This document provides an overview of UV-Visible spectroscopy. It discusses what spectroscopy and UV-Vis spectroscopy are, and describes the principle, instrumentation, and applications of UV-Vis spectroscopy. The main points covered include: UV-Vis spectroscopy measures absorption of UV and visible light by a sample; it is used to identify functional groups and determine concentrations; various solvents can be used depending on the sample; and applications range from pharmaceutical and environmental analysis to DNA/RNA and protein studies.

1. UV-Visible spectroscopy

2.  What is Spectroscopy?
 What is UV-Vis Spectroscopy?
 What is the main purpose of UV spectroscopy?
 Which solvent is used in UV spectroscopy?
 Principle of UV-Visible Spectroscopy
 Choice of solvents and solvent effect
 Difference/ derivative spectroscopy
 Applications of UV Visible spectroscopy

3.  Spectroscopy is a technique that is used to study the
interaction of matter and electromagnetic radiation.
 It involves the measurement of the energy absorbed
or emitted by a sample at different wavelengths.
 Spectroscopy can be used to identify the composition
of a sample, to study the physical and chemical
properties of a sample, and to determine the structure
of a molecule

4.  UV spectroscopy, also known as UV-visible spectrum (UV-
Vis also known as UV/Vis).
 UV-Vis spectroscopy is a technique used to measure the
absorption of ultraviolet (UV) and visible light by a sample.
 It is commonly used to determine the concentration of a
substance in a solution or to identify the functional groups
present in a molecule.
 The absorption of light by a sample is measured at different
wavelengths, and the resulting data is plotted as a spectrum.
 The absorption spectrum can be used to identify the presence
of specific functional groups or to determine the concentration
of a substance in a solution.

5.  The main purpose of UV spectroscopy is to determine the absorption or
transmission characteristics of a sample in the ultraviolet (UV) region of
the electromagnetic spectrum.
 By measuring the amount of light absorbed by a sample at different
wavelengths, researchers can learn about its chemical composition,
electronic structure, and other properties.
 UV spectroscopy is used in a wide range of applications, including:
 Identifying and quantifying the concentration of a specific compound in a
mixture
 Characterizing the purity of a sample
 Determining the electronic structure of a compound, such as the presence
of conjugated double bonds or aromatic ring systems
 Studying chemical reactions and reaction kinetics
 Analysis of biological macromolecules like proteins, DNA, and RNA
 Environmental analysis
 Pharmaceuticals and drug analysis

6.  The solvent used in UV spectroscopy depends on the nature of the sample
being analyzed and the specific application. Some common solvents used
in UV spectroscopy include:
 Water: Water is often used as a solvent for UV spectroscopy of polar
compounds, such as acids, bases, and biomolecules.
 Alcohols: Alcohols like methanol and ethanol are commonly used as
solvents for UV spectroscopy of organic compounds.
 Acetonitrile: Acetonitrile is a polar, aprotic solvent that is often used for
UV spectroscopy of polar compounds and for samples that are not soluble
in water or alcohols.
 Dichloromethane: Dichloromethane (DCM) is a non-polar solvent that is
often used for UV spectroscopy of non-polar compounds.
 Hexane: Hexane is also a non-polar solvent that is often used for UV
spectroscopy of non-polar compounds

7.  The Principle of UV-Visible Spectroscopy is based
on the absorption of ultraviolet light or visible light
by chemical compounds, which results in the
production of distinct spectra.
 Spectroscopy is based on the interaction between
light and matter.
 When the matter absorbs the light, it undergoes
excitation and de-excitation, resulting in the
production of a spectrum.

8.  As per the Beer-Lambert law, the greater the number
of absorbing molecules (that have the ability to
absorb light of a specific wavelength), the greater the
extent of absorption of the radiation.

9.  The photomultiplier tube is the extensively used
detector in UV-Vis instruments.
 It includes a photoemissive cathode (electrons are
emitted from the cathode when photons strike it),
several dynodes (a dynode emits multiple electrons
when one electron strikes it) and an anode.
 The UV-Vis spectrum can by recorded via the
following types of absorbance instruments:
a. Single beam spectrometer
b. Double beam spectrometer
c. Simultaneous spectrometer

10.  The absorption bands in ultraviolet spectrum are very broad as
compared with infrared or NMR spectrum.
 Ultraviolet spectra of compounds are usually determined either in
the vapour phase or in very dilute solution.
 Solvent must be transparent with in the wavelength
range being examined.
 It should not itself absorb radiation in the region under
investigationIt should be less polar, to minimum interaction with
solute molecule
 Spectrascopic (Analytical) grade solvents should be used.The most
commonly used solvent is 95% ethanol.
 It Is a cheap
 Good dissolving power
 Does not absorb radiation above 210nm
 Some other solvents which are transparent above 210 nm.Eg: n-
hexane, cyclohexane, methanol, water and ether

11.  DNA & RNA analysis
 Pharmaceutical analysis
 Bacterial culture
 Beverage analysis
 Characterization of smaller nanoparticles.
 Examination of structural protein changes by tracking changes in peak wavelength absorbance.
 Determination of battery composition
 Kinetic and monitoring studies of dyes and dye byproducts
 Absorbance of hemoglobin for the determination of concentration of hemoglobin in cancer research
 Measuring color index to monitor transformer oil
 Determination of concentration of proteins through direct measurement or colorimetric assays
 Study of enzymatic reactions
 Monitoring growth curve of bacterial cell suspensions
 Can be used as a detector for HPLC
 Quality control.
 Cosmetic industry.
 Petrochemistry.
 Pharmaceutical research.
 Optical components.
 Food and agriculture.
 Life sciences.
 Traditional chemistry.

12. Derivative spectroscopy
 another simplest method for an increasing a
selectivity is derivatisation of spectra. This operation
allows to remove spectral interferences and as a
consequence leads to increase selectivity of assay
 It involves the conversion of a normalspectrum to it's
first, second or higher derivative spectrum
 The normal absorption spectrum is reffered to as the
fundamental zero order or spectrum.

13.  The selectivity and accuracy of spectrophotometric
analysis of sample containing absorbing interference
may be markedly improved by the technique of
difference spectrophotometry.
 FEATURE OF DIFFERENCE
SPECTROPHOTOMETY:- The measured value is
the difference absorbance(AA) between two
equimolar solutions of the analyte in different
chemical forms which exhibit spectral characteristics.

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