UV VISIBLE

SPECTROSCOPY AND ITS

APPLICATIONS

HARPREET KAUR -19MSM0010

KINJAL SAHA -19MSM0005
KSHITIJA – 19MSM0061
POORNIMA -19MSM0049
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 INTRODUCTION
• The characterization of pure as well as mixtures of substances is achieved with physical
methods.
• Many techniques used, such as the determination of melting point, refractive index and
density.
• Optical spectroscopy in the UV/VIS range is widely applied in almost all market
segments and workplaces in research, production and quality control for the classification
and study of substances

WHAT IS SPECTROSCOPY?
Spectroscopy is study of the absorption and emission of light (any portion of
electromagnetic spectrum) and other radiation by matter , as related to the dependence of
these processes on the wavelength of the radiation. 2
ULTRAVIOLET-VISIBLE (UV-VIS) SPECTROSCOPY
• UV-VIS spectroscopy is type of absorption spectroscopy in which light of ultra-violet and
visible region (200-700 nm) is absorbed by the molecule. 
• Absorption of the UV-VIS
radiations results in the
excitation of the electrons
from the ground state to
higher energy state.

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USES OF UV-SPECTROSCOPY
• Determination of functional groups
• Detection of impurities
• Qualitative analysis and Quantitative analysis
of :-
1. Single compound without chromophore
2. Drugs with chromophore
• Helps to show the relationships between different
groups, detects the conjugation of the compound

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HISTORY
• Leading into World War II, in the 1930s, scientists found the phenomenon of absorption of
ultraviolet light on Vitamin A, while researching the content of vitamin in the army rations.
The research finally lead out the commercial UV-Vis spectrophotometer in the early 1940s
• In 1941, Beckman Instruments Inc. launched the first commercial UV-visible
spectrophotometer in the world, DU UV-Vis Spectrophotometer.
• It was the production version of the Model D prototype by Howard Cary and his
collegues
• Featured a molecular hydrogen lamp, a monochromator made of a Brazilian quartz prism,
and a UV-sensitive phototube
• The instrument allowed food analysis
• In 1947, Erwin Chargaff used this Beckman DU to determine that the ratio of nucleotide
monomers in DNA  5
LIGHT – ELECTROMAGNETIC RADIATION
Form of energy whose behaviour described by the properties of both wave(diffraction)
and particles(absorption and emission)
WAVE PROPERTIES :- Electromagnetic radiations consist of oscillating
perpendicular electric and magnetic fields to the direction of wave propagation
Wavelength:- distance between successive maxima or successive minima

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 PARTICLE PROPERTIES :- The interaction between the sample and the electromagnetic
radiations is easiest to understand if we assume the electromagnetic radiation consist of a
beam of energetic particles called PHOTONS
THE ABSORPTION SPECTROSCOPY
• Absorption of the UV-VIS radiations results in the excitation of the electrons from the
ground state to higher energy state ( ELECTRONIC TRANSITION)
• The energy of electromagnetic radiation absorbed is equal to the energy difference
between the ground state and higher energy states :-

ABSORPTION SPECTROSCOPY
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ELECTRONIC TRANSITION
• Transition from the highest occupied
molecular orbital (HOMO) to lowest
unoccupied molecular orbital (LUMO)
• The molecular orbital picture for the H-H
molecule consists of one bonding σ MO, and
a higher energy antibonding σ* MO. When
the molecule is in the ground state, both
If the molecule is exposed to light of a
electrons are paired in the lower-energy
wavelength with energy equal to ΔE(the
bonding orbital – this is the Highest HOMO-LUMO energy gap)  σ - σ*
Occupied Molecular Orbital (HOMO). The transition
antibonding σ* orbital, in turn, is the Lowest
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Unoccupied Molecular Orbital (LUMO).
• When a double-bonded molecule
π -π* transitions
such as ethene absorbs light, it
undergoes a π - π* transition. As
π- π* energy gaps are narrower
than σ - σ* gaps, it absorbs light
at longer wavelength

• UV-vis spectroscopy becomes useful to most organic and biological chemists is in the
study of molecules with conjugated π -π* systems. In these groups, the energy gap for
π -π* transitions is smaller than for isolated double bonds, and thus the wavelength
absorbed is longer.

• Molecules or parts of molecules that absorb light strongly in the UV-vis region are
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called CHROMOPHORES
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 TRANSMITTANCE AND ABSORBANCE
• The original intensity of light is Iₒ
• The intensity of light after transmission through the sample is I
•  The intensity of the transmitted light is attenuated by the sample solution due to
absorption of light at specific wavelengths. 
• Therefore, I< Iₒ
• Transmittance= Iₒ/I
• Absorbance is defined as the negative logarithm of the transmittance
• A= -log(T)
transmittance is recorded as a function of wavelength for a sample
• The light absorption is marked by a sharp decrease of the transmittance at the
particular wavelengths
• Generally, UV/VIS spectrum is graphically represented as absorbance Vs
wavelength. 
• The advantage being the height of the absorption peaks is directly proportional to the
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concentration of the species.
 PRINCIPLE
UV-VIS 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) = E*c*l 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 13
If no absorption has occurred, Transmittance = 1.0 and A= 0. Most spectrometers
display absorbance on the vertical axis, and the commonly observed range is from
0 (100% transmittance) to 2 (1% transmittance)
The wavelength of maximum absorbance is a characteristic value, designated
as λmax.

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 INSTRUMENTATION
COMPONENTS OF SPECTROPHOTOMETER :-
• Source
• Monochromator / Filter
• Sample holder/ Sample cells
• Detector
• Recorder

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 SOURCE :-
• A suitable light source which covers the UV/VIS spectrum of interest is used.

• In general, a lamp containing a gas such as xenon, or a combination of two different lamps
such as tungsten/deuterium is used.

• The electrical excitation of tungsten/deuterium at low pressure produces a continuous UV

spectrum.

• It is important that the power of the radiation source does not change abruptly over its
wavelength range.

• Both Deuterium and Tungsten lamps emit radiation in the range 160 - 375 nm.

• Quartz windows are used in these lamps, and quartz cuvettes are advisable to use, because
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glass absorbs radiation of wavelengths less than 350 nm.

Various UV radiation sources are
as follows:-
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp

Various Visible radiation sources

are as follows:-
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp 17
FILTERS OR MONOCHROMATOR :-
 A monochromator is used to select the wavelength at which an absorption
measurement is made.
 All monochromators contain the following component parts:
• An entrance slit
• A collimating lens
• A dispersing device (a prism or a grating)
• A focusing lens
• An exit slit
 There are two main choices for dispersing light into its different components: a prism, or
a diffraction grating. Most modern instruments employ gratings, because it is easier to
achieve high spectral resolution.
 Polychromatic radiation (radiation of more than one wavelength) enters the
monochromator through the entrance slit. 18
• The beam gets parallel , and then
strikes the dispersing element at an
angle. The beam is then split into its
component wavelengths by the
grating or prism.
• By moving the dispersing element
or the exit slit, radiation of only a
particular wavelength leaves the
monochromator through the exit slit.

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Sample holder/ Sample cells:-
• An appropriate sample holder is needed to hold the sample.
• Liquid samples are placed in cuvettes, which can be made of quartz,
borosilicate glass or acrylic plastic.
• Solid samples can be mounted into a suitable holder to be positioned in the
optical path of the spectrophotometer for measurement of the transmitted light
• A variety of sample cells available for UV region. The choice of sample cell is
based on:-
a) the path length, shape, size
b) the transmission characteristics at the desired wavelength
c) the relative expense
• The cell holding the sample should be transparent to the wavelength region to be
recorded.
• Quartz or fused silica cuvettes are required for spectroscopy in the UV region.
• Silicate glasses can be used for the manufacture of cuvettes for use between 350 and202000
nm.
DETECTORS
• The transmitted light after passed through the medium is sent to
detector .
• The detector takes the photon signal of transmitted light and
converts it into measurable signals i.e. The electromagnetic
radiation is converted into electrical signals .
• These electric signals are then amplified are processed for final
interpretation with the help of recorder.

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• Scanning spectrophotometer :- The principle is
based on that the measurement of the
transmittance value at each single wavelength.
The light is first dispersed into individual
wavelengths using a reflection grating. The
grating is rotated in order to individually select
each wavelength that is then sent through a
cuvette

• Array spectrophotometer :- The sample in the

cuvette simultaneously absorbs different
wavelengths of light. The transmitted light is then
diffracted by a reflection grating located
after the cuvette
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WORKING

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• There is two source of light- 1)Hydrogen or deuterium lamp to supply UV light ( 200-400 nm)
                                                    2) Tungsten lamp to supply visible light (400-800nm)

• This light from the source passes through the monochromator which absorbs all wavelengths of light but
allows only a single wavelength of light to come out of it with the help of a prism

• Question- How is colorimetry different from this spectroscopy?

Answer- In colorimetry one wavelength of light is chosen and only that is allowed to passed but in UV-
Visible spectroscopy we select a range of wavelength, so the monochromator absorbs all the other
wavelengths and allows only one wavelength at a time for the entire range

• This single wavelength light then passes through the beam splitter which splits the intensity of light to two
equal halves with equal intensities

• One beam passes through the reference (blank) and the other beam passes through the test sample

• Identical cuvettes are used because it follows beer lambert’s law, where l is the pathlength of the light which
should be the same in both reference and test sample

• The reference sample does not absorb any light hence the incident and transmitted light both have 24

intensities I0
• The test sample absorbs light only at a particular wavelength

• If test sample does not absorb light at a particular wavelength, then I= I 0 and absorbance is zero so
there is no peak observed 

• When light is absorbed at a particular wavelength, the transmitted light (I) has a intensity less than I0
and a absorbance value is observed as a peak 

• The intensity of the peak is directly proportional to the concentration of the sample

• Then the transmitted light from both the reference and test passes through the detector

• A photocell or a photodiode can be used as a detector. It compares the intensities of light coming
from reference as well as test sample

• A current is generated proportional to the difference in the intensities of reference and test sample

• The recorder gives the output of the spectrograph which shows the graph of absorbance vs
wavelength 25
Unique Features:
• Quartz overcoating protects the optics from the environment and allows cleaning
without damage to their reflective surface
• Sealed optics prevents exposure to corrosive environments
• Double choppers ensure that the sample and reference beam strike the detector
at the same point, removing any errors due to non uniformity of the detector
• Variable slits allow optimum control over data resolution. The spectral
bandwidth can be set down to 0.2 nm
• A phase locked wavelength drive prevents peak shifts and peak suppression at
high scan speeds
• The large sample compartment gives more flexibility in sample size 26
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APPLICATIONS OF UV-VISIBLE SPECTROSCOPY
1) Detection of Impurities:
• Additional peaks can be observed due to impurities and it can be compared with
standard raw material and measuring the absorbance at specific wavelength the
impurities can be detected
• In the food and beverage industry, UV/VIS spectrophotometry is used to monitor and
improve product quality and consistency, influence of packing material and stabilizers as
well as chemical deterioration and degradation processes can also be observed with this
method
• In bioprocessing and fermentation
• Benzene appears as a common impurity in cyclohexane, presence can be detected by its
absorption at 255nm 28
2) Structure elucidation of organic compounds:
Presence or absence of unsaturation and also hetero
atoms

3 ) Quantitative Analysis:
• Effectively been used in medical science for the routine
analysis of blood and urine samples

4) Qualitative Analysis:
• Identification is done by comparing the absorption
spectrum with the spectra of known compounds

5) Dissociation constants of acids and bases:

• The ratio of A-/HA can be determined
spectrophotometrically from the graph plotted between
absorbance and wavelength at different pH values
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6) Chemical Kinetics: monitor the change of the concentration of either the
reactant or the products by absorbance at a specific wavelength over time
• The UV radiation is passed through the reaction cell and the absorbance changes can be
observed.
• Investigate enzyme activity or re-action rates as well as the affinity of the enzyme-
substrate interaction

7) Quantitative analysis of pharmaceutical substances:

• Many drugs are either in the form of raw material or in the form of formulations. They
can be assayed by making a suitable solution of the drug in a solvent and measuring the
absorbance at specific wavelength.
• Ex- Diazepam tablet can be analyzed by 0.5% sulphuric acid in methanol at wavelength
at 250nm
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 8) Molecular weight determination can be done by preparing the suitable
derivatives of compounds
• Ex- Amine in amine picrate, known concentration of amine picrate is dissolved in a
liter of solution and OD is measured at 380nm then concentration of solution in gram
moles per liter is calculated

APPLICATION IN BIOMEDICAL SCIENCES

• UV-Visible spectroscopy is well suited to study
biological models of carcinogenesis and
therapeutic response in animal models due to a
good match in the penetration depth at its
wavelength and the size of animal tumors.

• Used in the protein detection and identification

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• Helps in determining early dysplastic changes in uterine
cervix which in turn helps in preventing the growth of
cervical cancer.

• Used in the breast cancer therapies as they showed that

malignant breast tissues are more highly absorbing and
scattering than the normal tissues.

• In the protein contamination detection :- surgical setting

where the decontamination of typically reusable medical
instruments

• Spectroscopy has proven to be invaluable in the fight

against cancers. Lung cancer detection is the use of
autofluorescence bronchoscopy
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RECENT ADVANCEMENTS
• Biomarkers in vivo for the diagnosis, prognosis and treatment of cancers
• Traditional biomarkers include features such as the tumor grade, size and/or the
number of local lymph nodes with metastasis
• Oxygenation and hypoxia : Hypoxic microenvironments have routinely been
identified in solid tumors of almost all tissues
• Angiogenesis and blood volume : tumor cells have a constant need for new blood
vessels to nourish their growth, solid tumors persistently sprout new segments of
vessels

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 REFERENCES
• MODERN ANALYTICAL CHEMISTRY by David Harvey, ‘Spectrometry’
• BIOPHYSICAL CHEMISTRY : Principles and Techniques by Nath and Upadhyay, ‘Spectroscopy’
• PRINCIPLE AND TECHNIQUES OF BIOCHEMISTRY AND MOLECULAR BIOLOGY by Keith Wilson
and John Walker, ‘Spectroscopic techniques’
• UV/VIS Spectrophotometry - Fundamentals and Applications , Publisher: Mettler-Toledo
• https://www.news-medical.net/life-sciences/Spectroscopy-Applications.aspx
• https://www.avantes.com/applications/application/item/1250-biomedical-applications
• http://www.uni-salzburg.at/fileadmin/oracle_file_imports/359201.PDF
• http://web.iitd.ac.in/~sdeep/Electronic.pdf
• https://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/uvspec.htm 34
THANK YOU

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