Absorbance

Absorbance is a measure of how much light at a specified wavelength is absorbed by a sample. In UV–visible spectroscopy, absorbance is usually reported as a unitless quantity, sometimes referred to as an absorbance unit.

Absorbance relates to transmittance, the fraction of incident light that passes through the sample. If I0 is the incident intensity and I the transmitted intensity, transmittance T = I/I0 and absorbance A = -log10(T) = log10(I0/I). Thus higher absorbance means less transmitted light.

Beer-Lambert law: A = ε l c. Here ε is the molar absorptivity (L·mol−1·cm−1), l is the optical path length in cm, and c is the solute concentration in mol/L. In a standard 1 cm cuvette, A is proportional to concentration for a given wavelength within a material’s linear range.

Measurement and calibration: To determine concentration from A, blank the instrument with the solvent, measure at the wavelength of maximum absorption, and use a calibration curve of A versus c. Keep within the linear range and dilute samples if necessary; use cuvettes of correct path length; ensure the solution is clear to minimize scattering.

Instrumentation and limitations: A spectrophotometer uses a light source, a wavelength selector, and a photodetector. Practical measurements can be affected by stray light, scattering, inner-filter effects, and fluorescence. At high concentrations, deviations from Beer-Lambert law occur, requiring dilution or correction.

Applications: Absorbance measurements are widely used to quantify concentrations of dyes, nucleic acids, proteins, and other analytes in chemistry and biology, as well as in environmental and clinical analyses.