Absorption refers to the reduction of the intensity of the light when it passes through the solution of a substance. An instrument called a spectrophotometer allows you to experimentally measure absorption of the solution placed in a cuvette. The Beer-Lambert Law determines the relationship between the concentration and absorption, expressed as Absorption = (Molar extinction coefficient) * (Path length) * (Concentration). The molar extinction coefficient is a characteristic of a substance--a protein, for example--that indicates how much the compound absorbs the light. The path length is the width of the cuvette.
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Divide the absorption value by the path length. A standard cuvette has the path length, or width, of 1 centimetre (cm). For example, if the absorption is 0.24, then 0.24/1 = 0.24.
Divide the value obtained in the previous step by the molar extinction coefficient to calculate the molar concentration of a compound. For example, if the molar extinction coefficient is 480, the concentration is 0.24/480 = 0.0005 mole per litre (L).
Multiply the molar concentration by the molecular mass of the compound to calculate the concentration in gram/L units. For example, if the compound has the molecular mass of 750g/mole, then 0.0005 mole/L * 750g/mole = 0.375g/L.
Tips and warnings
- Molar extinction coefficients for many substances (including proteins) are tabulated and can be found easily online.
- For proteins, this parameter can also be calculated based on the protein amino acid sequence using online tools.
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