PRESENTATION ON UV SPECTROSCOPY

BY

GROUP 5

COURSE: MEDICAL CHEMISTRY 1

COURSE CODE: PCH 402

GROUP MEMBERS
Amah Chisom Favour
Afon Adetola Grace
Chikelue Joshua
Go’ar Favour
Gwadza Doowuese Emmanuella BHU/19/01/05/0034
Okolie Grace Eninnor
Onyele Sixtus Chiemerie
Osham Sydney Anthony
Shaibu Zainab Ovayoza
Shittu Oreoluwa Peace
Sunday Favour Ndip
Tims-Ejigah Blessing Ojoma
BHU/19/01/05/0072
BHU/19/01/05/0024
BHU/19/01/05/0123
BHU/19/01/05/0026
BHU/19/01/05/0011
BHU/19/01/05/0095
BHU/19/01/05/0075
BHU/19/01/05/0107
BHU/19/01/05/0006
BHU/19/01/05/0010
BHU/19/01/08/0036
OUTLINE

● Introduction to UV spectroscopy

● Theory of UV spectroscopy

● Instrumentation of UV spectroscopy

● Data Interpretation and Spectral Analysis

● Applications of UV Spectroscopy

● Advancements and Future Trends

● Conclusion
INTRODUCTION
UV spectroscopy, also known as ultraviolet spectroscopy, is a widely used analytical technique
that involves the study of the interaction between ultraviolet (UV) light and matter. It is a non-
destructive method that provides valuable information about the electronic structure and
composition of molecules. When incident light strikes matter it can either be absorbed, reflected,
or transmitted.
The UV region of the electromagnetic spectrum ranges from approximately 10 to 400
nanometers (nm), with shorter wavelengths corresponding to higher energy. UV spectroscopy
primarily focuses on the range of 180 to 400 nm, as the shorter wavelengths are absorbed
strongly by air and are less commonly used for analysis. In UV spectroscopy, a sample is
exposed to UV light, and the absorption of light by the sample is measured. The absorption
occurs when the energy of the incident UV photons matches the energy difference between
electronic energy levels of the molecule. These energy differences are associated with the
electronic transitions of the molecule, such as the promotion of electrons from the ground state to
excited states. Ultraviolet spectrophotometers use a light source to illuminate a sample with light
across the UV to the visible wavelength range (typically 190 to 900 nm). The instruments then
measure the light absorbed, transmitted, or reflected by the sample at each wavelength.

THEORY OF UV SPECTROSCOPY
UV spectroscopy is a widely used analytical technique that involves the study of the interaction
between ultraviolet (UV) radiation and matter. It provides valuable information about the
electronic structure and chemical composition of substances. The theory of UV spectroscopy
revolves around the principles of electronic transitions, Beer-Lambert Law, and the interpretation
of UV spectra.
● Electronic Transitions: UV spectroscopy primarily focuses on the electronic transitions
that occur in molecules. Molecules absorb UV radiation when the energy of the photons
matches the energy difference between the electronic energy levels of the molecule.
These transitions involve the excitation of electrons from lower energy (ground) state to
higher energy (excited) states.
● Chromophores: Chromophores are the chemical groups or functional units within a
molecule responsible for UV absorption. They possess a π-electron system or a system of
alternating single and double bonds that can undergo electronic transitions. Examples of
common chromophores include conjugated systems like double bonds, aromatic rings,
carbonyl groups, etc.
 ● Absorption Spectrum: The absorption spectrum in UV spectroscopy provides information
about the absorption of UV radiation by a sample as a function of wavelength. It is
represented as a plot of the absorbance or the logarithm of the ratio of incident and
transmitted intensities (I/I₀) versus the wavelength. The spectrum typically exhibits
characteristic peaks or bands corresponding to specific electronic transitions.
● Beer-Lambert Law: The Beer-Lambert Law describes the relationship between the
concentration of a substance in a sample, the path length of the sample, and the
absorption of light. It states that the absorbance (A) of a sample is directly proportional to
the concentration (c) of the absorbing species and the path length (l) traversed by the light
according to the equation: A = ɛcl, where ɛ is the molar absorptivity (extinction
coefficient) of the substance.
● Quantitative Analysis: UV spectroscopy can be used for quantitative analysis by
measuring the absorbance of a sample at a specific wavelength and relating it to the
concentration of the analyte using the Beer-Lambert Law. Calibration curves are
constructed using standard solutions of known concentrations to determine the
concentration of unknown samples.
● Spectral Interpretation: The interpretation of UV spectra involves analyzing the positions,
intensities, and shapes of the absorption peaks or bands. Factors such as the nature of the
chromophore, the presence of conjugation, electronic effects, and molecular environment
influence the spectral features. Comparisons with reference spectra or databases aid in the
identification of compounds or functional groups.

INSTRUMENTATION OF UV SPECTROSCOPY
The instrumentation of UV spectroscopy typically involves several key components. Here's an
overview of the main elements:
● Light Source: UV spectrophotometers use a light source that emits radiation in the
ultraviolet and visible range. Deuterium lamps (UV range) and tungsten or quartz-
halogen lamps (visible range) are commonly employed in UV spectroscopy. These lamps
provide a broad spectrum of wavelengths for analysis.
● Monochromator: The monochromator is responsible for selecting a specific wavelength
or a narrow range of wavelengths from the light source. It consists of a prism or a
diffraction grating, which disperses the incoming light into its constituent wavelengths.
By adjusting the position of the prism or grating, a desired wavelength is isolated.
● Sample Compartment: The sample compartment holds the sample to be analyzed. It
typically consists of a quartz cuvette with optically transparent windows that allow the
passage of UV and visible light. The sample compartment should be free from
contaminants that may interfere with the analysis.
● Photodetector: The photodetector is responsible for measuring the intensity of light after
it passes through the sample. In UV spectroscopy, photomultiplier tubes (PMTs) or
 photodiodes are commonly used as detectors. They convert the light energy into an
electrical signal that can be measured and analyzed.
● Signal Processor and Display: The electrical signal from the photodetector is processed
and amplified by electronic circuits. The signal is then sent to a display unit, such as a
computer or a digital readout, where the absorbance or transmittance values are
presented.
● Data Acquisition and Analysis: Modern UV spectrophotometers are often equipped with
software or computer interfaces that enable data acquisition, storage, and analysis. These
systems allow for easy manipulation of the collected spectral data, including baseline
correction, peak identification, and quantification.
● Control Mechanism: UV spectrophotometers have various control mechanisms to adjust
parameters such as wavelength, path length, and scan speed. These controls allow the
user to optimize the instrument settings for different samples and experimental
requirements.

DATA INTERPRETATION AND SPECTRAL ANALYSIS

A. Spectral range of uvThe UV region covers the wavelength range 100-400 nm and is divided
into three bands: UVA (315-400 nm) UVB (280-315 nm) UVC (100-280 nm).
B. Beer lambert’s law and quantitative analysisWe use Beer Lambert Law to conduct a
qualitative and quantitative analysis of biological and dosimetric materials that may contain
organic or inorganic materials. We can determine the concentration of various substances in cell
structures by measuring their absorbing spectra in the cell.
Beer lambert’s law: States that the absorbance of a solution is directly proportional to the
concentration of the absorbing material present in the solution and path length.
C. Chromophores and characteristics absorption: Molecules or parts of molecules that absorb
light strongly in the UV-vis regionare called chromophores. When a double-bonded molecule
such as ethene (common name ethylene) absorbs light, it undergoes a π - π* transition
D. Factors affecting the appearance of uv spectra:The temperature, concentration and pH of the
sample solution affect the position and shape of UV-Vis absorption bands. Recording the spectra
at low temperature gives sharp absorption bands, whereas high temperature causes the
broadening of UV-bands.

Effect on sample pH
1)The pH of the sample solution can also have a significant effecton absorption spectra.
2)The absorption spectra of certain aromatic compounds such as: phenols and anilines change on
changing the pH of the solution.
Effect on sample concentration According to Lambert-Beer law it might be expected that the
sample concentration is directly proportional to the intensity of the absorption. But at high
concentrations, molecular interactions can take place causing changes to the position and shape
of absorption bands. Such effects need to be identified and taken into consideration for
quantitative work.

Effect on temperature
The effect of temperature is less pronounced. However, simple thermal expansion of the
solution may be sufficient to change band intensity. Therefore to get more accurate results the
spectrum needs to taken at a specified or constant temperature.
The following three criteria may be given as general effect of temperature on solution spectra.
1)Band sharpness increases with decreasing temperature.
2) Position of absorption maximum does not move or moves very little towards the longer
wavelength side, with decreasing temperature.
3)The total absorption intensity is approximately independent of the temperature.

How to find unknown concentrations in uv spectroscopy:

By mixing a series of known concentration solutions of the dye and measuring absorbance
ultraviolet-visible (UV-Vis) light for each, a calibration curve can be made to determine the
unknown drink concentration. Evaluate and apply the concept of solution concentration.

APPLICATION OF UV SPECTROSCOPY
UV-Visible spectroscopy is a well-established analytical technique used in the pharmaceutical
industry for testing in the research and quality control stages of drug development. They are also
used in different industries like:
1. Pharmaceutical analysis and drug development.
2. Environmental monitoring.
3. Chemical and biological plant industry.

1. Pharmaceutical analysis And drug development. UV-Vis spectrophotometry quantifies

the amount of UV or visible light absorbed by a compound, allowing researchers to
objectively assess a number of key stability indicators, including determination of active
pharmaceutical ingredients (API) and identification of contaminants. Also, UV imaging
 provides spatially and temporally resolved absorbance measurements, which are highly
useful in pharmaceutical analysis. In drugs development they help in the quantification of
impurities.
2. Environmental Monitoring UV-VIS spectroscopy is an effective method to determine
the different types of chlorine residues. This identification of different chlorine is crucial
because free residual chlorine is used effectively when detected in the drinking water.
Fluoride is another common additive present in drinking water. This spectroscopy is also
a versatile tool for the study of structure and interactions in environmental media such as
air, soil, and water as well as monitoring the metabolic responses of living organisms to
an ever changing environment.
3. Chemical and biological industryIn 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. In biology UV spectrophotometer is widely used in medical sciences for
the analysis of blood and urine samples. Quantitation of hemoglobin in the blood sample
is the key diagnostic parameter for various diseases such as dehydration, polycythemia,
and anemia.

UV spectroscopy is also used in:

● FORENSIC SCIENCE AND COUNTERFEIT DETECTION: It can also be used for the
study of carbon monoxide poising by analysis of carboxyhemoglobin in forensic Blood
Samples. Determination of metals in forensic samples can also done with UV visible
spectroscopy. Lead, arsenic are sometimes encountered at industrial work places and
many time used as poisons. In counterfeit detection it is the most commonly used method
to expose fraudulent bills. A UV detector verifies the UV marks on authentic notes by
exposing them to ultraviolet light.
● FOOD AND BEVERAGE INDUSTRY: it can be used in the food and beverage segment
in the enzymatic determination of carbohydrates like starch, glucose, sucrose, fructose,
and total dietary fiber.
● BIOMEDICAL AND CLINICAL DIAGNOSTICS: It is widely used in medical sciences
for the analysis of blood and urine samples. Quantitation of hemoglobin in the blood
sample is the key diagnostic parameter for various diseases such as dehydration,
polycythemia, and anemia.

ADVANCEMENTS AND FUTURE TRENDS

● Miniaturization and Portability: Advances in technology have led to the development of
smaller and more portable UV spectrophotometers. These compact devices allow for on-
site and real-time analysis in various applications, such as environmental monitoring,
food safety, and quality control.
● Enhanced Sensitivity: Improving the sensitivity of UV spectroscopy is a crucial aspect
for detecting trace amounts of analytes. New developments in instrumentation, such as
higher-energy light sources, improved detectors, and optimized optics, have significantly
increased the sensitivity of UV spectrophotometers.
● Multidimensional UV Spectroscopy: Multidimensional UV spectroscopy involves the
simultaneous collection of absorption data at multiple wavelengths and under different
experimental conditions. This approach provides more comprehensive information about
complex samples, allowing for better characterization and analysis of mixtures.
● Time-Resolved UV Spectroscopy: Time-resolved UV spectroscopy involves studying the
dynamic behavior of molecules over short time intervals. By using pulsed light sources
and ultrafast detection techniques, researchers can monitor rapid processes such as
chemical reactions, protein folding, and enzymatic kinetics.
● Nanotechnology Applications: The integration of UV spectroscopy with nanotechnology
has opened up new possibilities in various fields. For example, plasmonic nanoparticles
can be used to enhance the sensitivity of UV spectroscopy by amplifying the
electromagnetic field around the sample, enabling the detection of even lower
concentrations of analytes.
● Data Analysis and Chemometrics: With the increasing complexity of data generated by
UV spectroscopy, advanced data analysis techniques and chemometric methods are
becoming more important. Machine learning algorithms and statistical tools are
employed to extract valuable information, classify samples, and predict properties based
on UV spectra.
Key Points

● UV-Visible Spectroscopy is absorption spectroscopy and it involves the measurement of

the absorption, transmission and reflection of ultraviolet (UV) and visible light by
molecules.
● In UV-Visible spectroscopy, compounds absorb light falling in the UV-Visible range of
the electromagnetic spectrum.
● UV-Visible Spectroscopy is measured by an instrument called UV-Visible
Spectrophotometer.
● The UV-Visible Spectrophotometer measures absorbance.
● Beer-Lambert Law relates the concentration of the absorber with the absorbance of the
solution.
● Part of a molecule (usually an organic compound) that absorbs in the visible region gives
colour to the compound and is called a chromophore.
● It finds applications in diverse fields, including pharmaceuticals, environmental analysis,
and biochemical research.

CONCLUSION
In summary, UV spectroscopy is a versatile analytical tool that contributes significantly to
scientific research and various industries. Its ability to provide valuable insights into molecular
structure, concentration determination, reaction monitoring, and quality control makes it an
essential technique in fields ranging from pharmaceuticals and chemistry to environmental
science and materials research.