Testing:
(1) A beam of light of known intensity is split and directed through 2 cells, and the intensity of the light is measured and compared.
(2) One cell holds solvent containing the compound (sample cell), while the second cell contains only pure solvent (reference cell).
(3) When only solvent is present without added compound, the transmission is 100% (sample cell same as reference cell).
percent transmission =
= - log10 ((intensity of light passing through sample cell) / (intensity of light passing through the reference cell))
absorbance =
= - log10 (percent transmission)
= 2 - log10 (percent transmission)
A series of standards of known concentration are tested and the percent transmission recorded
(1) the percent transmission shows a semilogarithmic plot when plotted versus the concentration of the compound, showing a decline as the concentration increases
(2) the absorbance is linear when plotted versus concentration of compound, and increases as the concentration increases
Once a series of standards has been measured, then a sample containing an unknown concentration of that compound can be found, by comparing the percent transmission (or absorbance) for that sample versus that of the standard curve.
