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.
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.