UV-Vis (Ultraviolet-Visible) spectroscopy is an analytical technique that measures the absorption of light in the ultraviolet (190-400 nm) and visible (400-800 nm) regions of the electromagnetic spectrum. It is widely used for quantitative analysis, kinetic studies, and characterization of electronic transitions in molecules.
Principle of UV-Vis Absorption
When light passes through a sample, molecules absorb photons whose energy matches the energy gap between electronic orbitals, promoting electrons from the ground state to an excited state. The amount of light absorbed at each wavelength is recorded as an absorbance spectrum, and the wavelength of maximum absorption (λmax) is characteristic of each chromophore. The Beer-Lambert Law (A = εcl) relates absorbance (A) to molar absorptivity (ε), path length (c), and concentration (l), enabling quantification.
Instrumentation
A UV-Vis spectrometer comprises several components. A deuterium lamp provides light for the UV range and a tungsten-halogen lamp for the visible range. A monochromator (diffraction grating or prism) selects a narrow band of wavelengths. The sample compartment holds a quartz cuvette for UV measurements or a glass cuvette for visible-range measurements, and a detector (photomultiplier tube or photodiode array) converts transmitted light into an electrical signal. Double-beam instruments split the light into sample and reference beams to correct for solvent and lamp fluctuations.
Applications
UV-Vis spectroscopy is used for quantitative analysis of nucleic acids (A260), proteins (A280), and enzyme kinetics, determination of pKa values by measuring absorbance changes with pH, quality control of pharmaceuticals, food colorants, and water pollutants, and characterization of transition metal complexes and conjugated organic compounds.
Advantages and Limitations
- Advantages: Fast, non-destructive, requires small sample volumes, and is relatively inexpensive.
- Limitations: Only measures chromophoric species; samples must be transparent and free of scattering particles.
