Lambert-Beer's law
The law was first developed by by Pierre Bouguer before 1729. It was later attributed to Johann Heinrich Lambert who cited Bouguer’s findings. The law included path length as a variable that affected absorbance. Later, Beer extended in 1852 the law to include the concentration of solutions, thus giving the law its name Beer-Lambert Law.
Definition & Equation
- The Beer-Lambert law states that the quantity of light absorbed by a substance dissolved in a fully transmitting solvent is directly proportional to the concentration of the substance and the path length of the light through the solution.
- Absorbance [A (λ)] of a substance at a particular wavelength of electromagnetic radiation, λ, is proportional to the concentration, c, of the absorbing substance and to the length of the path, l, of the electromagnetic radiation through the sample containing the absorbing substance.
- The law is written as follows:
A(λ) = e(λ) l c. The proportionality constant e (λ) is called the absorptivity of the substance at the wavelength λ. e (λ) is called the molar absorptivity if the concentration is measured in moles/liter.
Derivation of Law
A spectrophotometer is an apparatus that measures the intensity, energy carried by the radiation per unit area per unit time, of the light entering a sample solution and the light going out of a sample solution. The two intensities can be expressed as transmittance: the ratio of the intensity of the exiting light to the entering light or percent transmittance (%T). Different substances absorb different wavelengths of light. Therefore, the wavelength of maximum absorption by a substance is one of the characteristic properties of that material. A completely transparent substance will have It = I0 and its percent transmittance will be 100. Similarly, a substance which allows no radiation of a particular wavelength to pass through it will have It = 0, and a corresponding percent transmittance of 0.
Transmittance
T = It / I0
% Transmittance: %T = 100 T
Absorbance
A = log10 (I0 / It)
A = log10 (1 / T) = -log10 (T)
A = log10 (100 / %T)
A = 2 - log10 (%T)
Transmittance for liquids, is usually written as:
T = I / I0=10-αl =10Σlc'',
Transmittance for gases they are written as:
T=I/Io=10-α´l=e-σlN
I0 and I are the intensity (or power) of the incident light and the transmitted light, respectively.
Absorbance for liquids, is written as:
A =-log10 = (I/I0)
Absorbance for gases, it is written as:
A´=-ln(I/I0)
Deviations to the Law
The Beer-Lambert law maintains linearity under specific conditions only. The law will make inaccurate measurements at high concentrations because the molecules of the analyte exhibit stronger intermolecular and electrostatics interactions which is due to the lesser amount of space between molecules. This can change the molar absorptivity of the analyte. Not only does high concentrations change molar absorptivity, but it also changes the refractive index of the solution causing departures from the Beer-Lambert law.
Applications
Beer-Lamberts law is applied to the analysis of a mixture by spectrophotometry, without the need for extensive pre-processing of the sample. Examples include the determination of bilirubin in blood plasma samples. The spectrum of pure bilirubin is known thus the molar absorbance is known. Measurements are made at one specific wavelength almost unique for bilirubin and another measurement at a second wavelength so interferences or deviations can be eliminated or corrected. Generally, it can be used to determine concentrations of s particular substance, or determine the molar absorptivity of a substance.
