UV VISIBLE SPECTROSCOPY PRINCIPLE, INSTRUMENTATION, APPLICATIONS

1. UV-VIS
SPECTROSCOPY
Kad Dhanashree R.
Modern College of Pharmacy (for ladies)
Moshi, Pune
spectrophotometer

2. ABSTRACT
 Spectroscopy is the study of interaction of electromagnetic radiation
with matter.
 Basic principle of spectroscopy is the Beer-Lambert’s law.
 In UV-VIS Spectroscopy, a continuum range of wavelengths from
200nm to 800nm is used.
 Spectrophotometer device is used in UV-VIS Spectroscopy. See
figure1.
 Components of spectrophotometer
 Sources of light(200nm to 800nm).
 Monochromator.
 Sample solution in cuvette.
 Detectors.
 Readout devices.
 Types of spectrophotometer Single and double beam instruments.
 Applications of UV-VIS Spectroscopy Qualitative &Quantitative
analyses.
FIG 1.Spectophotometer.

3. INTRODUCTION
 Spectroscopy is the branch of science which deals
with interaction of electromagnetic radiation with
materials. In other words it is an analytical method for
qualitative and quantitative analysis by use of light.
 The Lambert Beer law in 1852 made the basis for the
quantitative evaluation of absorption measurements.

4. 1.SPECTROSCOPY
 When an Electromagnetic radiation is incident on a
matter, phenomena like reflection, transmission,
absorption ,are occurring.
 Spectroscopy is the study of interaction of
electromagnetic radiation with matter based on the
Bohr-Einstein frequency relationship E=hv , here h
is the proportionality constant called Planck’s
constant (6.626 x 10-34 J s) and v is frequency.

5.  Measurement of radiation intensity as a function of
wavelength is described by spectroscopy, as shown in
figure 2.
 All forms of spectroscopy use part of the
electromagnetic radiation to give us information about the
materials.
FIG.2.Spectroscopy graph.

6. 1.1 SPECTRUM
 The spectrum is formed by electromagnetic waves
and the wavelength is varies. See figure 3.
 When a narrow beam of light is allowed to pass
through a prism/grating, it is dispersed into seven
colors from red to violet and the
band is called Spectrum..
See figure 4.
FIG.3.Electromagnatic spectrum.
FIG.4.Glase prism dispersion

7. 1.2 PRINCIPLE
Basic principle of spectroscopy is the Beer-Lambert’s law.
1.2.1 BEER LAW
 Beer's law stated that absorbance is proportional to the
concentrations of the material sample.
1.2.2 LAMBERT LAW
 Lambert's law stated that absorbance of a material is
directly proportional to its thickness (path length).

8.  The modern derivation of the Beer–Lambert law combines the
two laws and correlates the absorbance to both the
concentrations and the thickness of the material.
FIG 5. Beer–Lambert law.

9. 2. UV-VISIBLE SPECTROSCOPY
 Ultraviolet–visible spectrum can be generated when
ultraviolet light and visible light(200-800nm) are
absorbed by materials. The spectrum can be used to
analyze the composition and the structure of the
materials. For a particular wavelength in the ultraviolet–
visible ranges, the absorption degree is proportional to
the components of the materials. Therefore, the
characteristics of the materials are quantitatively
reflected by the spectrum, which changes with the
wave-length.
 Ultraviolet–visible spectrum consists of an
absorption spectrum. An absorption spectrum gives
information about the molar absorptivity, concentration
of the sample, optical path length.

10. 2.1 INSTRUMENTATION
 2.1.1 SOURCE of LIGHT.
 2.1.2 MONOCHROMATOR.
 2.1.3 SMPLE SOLIOTION in CUVETTE.
 2.1.4 PHOTO DETECTOR.
 2.1.5 READOUT DEVICE.
FIG 6.Components of spectrophotometer.

38. The monochromator unit consists of :
•Entrance slit: defines narrow beam of radiation from
source.
•Collimating mirror:(polished surface) collimates the
lights.
•Diffraction grating or Prism (make of quartz):
disperses the light into specific wavelength.
•Focusing mirror: captures the dispersed light &
sharpens the same to the sample via exit slit
•Exit slit: allows the corrected wavelength of light to the
sample .

59. Advantages of UV-Vis spectroscopy
Versatility: UV-Vis spectroscopy can be used to study a wide range of samples, including liquids,
gases, and solids. It can also be used to study samples in different states, such as solutions,
suspensions, and emulsions.
Non-destructive: The procedure is nondestructive, allowing the sample to be reused or processed
or analysed further.
Rapid: Rapid measurements allow for simple integration into experimental methods.
Sensitivity: UV-Vis spectroscopy is a highly sensitive technique that can detect even small
concentrations of a substance in a sample.
Easy to use: The instruments are simple to operate and require minimal training prior to use.
Minimal processing: In general, data analysis needs minimum processing, hence requiring
minimal user training.
Quantitative analysis: UV-Vis spectroscopy can be used to determine the concentration of a
substance in a sample, which makes it useful for quantitative analysis.
Non-destructive: UV-Vis spectroscopy does not damage or alter the sample being studied, making
it a non-destructive technique.
High-throughput: UV-Vis spectroscopy can be used to analyze multiple samples simultaneously,
which makes it useful for high-throughput applications.
Low cost: UV-Vis spectroscopy equipment is relatively low cost and easy to operate and maintain.
Widely available: UV-Vis spectroscopy is widely available in research and industrial laboratories,
making it easily accessible for a wide range of applications.
High-resolution: UV-Vis spectroscopy can be used to study samples at high-resolution, which
allows for detailed analysis of the sample.
Wide range of applications: UV-Vis spectroscopy has a wide range of applications, including in
analytical chemistry, biochemistry, materials science, and in industries such as Pharmaceuticals,
food, water and environment analysis, etc.

60. Major disadvantages of uv-vis spectroscopy are:
1. Not linear for high concentration.
2. Does not work with compounds that do not absorb
light at this wavelength region.
3. Generates significant heat and requires external
cooling.

61. 2. Pharmaceutical analysis
In the pharmaceutical sector, UV-Vis spectroscopy is one of the most prevalent applications.
Specifically, analysing UV-Vis spectra with mathematical derivatives enables the resolution of
overlapping absorbance peaks in the original spectra to identify individual pharmaceutical
drugs.
By applying the first mathematical derivative to the absorbance spectra, benzocaine, a local
anaesthetic, and chlortetracycline, an antibiotic, can be recognised simultaneously in
commercial veterinary powder formulations.
By constructing a calibration function for each component, simultaneous quantification of
both chemicals was feasible over a concentration range of micrograms per millilitre.
3. Bacterial culture
UV-Vis spectroscopy is utilised frequently in bacterial cultivation. To assess cell
concentration and monitor growth, 600 nm OD readings are routinely and rapidly acquired at
a wavelength of 600 nm.
600 nm is widely employed and recommended because to the optical properties of bacterial
growth conditions in which they are cultivated and to avoid injuring the cells when they are
needed for further investigation.
Applications of UV-Vis spectroscopy
UV-Vis has been adapted to numerous situations and purposes such as but not limited:
1. DNA and RNA analysis
The rapid verification of the purity and concentration of RNA and DNA is a ubiquitous
application. Table 1 provides a summary of the wavelengths employed in their analysis and
what they represent. When preparing DNA or RNA samples for downstream applications
such as sequencing, it is sometimes crucial to ensure that neither sample is contaminated
with the other or with protein or chemicals derived from the isolation procedure.

62. 4. Beverage analysis
Another common application of UV-Vis spectroscopy is the determination of specific chemicals
in beverages. Caffeine content must be under specified legal limits which can be measured
with UV light.
Certain types of colourful compounds, such as the anthocyanin present in blueberries,
raspberries, blackberries, and cherries, can be easily identified using UV-Vis absorbance by
matching their known peak absorbance wavelengths in wine.
Other applications
Analytical chemistry: UV-Vis spectroscopy is used to determine the concentration of a
substance in a sample, to identify unknown compounds, and to monitor chemical reactions.
Biochemistry: UV-Vis spectroscopy is used to study the structure and function of
biomolecules, such as proteins, nucleic acids, and pigments.
Environmental science: UV-Vis spectroscopy is used to monitor the quality of water and air,
to detect pollutants and to study the photochemistry of atmospheric gases.
Food industry: UV-Vis spectroscopy is used to measure the concentration of food ingredients,
to monitor the quality of food products, and to detect contaminants.
Materials science: UV-Vis spectroscopy is used to study the electronic and optical properties
of materials, such as semiconductors, dyes, and pigments.
Organic chemistry: UV-Vis spectroscopy is used to study the electronic structure of organic
molecules, to identify functional groups and to study the mechanism of chemical reactions.
Medical research: UV-Vis spectroscopy is used to study the properties of blood, to monitor the
level of glucose in blood, and to study the photochemistry of biological systems.
Forensics: UV-Vis spectroscopy is used to analyze trace evidence, such as fibers and paint, to
identify the source of a sample.
Industrial process control: UV-Vis spectroscopy is used to monitor the progress of chemical
reactions in industrial processes, to optimize conditions and to control the quality of the final
product.