Concentration Determination Using Beer-Lambert Law

Definition

The Beer-Lambert law for the absorption, A, of light by a sample of concentration C is

$$ A=\upepsilon C\mathrm{\ell} $$

(1)

where ℓ is the length of the sample through which the light passes and ε is known as the (wavelength dependent) extinction coefficient. If C is measured in mol dm⁻³ and ℓ is measured in cm, then ε has units of mol⁻¹ dm³ cm⁻¹. If ε and ℓ are known, the absorbance at a given wavelength can then be used to determine concentration. Ideally, one uses a point of maximum absorbance as the absorbance signal will be less affected by small errors in wavelength calibration. However, sometimes this is not possible.

For DNA, one usually uses the absorbance signal at or near 260 nm (wherever the maximum is for a particular DNA). For DNA, the linear relationship between concentration and absorbance breaks down when the absorbance of a 1-cm path length solution exceeds ∼ 1.5–2 absorbance units, presumably due to DNA-DNA interactions. Some DNA extinction coefficients...