Photometry: Colorimeter
and Spectrophotometer^5
Photometry deals with the study of phenomenon of light absorption by molecules in
solution. The specificity of a compound to absorb light at a particular wavelength is
useful in quantitative measurements. When a light beam of particular wavelength is
passed through a solution, some amount of light is absorbed by the solution, and
consequently the intensity of light that comes out of solution is diminished. The
phenomenon of absorption of light by a solution follows Beer-Lambert’s law. Beer’s
law states that amount of transmitted light decreases exponentially with increase in
the concentration of absorbing medium. In other words, absorbance is directly
related to the concentration of the absorbing medium. Lambert’s law states that
amount of transmitted light decreases exponentially with increase in thickness of
absorbing medium. The two laws can be combined, hence
I¼I 0 εclwhere
I¼Intensity of transmitted light
I 0 ¼Intensity of incident light
ε¼Molar absorption coefficient
c¼Concentration of absorbing substance (g/dl)
l¼Thickness of absorbing medium or solution
When thickness of absorbing medium is kept constant, then intensity of incident
light depends only upon the concentration of absorbing medium, or we can say that
Beer’s law is operative. The ratio of transmitted light to incident light is called
transmittance, whereas absorbance or optical density can be calculated as
#Springer Nature Singapore Pte Ltd. 2018
V. Kumar, K. D. Gill,Basic Concepts in Clinical Biochemistry: A Practical Guide,
https://doi.org/10.1007/978-981-10-8186-6_5
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