to the high operating temperature.
 Advantages:
• Have long half life,Stable, cheap, easy to use.
Disadvantages:
• Intensity at lower wavelength region in very feeble.
• Need voltage control for stable radiation.

36
C. Xenon Discharge Lamp
This lamp produces intense radiation by passage of current through an atmosphere xenon.
The spectrum is continuous over the range between 200 - 600nm.
Xenon gas is stored under pressure in the range of 10 - 30 atm.
The excess xenon lamp possess two tungsten electrodes separated by about 8nm.
When an intense arc is formed between two tungsten electrodes by applying a low voltage
the UV light is produced.
The intensity of UV radiation produced by xenon discharge lamp is greater than that of
hydrogen lamp.
Advantages:
• The intensity of UV radiation produced are greater than that of hydrogen lamp.
• Emit both UV and visible wave length.

39
Collimating System:
The radiation emitted by the source is collimated (made parallel) by lenses, mirrors and slits.
1. Lenses:
Materials used for the lenses must be transparent to the radiation being used. Ordinary
silicate glass transmits between 350 to 3000 nm.
2. Mirrors:
These are used to reflect, focus or collimate light beams in spectrophotometer.
To minimize the light loss, mirrors aluminized on their front surfaces.
3. Slits:
Slit is an important device in resolving polychromatic radiation into monochromatic
radiation.
To achieve this, entrance slit and exit slit are used.
The width of slit plays an important role in resolution of polychromatic radiation.

40
Wavelength Selector:
Most of the spectroscopic analysis radiation that consist of a limited narrow continuous
group of wave length is called as band.
Ideally the output from a wavelength selector would be a radiation of single wavelength /
frequency.
A narrow band width represents better performance.
Dispersive devices or wavelength selectors ae classified broadly into two categories:
1. Filters
2. Monochromators
1. Filters:
• Filters are the devices which allow selective transmission of light of specific wavelengths.
Selection of filters is usually done on a compromise between peak transmittance and band
pass width.
• Advantages: These are simpler in construction and cheaper in cost.
• Disadvantages: Accuracy is very low. Types of filters used in Spectroscopy

41
Types of filters used in Spectroscopy:
a. Absorption filters
b. Interference filters
a) Absorption filters – Absorption filters are simple filters which Works by selective
absorption of unwanted radiation and transmits the radiation which is required.
Examples Glass and Gelatin filters.
b) Interference filters- Works on the interference phenomenon, causes rejection of
unwanted wavelength by selective reflection. It is constructed by using two parallel
glass plates, which are silvered internally and seperated by thin film of dielectric
material of different (CaF2, SiO, MgF2) refractive index. These filters have a band pass
of 10- 15nm with peak transmittance of 40-60%.
2. Monochromators:All monochromators contain the following component parts,
a. An entrance slit,
b. A collimating lens,
c. A dispersing device (a prism or a grating),
d. A focusing lens,
e. An exit slit,

42
Types of Monochromators
Prism Type
Dispersive
Type
(Refraction)
Littrow Type
(Reflection)
Grating Type
Diffraction
Type
Transmission
Type

43
Prism Type:
• Prism is made from glass, Quartz or fused silica.
• Quartz or fused silica is the choice of material of UV spectrum.
• When white light is passed through glass prism, dispersion of polychromatic light in
rainbow occurs. Now by rotation of the prism different wavelengths of the spectrum can
be made to pass through in exit slit on the sample.
• The effective wavelength depends on the dispersive power of prism material and the
optical angle of the prism.
1. Dispersive Type:
The source of light through entrance slit falls on a collimator.
The parallel radiation from collimator are dispersed into different colours or wavelengths
& by using another collimator, the images of entrance slit are reformed.
The reformed ones will be either violet, indigo, blue, green, yellow, orange or red.
The required radiation on exit slit are be selected by rotating the prism or by keeping the
prism stationary & moving the exit slit.

44
2. Littrow Type:
The principle of working is similar to the refractive type except that, a reflective surface is
present on one side of the prism.
It has optical angle 3 and its one surface is aluminized with reflected light back to pass
0̊
through prism and to emerge on the same side of the light source that is light doesn’t pass
through the prism on other side.
Hence the dispersed radiations gets reflected & can be collected on the source of light.

45
Grating Type:
Are most effective one in converting a polychromatic light to monochromatic light.
As a resolution of +/- 0.1 nm could be achieved by using gratings, they are commonly
used in spectrophotometers.
1. Diffraction type:
More refined dispersion of light is obtained by means of diffraction ratings.
These consist of large number of parallel lines (grooves) about 15000-30000/ inch is ruled
on highly polished surface of aluminium.
These gratings are replica made from master gratings by coating the original master
grating with epoxy resin and are removed after setting.
To make the surface reflective, a deposit of aluminium is made on the surface.
In order to minimize to greater amounts of scattered radiation and appearance of
unwanted radiation of other spectral orders, the gratings are blazed to concentrate the
radiation into a single order.

46
2. Transmission type:
It is similar to diffraction grating but refraction takes place instead of reflection.
Refraction produces reinforcement.
This occurs when radiation transmitted through grating reinforces with the partially
refracted radiation.
Transmission Type Diffraction Type

47
Sample cells/Holders:
The cells or cuvettes are used for handling liquid samples.
The cell may either be rectangular or cylindrical in nature.
For study in UV region; the cells are prepared from quartz or fused silica.
For study in Visible region; Color corrected fused glass is used
The surfaces of absorption cells must be kept scrupulously clean.
No fingerprints or a touch should be present on cells.
Cleaning is carried out washing with distilled water or with dialcohol, acetone.
The thickness of the cell is generally 1cm, call maybe be rectangular in shape or
cylindrical with flat ends.
Two opposite faces of the cuvettes are slightly translucent and the other two faces
are clear.

48
Detectors:
Device which converts light energy into electrical signals, that are displayed on readout
devices.
The transmitted radiation falls on the detector which determines the intensity of radiation
absorbed by sample.
The following types of detectors are employed in instrumentation of absorption
spectrophotometer:
a)Photovoltaic cell
b) Phototubes/Photo emissive tube
c) Photomultiplier tube
d) Silicon Photodiode

49
1. Photo Tubes/ Photo emissive Tubes:
 Photo tubes are also known as Photo emissive tubes.
A photo tube consists of an evacuated glass tube.
 There is a light sensitive cathode inside it.
 The inner surface of the cathode is coated with a light
sensitive layer such as potassium oxide or silver oxide.
 When radiation is incident upon the cathode, electrons are
emitted.
Electrons are emitted which are attracted to anode causing
current to flow.
And by this process current is amplified and recorded.

50
2. Photomultiplier tube :
The photomultiplier tube is a commonly used detector in UV spectroscopy.
It consist of a cathode covered with a photo emissive material, which emits electrons when exposed
to radiation.
The tube contain additional electrodes called dynodes.
Dynodes is maintained at a potential 90v more positive than the cathode and so the electrons
emitted by the cathode are attracted towards it.
Each photo electron striking the dynodes causes emission of several additional electrons.
These electrons are then accelerated towards the second dynode, which is at 90v more than dynode
1.
Again several electrons are emitted for each electron.
Repetition of the process leads to formation of 10^6 to 10^7 electrons for each photon striking the
cathode.
Ideal for sensing weak light intensities.

51
Cross sectional view of photomultiplier tube

52
3. Photo Voltaic cell:
The detector has a thin film metallic layer coated with silver
or gold and acts as an electrode.
It also has a metal base plate which acts as another electrode.
These two layers are separated by a semiconductor layer of
selenium.
When light radiation falls on selenium layer, electrons
become mobile and are taken up by transparent metal layer.
This creates a potential difference between two electrodes
and causes the flow of current.
When it is connected to galvanometer, a flow of current
observed which is proportional to the intensity and
wavelength of light falling on it.

53
APPLICATIONS
1. Chemical identification and quantification: UV-Vis spectroscopy can confirm
chemical identity and quantify the purity of drugs/drug ingredients.
2. Quantification of impurities: UV-Vis is commonly utilized in pharmaceutical
monographs for quantifying impurities in drug ingredients and drug products.
3. Dissolution testing: UV-Vis spectroscopy has long been a method for analyzing the
results of dissolution testing of solid oral dosage forms like tablets.
4. Development of Active Pharmaceutical Ingredients (APIs): From scans to stop-flow
kinetics, UV-Vis is a common technique in drug discovery and development.
5. Protein analysis and measurements: Simple techniques enable quantification and
qualification of biomaterials from protein to bacteria and nucleic acids.
6. 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.

54
7. Quantitative Analysis: UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This determination is based on
Beer’s law which is as follows.
A = εLc
8. 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.
9. 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.
10. Detection Of Functional Groups: This technique is used to detect the presence or
absence of functional group in the compound.
11. As HPLC Detector: A UV/Vis spectrophotometer may be used as a detector for HPLC.

55
12. Spectrophotometric titrations:
Generally, in normal titrimetric methods, the equivalence point of a reaction is visually detected
by the colour produced by an indicator (as in acid-base titrations etc.) or by the colour of the
reactant (permanganate etc).
Such titrations give results within one per cent accuracy.
In titrations, if the colour change is gradual or the colour change does not contrast sharply,
accurate results are not obtained.
All such problems can be overcome if photometric or spectrophotometric titrations are carried
out.
In spectrophotometric titrations, a spectrophotometer is used to determine the equivalence point.
The method consists in placing the titration vessel directly in the path of the light of the
instrument.
The absorbance of the solution is then determined after adding titrant and a plot of absorbance
as a function of the volume of the tritrant is made.

56
13. Single component Analysis:
Each molecule absorbs UV-Vis light at characteristic wavelengths.
By measuring the absorbance of a solution at a specific wavelength, one can determine the
concentration of the molecule of interest using Beer-Lambert law.
Application:
Used for quantifying the concentration of a specific molecule in a solution when no other
molecules absorb light at the chosen wavelength.
14. Multi component Analysis:
In mixtures with multiple absorbing components, each component contributes to the overall
absorbance.
Advanced techniques and mathematical treatments (like chemometrics) can be used to
deconvolve the spectrum and determine concentrations of individual components.
Used for analyzing mixtures where multiple species are present and can absorb UV-Vis light
simultaneously.

UV- Visible Spectrophotometry: Introduction, Spectral Shifts, Instrumentation and Applications.pptx

More Related Content

What's hot

Similar to UV- Visible Spectrophotometry: Introduction, Spectral Shifts, Instrumentation and Applications.pptx

More from JaskiranKaur72

Recently uploaded

UV- Visible Spectrophotometry: Introduction, Spectral Shifts, Instrumentation and Applications.pptx