Understanding UV-Vis Spectrophotometry

UV-Visible (UV-Vis) spectrophotometry is a widely used analytical technique for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds, and biological macromolecules.

1. The Beer-Lambert Law

The principle behind UV-Vis spectrophotometry is the Beer-Lambert law, which states that the absorbance of a solution is directly proportional to the concentration of the absorbing species and the path length of the light through the solution. The formula is A = εbc, where A is absorbance, ε is the molar absorptivity, b is the path length, and c is the concentration.

2. Instrument Components

A basic UV-Vis spectrophotometer consists of a light source (deuterium lamp for UV, tungsten lamp for visible), a monochromator to select the wavelength, a sample holder (cuvette), and a detector. Proper handling of cuvettes is crucial to avoid fingerprints and scratches that can interfere with the reading.

3. Calibration and Validation

Instrument performance must be regularly verified. This includes checking wavelength accuracy with materials like holmium oxide, photometric accuracy with potassium dichromate standards, and stray light with cutoff filters.