Ultraviolet-Visible Spectroscopy

Ultraviolet-visible spectrophotometer (UV-Vis) is an instrument that measures the absorption of light by a substance in the wavelength range of 200–800 nm. Molecules that absorb light in the UV (200–400 nm) and visible (400–800 nm) regions exhibit characteristic spectral responses. UV-Vis spectroscopy is directly or indirectly used in approximately 80% of chemical analyses. It is particularly preferred in biochemistry laboratories for determining protein and nucleic acid concentrations, in environmental laboratories for water quality analysis, and in the pharmaceutical industry for purity testing.

The theoretical basis of this method is the Beer-Lambert law, and absorbance is defined by the following formula:

Where:

• A: Absorbance (dimensionless)
• ε: Molar absorptivity coefficient (L·mol⁻¹·cm⁻¹)
• l: Path length of the cuvette (cm)
• c: Concentration of the substance in solution (mol·L⁻¹)

The molar absorptivity (ε) value indicates how strongly a substance absorbs light at a specific wavelength and determines the analytical sensitivity of the selected analyte.

^UV-VIS (ARUM)

In a UV-Vis spectrophotometer, light emitted from a source is filtered by a monochromator to select a specific wavelength. This light passes through a cuvette containing the sample, and the reduction in light intensity (transmittance) is measured. This reduction corresponds to the amount of light absorbed by the sample.

The relationship between absorbance and concentration is linear, and for a substance with ε = 10⁴ L/mol·cm, analysis with over 95% accuracy is achievable. Electronic transitions in the UV region typically occur as π→π* and n→π* transitions, which are generally associated with conjugated double bonds or structures containing heteroatoms.

The wavelength resolution of monochromators is typically 0.1 nm, providing high resolving power for selective analyses.

1. Light Source:
2. 1. Deuterium lamp: Operates in the 200–400 nm range and provides 90% light efficiency.
   2. Tungsten-halogen lamp: Provides visible light in the 320–800 nm range.
2. Monochromator:
3. Works with prism or grating systems to produce selective light.
4. Sample Cuvette:
5. Cuvettes made of quartz material offer 99% transmittance in the UV region. The standard path length is 1 cm.
6. Detector:
7. 1. For the UV region: Photomultiplier tube (PMT) is used, offering light sensitivity down to 10⁻⁴ lux.
   2. For visible and near-infrared regions: InGaAs and PbS detectors are preferred.

UV-Vis spectrophotometry is used in a wide range of analytical applications:

• Protein analysis: Absorption at 280 nm due to aromatic amino acids in proteins can be determined with 95% accuracy.
• DNA/RNA quantification: Nucleic acid concentration is determined by measuring absorbance at 260 nm.
• Pharmaceutical analysis: Purity testing of pharmaceuticals can be performed with 98% sensitivity.
• Environmental analysis: In various environmental laboratories in Türkiye, UV-Vis instruments are used to analyze ions such as nitrate, nitrite, and phosphate in water samples.
• Reaction kinetics: Reaction rates are determined by monitoring the change over time of colored or UV-active intermediates.

• Single-beam System: Light passes through the sample and is measured directly; economical but lacks reference correction.
• Double-beam System: Uses reference and sample light paths simultaneously; these systems provide 20% higher accuracy and stability.
• UV-Vis-NIR Instruments: Operate in the 190–3200 nm range. The NIR region is particularly used to determine the optical band gap of polymers.

Advantages:

• High analysis speed
• Sensitivity of 0.01 AU
• Low sample volume requirement
• Suitable for quantitative analysis

Limitations:

• Only molecules containing chromophores can be analyzed.
• Spectral overlap may occur in complex mixtures, potentially reducing measurement accuracy by up to 30%.