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DESCRIPTION
4.3. PRINCIPLE OF OPERATION
Absorption of light is a typical phenomenon of interaction between electromagnetic radiation and matter.
A spectrophotometer separates electromagnetic radiation (white light) into its component wavelengths and selectively measures the
intensity of the radiation after it passes through a sample.
The white light is passed through a prism to disperse the light into bands of color. These bands of color make up the visible light
spectrum and correlate to the wavelength.
When a light beam crosses a substance, some of the radiation may be absorbed by atoms, molecules or crystal lattices.
If pure absorption occurs, the fraction of light absorbed depends both on the optical path length through the matter and on the
physical-chemical characteristics of the substance according to the Beer-Lambert Law:
T= I/I
o
-log I/I
o
= e
l
c d
or
A = e
l
c d
T = transmittance
A = absorbance
I
o
= intensity of incident light beam
I = intensity of light beam after absorption
e
l
= molar extinction coefficient at wavelength l
c = molar concentration of the substance
d = optical path through the substance
The concentration "c" can be calculated from the absorbance of the substance as the other factors are constant.
Photometric chemical analysis is based on specific chemical reactions between a sample and reagent to produce a light-absorbing
compound.
Wavelength (nm) Absorbed Color Transmitted Color
400 violet yellow-green
435 blue yellow
495 green purple
560 yellow blue
650 orange greenish blue
800 red bluish green
I
I
o
Incident light
beam
Transmitted light beam
(light beam after
absorption)
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