Spectroscopy: study guide

Key ideas

Absorption spectroscopy measures how much light a sample absorbs. The Beer–Lambert law makes absorbance proportional to concentration, which is the basis of most colorimetric and UV assays.

• Transmittance is T = I ÷ I₀, and absorbance is A = −log₁₀ T. An absorbance of 1 means 10 % of the light gets through, and 2 means 1 %.
• The Beer–Lambert law is A = εbc, where ε is the molar absorptivity, b is the path length in cm and c is the concentration in mol/L.
• A calibration curve of absorbance against known concentrations lets you read off an unknown. It works best for absorbances below about 1.
• Always blank the instrument with the solvent or buffer so that only the sample’s absorbance is measured.

Key equations

• A = εbc
• A = −log₁₀ T
• T = I ÷ I₀
• E = hc ÷ λ

Worked example

A solution has ε = 6220 L/(mol·cm), a 1.00 cm cuvette, and A = 0.62. Then c = A ÷ (εb) = 0.62 ÷ 6220 = 1.0 × 10⁻⁴ mol/L. The transmittance is T = 10^(−0.62) = 0.24, so 24 % of the light is transmitted.

Common mistakes

• Using percent transmittance in place of absorbance in A = εbc.
• Forgetting the path length when the cuvette is not 1 cm.
• Trusting readings above an absorbance of about 2, where the instrument is no longer linear.

Practice

Test yourself with practice questions on this topic: Spectroscopy