spectrofotometers

A spectrophotometer is an analytical instrument that measures the amount of light absorbed by a sample as a function of wavelength, enabling quantitative and qualitative analysis of chemical substances. Modern instruments commonly cover ultraviolet, visible, and near-infrared ranges.

In operation, light from a source passes through a wavelength selector to define a narrow band, then through the sample. A detector measures transmitted light, and the instrument compares it with a reference or blank to determine absorbance or transmittance. Double-beam instruments split the light into a sample and reference path for continual correction of fluctuations.

Typical components include a light source (such as a deuterium or halogen lamp), a monochromator (prism or diffraction grating), a sample holder (cuvette), a photodetector, and an electronics readout. Some models are single-beam; others are double-beam, often with automatic baseline correction and software for data acquisition.

The principal relationship is Beer-Lambert law: A = εlc, where A is absorbance, ε is molar absorptivity, l is the path length, and c is concentration. Measurements are usually reported as absorbance or percent transmittance. Calibration with standards allows determination of unknown concentrations.

Applications span chemistry, biochemistry, clinical diagnostics, environmental monitoring, and food and pharmaceutical analysis. They require proper blanks, cuvettes of known path length, and attention to baseline drift and stray light.

History: The method is named for Beer and Lambert, who established the relationship between absorbance and concentration in the 19th century; modern spectrophotometers were developed in the 20th century. Limitations include sensitivity to stray light, sample turbidity, and the need for appropriate standards and wavelength accuracy.