Biochemical Techniques and Instrumentation

1.1.4. Basic laws of light absorption

The absorbance of photon by the matter is given by an extinction coefficient and it is dependent on the wavelength λ of the photon. If a light with intensity, Io passes through a sample and the path length or thickness (d), the intensity I drops along the pathway. This characteristic absorption parameter is the extinction coefficient x. This yields the correlation I = Io e ^–xd. The ratio of T = I / Io is called the transmission.

According to the Beer-Lambert law:

log Io / I = log 1 / T = ε x c x d = A

where Io = intensity of incident light, I = intensity of transmitted light, d = 1 cm, c = 1 mol dm^-3 and ε = 1 dm³mol‑cm^-1, ε is the molar absorption coefficient. A is the absorbance of the sample.

Beers-Lamberts Law states that the absorption is proportional to the concentration of absorbing substance and to the path length.

Beer’s law is valid for low concentrations only. Higher concentrations lead to association of molecules and cause deviations.