UVVisspektrofotometri

UV-Vis spectrophotometry is an analytical technique that measures how a sample absorbs or transmits ultraviolet and visible light. It is commonly used to determine the concentration of absorbing species in solution by quantifying the amount of light that passes through the sample at selected wavelengths. The data are usually expressed as absorbance (A) or percent transmittance (T).

Principle and calibration: The measurement relies on the Beer-Lambert law, A = εbc, where ε is the molar absorptivity, b is the optical path length, and c is the concentration. In practice, calibration with standards of known concentration is used to relate absorbance to concentration for unknown samples. The law works best for dilute, non-scattering solutions; deviations can occur with high concentrations or turbid samples.

Instrumentation and configuration: A UV-Vis spectrophotometer typically includes a light source (deuterium for the UV range and a tungsten halogen for the visible range), a monochromator (grating or prism) to select the wavelength, a sample holder or cuvette, and a detector (e.g., photodiode or photomultiplier). Instruments can be single-beam or double-beam, and many modern units are diode-array spectrophotometers capable of recording full spectra quickly.

Applications and data: Measurements are performed by recording absorbance at specific wavelengths or by scanning across a spectral range to obtain a spectrum. Common applications include quantifying nucleic acids and proteins, analyzing pharmaceutical compounds, environmental monitoring, and various colorimetric assays. Limitations include scattering and turbidity, stray light, solvent absorption, and the need for accurate path length and cuvette quality. Advantages include speed, simplicity, and minimal sample preparation.