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Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". [1] Alternatively, for samples which scatter light, absorbance may be defined as "the negative logarithm of one minus absorptance, as measured on a uniform sample". [2]
The absorbance of a material that has only one absorbing species also depends on the pathlength and the concentration of the species, according to the Beer–Lambert law =, where ε is the molar absorption coefficient of that material; c is the molar concentration of those species; ℓ is the path length.
This should not be confused with "absorbance". Spectral hemispherical absorptance: A ν A λ — Spectral flux absorbed by a surface, divided by that received by that surface. This should not be confused with "spectral absorbance". Directional absorptance: A Ω — Radiance absorbed by a surface, divided by the radiance incident onto that surface.
Hemispherical transmittance of a surface, denoted T, ... A is the absorbance. The Beer–Lambert law states that, for N attenuating species in the material sample, = ...
Variable pathlength absorption spectroscopy uses a determined slope to calculate concentration. As stated above this is a product of the molar absorptivity and the concentration. Since the actual absorbance value is taken at many data points at equal intervals, background subtraction is generally unnecessary.
The decadic absorbance of a scattering sample is defined as −log 10 (R+T) or −log 10 (1−A). For a non scattering sample, R = 0, and the expression becomes −log 10 T or log( 1 / T ), which is more familiar. In a non-scattering sample, the absorbance has the property that the numerical value is proportional to sample thickness.
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The ratio of the absorbance at 260 and 280 nm (A 260/280) is used to assess the purity of nucleic acids. For pure DNA, A 260/280 is widely considered ~1.8 but has been argued to translate - due to numeric errors in the original Warburg paper - into a mix of 60% protein and 40% DNA. [ 6 ]