UV-Vis Spectroscopy – Principle, Instrumentation, Applications, Advantages & Limitations

UV-Vis Spectroscopy – Principle, Instrumentation, Applications, Advantages & Limitations

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What is UV-Vis Spectroscopy?

  • UV-Vis Spectroscopy (Ultraviolet–Visible Spectroscopy) is a widely used analytical technique that studies how a sample absorbs or transmits ultraviolet (190–400 nm) and visible light (400–800 nm).
  • It is also called absorption spectroscopy or reflectance spectroscopy.
  • Since it involves electron transitions between energy levels, it is also known as electronic spectroscopy.
  • UV-Vis spectroscopy is simple, cost-effective, versatile, and non-destructive – meaning the same sample can be reused after analysis.

Thus, UV-Vis spectroscopy tells us how much light a substance absorbs, which helps in identifying compounds, checking purity, and estimating concentration.

UV-Vis Spectroscopy- Light Spectrum
Figure: Light Spectrum

Principle of UV-Vis Spectroscopy

  • UV-Vis spectroscopy works on the principle of electronic transitions.
  • When light of a specific wavelength hits a molecule, its electrons get excited from a lower energy level (ground state) to a higher energy level (excited state).
  • The amount of energy absorbed = the difference between the two energy levels.
  • Only light with the right energy (wavelength) will be absorbed.

This absorption pattern gives an absorption spectrum, unique for each substance, which can be used for identification and quantification.

Beer–Lambert Law

The Beer–Lambert Law forms the foundation of UV-Vis spectroscopy:

A=ε⋅c⋅l

Where:

  • A = Absorbance
  • ε = Molar absorptivity (constant for a given substance at a specific wavelength)
  • c = Concentration of the solution
  • l = Path length of the cuvette (cm)

Key Point: Absorbance is directly proportional to concentration. This means UV-Vis spectroscopy can be used to measure unknown concentrations by comparing with standards.

Figure: UV-Vis Spectroscopy
Figure: UV-Vis Spectroscopy

Wavelength Ranges in UV-Vis

  • Far UV region: < 200 nm
  • Near UV region: 190–400 nm
  • Visible region: 400–800 nm

Shorter wavelength = higher energy & frequency (UV range)
Longer wavelength = lower energy & frequency (visible range)

Components of UV-Vis Spectroscopy

A typical UV-Vis spectrophotometer has the following parts:

1. Light Source

  • Provides radiation in both UV and visible ranges.
  • Common sources:
    • Deuterium lamp → UV light (190–400 nm).
    • Tungsten/Halogen lamp → Visible light (400–800 nm).
    • Xenon lamp → Broad spectrum, but costly.

2. Wavelength Selector (Monochromator)

  • Separates light into specific wavelengths.
  • Types:
    • Prism (non-linear dispersion).
    • Diffraction grating (linear dispersion, widely used).

3. Sample Holder (Cuvette)

  • Holds the sample solution.
  • Quartz cuvettes → for UV region.
  • Glass/plastic cuvettes → for visible region.
  • Types of instruments:
    • Single-beam spectrophotometer → compares sample vs blank separately.
    • Double-beam spectrophotometer → splits light into two beams (sample & reference simultaneously), more accurate.

4. Detector

  • Detects transmitted light and converts it into an electrical signal.
  • Examples: Photodiodes, Photomultiplier tubes (PMTs).
  • Must be sensitive, linear, and low-noise.

5. Readout System

  • Displays data as absorbance or transmittance.
  • Modern systems are computerized and provide detailed spectra.

Working of UV-Vis Spectroscopy

  1. Calibration with a blank (solvent without solute).
  2. Sample solution placed in cuvette.
  3. Selected wavelength light passes through the sample.
  4. Part of light absorbed, part transmitted.
  5. Detector measures transmitted intensity (I).
  6. Absorbance calculated using:

A=−log(T)=−log(I/I₀​)

Where:

  • I₀ = intensity of incident light
  • I = intensity of transmitted light

Applications of UV-Vis Spectroscopy

Biological & Biochemical Applications

  • DNA and RNA quantification – purity checked using absorbance ratio at 260/280 nm.
  • Protein analysis – absorbance at 280 nm.
  • Bacterial culture monitoring – growth tracked by measuring optical density (OD) at 600 nm.

Pharmaceutical Applications

  • Drug discovery and quality control.
  • Detecting impurities in drugs.
  • Dissolution testing of tablets.
  • Identification of active compounds.

Food & Beverage Industry

  • Analysis of nutritional content.
  • Quality control in juices, wine, beer, honey, oils.
  • Detecting contaminants or adulterants.
  • Evaluating color and flavor compounds (e.g., anthocyanins in berries).

Environmental Science

  • Monitoring pollutants, dyes, and byproducts in water treatment.
  • Studying kinetics of dye removal in wastewater.

Industrial Applications

  • Cosmetic industry → testing dyes, antioxidants, and photostability.
  • Petrochemical industry → analyzing crude oil, sulfur content, aromatic compounds.
  • Material science → nanoparticle characterization.

Medical & Clinical Uses

  • Hemoglobin estimation in cancer research.
  • Protein denaturation studies.
  • Vitamin and antioxidant quantification.

Advantages of UV-Vis Spectroscopy

  • Simple and easy to operate.
  • Non-destructive – sample can be reused.
  • Cost-effective and widely available.
  • Provides fast and accurate results.
  • Can be applied to solids, liquids, powders, and films.

Limitations of UV-Vis Spectroscopy

  • Cannot give detailed structural information.
  • Interference from impurities or stray light may affect accuracy.
  • Limited to compounds that absorb in UV-Vis range.
  • Requires transparent and clean cuvettes.

Precautions

  • Always use a blank solution for calibration.
  • Use clean, scratch-free cuvettes.
  • Avoid stray light or bubbles in the sample.
  • Maintain lamp stability and replace faulty light sources.
  • Ensure proper wavelength selection for accurate results.

FAQs on UV-Vis Spectroscopy

Q1. Why is quartz cuvette used for UV measurements?
Because ordinary glass absorbs UV light.

Q2. What is the wavelength range of UV-Vis spectroscopy?
190–800 nm.

Q3. What law is the basis of UV-Vis spectroscopy?
Beer–Lambert’s Law.

Q4. Can UV-Vis spectroscopy be used for DNA/RNA analysis?
Yes, it measures purity and concentration of nucleic acids.

Q5. What is the difference between single-beam and double-beam spectrophotometers?
Single-beam compares sample vs blank separately, while double-beam measures both simultaneously.

Conclusion

UV-Vis Spectroscopy is one of the most important analytical tools in biology, chemistry, pharmaceuticals, food science, and environmental studies. By studying how compounds absorb light in the 190–800 nm range, scientists can determine concentration, purity, and molecular properties.

In short: UV-Vis spectroscopy is a simple, fast, and reliable method to analyze and quantify compounds in both research and industry.

References

  1. https://www.smacgigworld.com/blog/applications-uv-vis-spectroscopy.php
  2. https://suntrics.com/tech-blogs/uv-vis-spectroscopy/
  3. https://www.mt.com/in/en/home/applications/Application_Browse_Laboratory_Analytics/uv-vis-spectroscopy/uvvis-spectroscopy-explained.html
  4. https://edu.rsc.org/download?ac=12904
  5. https://www.technologynetworks.com/analysis/articles/uv-vis-spectroscopy-principle-strengths-and-limitations-and-applications-349865
  6. https://www.agilent.com/cs/library/primers/public/primer-uv-vis-basics-5980-1397en-agilent.pdf
  7. https://byjus.com/chemistry/spectrophotometer-principle/
  8. https://www.slideshare.net/mariomS7/uvvis-spectroscopy
  9. https://www.labcompare.com/10-Featured-Articles/592706-Tech-Compare-Single-vs-Double-Beam-Spectrophotometers/
  10. https://www.hunterlab.com/blog/single-beam-vs-double-beam-spectrophotometer/
  11. https://www.slideshare.net/msakhan61/uv-visible-spectroscopy-principles-and-instrumentation

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