UV Spectroscopy – Definition, Principle, Instrumentation, Steps, Applications, Advantages & Limitations

Table of Contents

Introduction to UV Spectroscopy

Spectroscopy is the study of the interaction between matter and electromagnetic radiation.
In UV spectroscopy, light from the ultraviolet (UV) region (200–400 nm) is absorbed by molecules, causing electronic excitation (promotion of electrons from ground state to higher energy states).
Molecules with π-electrons (like in double bonds) or non-bonding electrons (n-electrons) absorb UV light strongly.
The technique produces a unique absorption spectrum that serves as a molecular fingerprint for qualitative and quantitative analysis.

In simple terms: UV Spectroscopy tells us about the structure, purity, and concentration of compounds by analyzing how they absorb UV light.

**Figure:** UV Spectrophotometer used in UV Spectroscopy. Image source: richmondscientific

Principle of UV Spectroscopy

When a molecule absorbs UV light, its electrons are excited from ground state (low energy) to excited state (high energy).
The wavelength of absorbed light depends on how easily electrons can be excited.
Molecules with conjugated double bonds absorb at longer wavelengths.
The absorption spectrum helps identify compounds and functional groups.

Types of Electronic Transitions in UV Spectroscopy

σ → σ* (highest energy transition)
- Involves excitation of sigma bond electrons.
- Requires strong UV light of shorter wavelength (<200 nm).
n → σ*
- Involves excitation of non-bonding electrons (lone pairs) to antibonding σ-orbitals.
- Absorption occurs in 150–250 nm region.
π → π*
- Involves excitation of pi electrons in double bonds to antibonding π-orbitals.
- Found in molecules with C=C, C=O, and aromatic systems.
n → π*
- Involves excitation of lone pairs to π* antibonding orbitals.
- Seen in carbonyl groups (C=O) and compounds with heteroatoms.
- Requires less energy, absorbs at longer wavelengths.

Order of energy required: σ → σ* > n → σ* > π → π* > n → π*

Components of UV Spectroscopy

A UV spectrophotometer has the following main parts:

Radiation Source
- Provides UV light (200–400 nm).
- Common sources:
  - Deuterium lamp – for continuous UV radiation.
  - Hydrogen lamp – alternative source.
Monochromator
- Splits light into individual wavelengths.
- Uses prisms or diffraction gratings.
Sample Holder (Cuvette)
- Usually made of quartz (since glass absorbs UV).
- Holds liquid samples in path of UV light.
Detector
- Measures transmitted light after passing through sample.
- Common detectors:
  - Photomultiplier tube
  - Photodiode array detectors
Recorder/Display Unit
- Converts detector signals into a spectrum.
- Produces a graph of absorbance vs wavelength.

Steps in UV Spectroscopy

Sample Preparation
- Dissolve sample in a solvent transparent to UV light (e.g., ethanol, hexane).
- Place in a quartz cuvette.
Radiation Exposure
- Pass UV light through the sample.
- Some light is absorbed, the rest is transmitted.
Detection
- Detector measures transmitted light.
Data Analysis
- The spectrophotometer generates a graph (spectrum) showing peaks at absorbed wavelengths.
- The position and intensity of peaks provide structural information.

Factors Affecting UV Absorption

Conjugation: Longer conjugation → absorption shifts to longer wavelengths (bathochromic shift).
Polarity of Solvent: Polar solvents stabilize excited states → shift absorption peaks.
Substituents: Electron-donating groups shift absorption to longer wavelengths.
pH: Ionization of molecules can change absorption.

Applications of UV Spectroscopy

1. Detection of Impurities

Extra peaks in the spectrum indicate impurities.
Comparison with pure standard helps identify contamination.

2. Structure Elucidation of Organic Compounds

Identifies functional groups and conjugated systems.
Helps determine unsaturation and presence of heteroatoms.

3. Qualitative and Quantitative Analysis

Qualitative: Identification by comparing spectra with reference.
Quantitative: Based on Beer-Lambert’s law (Absorbance = εcl).

4. Drug Analysis in Pharmaceuticals

Used to assay raw materials and drug formulations.
Quick method for quality control of medicines.

5. Kinetics of Reactions

Absorbance changes with time → used to study reaction rates.

6. Molecular Weight Determination

By preparing derivatives and analyzing absorbance, approximate molecular weight can be determined.

7. Chromatography Detector

UV detectors are widely used in HPLC for compound detection.

Advantages of UV Spectroscopy

Simple and rapid technique.
Non-destructive method.
Requires very small sample.
High sensitivity.
Applicable to both qualitative and quantitative analysis.
Can detect impurities at low concentrations.

Limitations of UV Spectroscopy

Only works for compounds with chromophores.
Provides limited structural information (not as detailed as NMR or IR).
Overlapping spectra may complicate interpretation.
Sample must be soluble in UV-transparent solvent.

FAQs on UV Spectroscopy

Q1. What is UV Spectroscopy?
Ans: It is an analytical method that studies how molecules absorb ultraviolet light (200–400 nm) to identify and analyze compounds.

Q2. What is the principle of UV Spectroscopy?
Ans: Molecules absorb UV radiation, causing electronic excitation of π and n electrons to higher energy states.

Q3. What type of transitions are studied in UV Spectroscopy?
Ans: σ → σ*, n → σ*, π → π*, and n → π*.

Q4. Why is quartz used in UV cuvettes instead of glass?
Ans: Glass absorbs UV light, while quartz is transparent to it.

Q5. What are the applications of UV Spectroscopy?
Ans: Drug analysis, impurity detection, reaction kinetics, structure elucidation, and as detectors in chromatography.

Q6. What is Beer-Lambert’s law in UV Spectroscopy?
Ans: It relates absorbance (A) to concentration (c), path length (l), and molar absorptivity (ε): A = εcl.

Q7. Can UV Spectroscopy be used for quantitative analysis?
Ans: Yes. It is widely used for quantitative estimation of compounds using Beer-Lambert’s law.

Q8. What are chromophores and auxochromes in UV Spectroscopy?
Ans:

Chromophores: Groups responsible for absorption (C=C, C=O, aromatic rings).
Auxochromes: Groups that modify absorption intensity or wavelength (–OH, –NH2).

Q9. What is a bathochromic shift?
Ans: Also called red shift, it is the shift of absorption to longer wavelength due to conjugation or solvent effects.

Q10. What is a hypsochromic shift?
Ans: Also called blue shift, it is the shift of absorption to shorter wavelength, usually due to loss of conjugation.

Q11. Can UV spectroscopy detect impurities in drugs?
Ans: Yes. It is one of the most reliable methods for detecting impurities in pharmaceuticals.

Q12. What solvents are used in UV Spectroscopy?
Ans: Ethanol, hexane, cyclohexane, and water (if transparent in the UV region).

Q13. What are the advantages of UV Spectroscopy?
Ans: Rapid, simple, sensitive, non-destructive, and requires small sample volume.

Q14. What are the limitations of UV Spectroscopy?
Ans: Cannot analyze compounds without chromophores, less detailed structural information compared to IR or NMR.

Q15. Why is UV Spectroscopy important in pharmaceuticals?
Ans: It ensures drug purity, potency, and quality control during manufacturing.

Conclusion

UV Spectroscopy is a fundamental analytical tool in chemistry, biology, and pharmaceuticals.
It works on the principle of electronic transitions caused by UV light absorption.
Its applications range from impurity detection and drug analysis to chromatography detection and reaction kinetics.
Although limited in providing detailed structural data, its simplicity, sensitivity, and speed make it indispensable.

In short: UV Spectroscopy is a fast, reliable, and widely used technique for studying molecular properties through light absorption.