(02) UV Visible Spectroscopy | Instrumental Methods of Analysis | Ultraviolet Spectroscopy | B.Pharm

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UV-visible spectroscopy is a technique used to analyze the interaction between molecules and light within the ultraviolet (UV) and visible regions of the electromagnetic spectrum. It primarily focuses on the absorption of light by molecules, providing valuable information about the electronic structure of compounds.

Principle:
Absorption of Light: UV-visible spectroscopy measures the absorption of light by molecules in the UV and visible regions. When molecules absorb photons of light, they transition from lower-energy electronic states to higher-energy states.
Instrumentation:
UV-Visible Spectrophotometer: This instrument consists of a light source, monochromator (which selects specific wavelengths of light), sample holder, and detector. It measures the amount of light absorbed by a sample at different wavelengths.

How it Works:
1. Sample Preparation: The sample is usually dissolved in a suitable solvent to form a clear solution. The solvent itself should not absorb significantly in the UV-visible range.
2. Measurement: The sample solution is placed in the spectrophotometer. Light of a specific wavelength or a range of wavelengths is passed through the sample.
3. Absorbance Measurement: The spectrophotometer measures the amount of light absorbed by the sample at each wavelength.
The absorbance (A) is determined based on the ratio of incident light intensity to transmitted light intensity using Beer-Lambert's Law:
A = -log (I/I₀) = ε * c * L,
where I is the transmitted light intensity, I₀ is the incident light intensity, ε is the molar absorptivity, c is the concentration of the absorbing species, and L is the path length.

Applications:
1. Quantitative Analysis: UV-visible spectroscopy is commonly used for quantitative analysis to determine the concentration of a substance in solution.
2. Qualitative Analysis: It helps in identifying compounds based on their characteristic absorption spectra in the UV-visible range.
3. Chemical Kinetics: Monitoring changes in absorbance over time helps study reaction rates and mechanisms.
4. Determining Purity: Assessing the purity of substances by comparing spectra of pure and impure samples.

Uses in Various Fields:
1. Pharmaceuticals: Analyzing drug concentrations, purity, and stability.
2. Environmental Science: Monitoring pollutants and analyzing environmental samples.
3. Biochemistry: Studying biomolecules like proteins, nucleic acids, and enzymes.
4. Materials Science: Investigating optical properties of materials and coatings.
5. UV-visible spectroscopy is a versatile analytical technique widely used in research, quality control, and various scientific disciplines due to its simplicity, sensitivity, and ability to provide valuable information about molecular electronic transitions.

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