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UV-Vis Spectroscopy Notes

UV-Visible (UV-Vis) spectroscopy is an analytical technique that measures the absorbance of UV and visible light by chemical substances, aiding in qualitative and quantitative analysis. It operates on the principle of electronic transitions and follows Beer-Lambert Law to determine concentration and identify compounds. Key components include a light source, monochromator, sample holder, detector, and data processor, with applications in various fields such as pharmaceuticals, environmental monitoring, and biochemistry.

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25 views2 pages

UV-Vis Spectroscopy Notes

UV-Visible (UV-Vis) spectroscopy is an analytical technique that measures the absorbance of UV and visible light by chemical substances, aiding in qualitative and quantitative analysis. It operates on the principle of electronic transitions and follows Beer-Lambert Law to determine concentration and identify compounds. Key components include a light source, monochromator, sample holder, detector, and data processor, with applications in various fields such as pharmaceuticals, environmental monitoring, and biochemistry.

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UV-Visible (UV-Vis) Spectroscopy

Definition:
UV-Vis spectroscopy is an analytical technique used to measure the absorbance or
transmittance of UV (200–400 nm) and visible (400–800 nm) light by a chemical substance,
which helps in qualitative and quantitative analysis.

1. Principle
The UV-Vis spectroscopy principle is based on electronic transitions. When a molecule
absorbs ultraviolet or visible light, electrons in the molecule are excited from a lower
energy level (ground state) to a higher energy level (excited state). The amount of light
absorbed at each wavelength is measured, and the absorption pattern provides information
about the structure and concentration of the molecule.

The absorption follows Beer-Lambert Law:

A=εcl

Where:
A = Absorbance
ε = Molar absorptivity (L mol⁻¹ cm⁻¹)
c = Concentration (mol L⁻¹)
l = Path length (cm)

2. Working
A beam of UV or visible light passes through a sample. If the sample contains molecules that
can absorb light in this range, part of the light will be absorbed and the rest will pass
through. The spectrophotometer measures the intensity of the light before (I₀) and after (I)
passing through the sample.

The instrument calculates absorbance (A) using:

A = log(I₀ / I)

The absorbance at specific wavelengths can be used to identify the compound and
determine its concentration.
3. How Does a UV-Vis Spectrophotometer Work?
A UV-Vis spectrophotometer works by:
- Emitting light from a light source.
- Passing the light through a monochromator (which selects the desired wavelength).
- Splitting the light into two beams: one passes through a reference sample (usually
solvent), and the other through the test sample.
- Measuring the difference in light intensity between the two beams using a detector.
- The instrument converts the data into an absorbance spectrum, showing how much light
was absorbed at each wavelength.

4. Components of UV-Vis Spectrophotometer


1. Light Source:
- Deuterium lamp (for UV region: 200–400 nm)
- Tungsten-halogen lamp (for Visible region: 400–800 nm)

2. Monochromator:
- Splits light into different wavelengths and selects the desired one using prisms or
diffraction gratings.

3. Sample Holder (Cuvette):


- Holds the liquid or solid sample. Typically made of quartz (UV) or glass (Visible).

4. Detector:
- Converts transmitted light into electrical signals (Photodiode or Photomultiplier Tube).

5. Data Processor and Display:


- Converts signals into readable absorbance spectra.

5. Applications of UV-Vis Spectroscopy


- Quantitative Analysis: Determining concentrations of solutions (using Beer-Lambert Law).
- Qualitative Analysis: Identifying compounds based on their characteristic absorbance
spectra.
- Molecular Structure Analysis: Studying electronic transitions in conjugated systems.
- Pharmaceutical Industry: Purity testing, drug formulation, and dissolution studies.
- Environmental Monitoring: Detecting pollutants in water and air.
- Biochemistry: Protein and nucleic acid quantification (e.g., DNA absorbs at 260 nm).
- Food Industry: Color measurements and additive detection.

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