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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.
where l is the optical path length, ε is a molar absorbance at unit path length and c is a concentration. More than one of the species may contribute to the absorbance. In principle absorbance may be measured at one wavelength only, but in present-day practice it is common to record complete spectra.
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.
Molar extinction coefficient, how strongly a substance absorbs light at a given wavelength, per molar concentration; Optical extinction coefficient, the imaginary part of the complex index of refraction
Molar concentration or molarity is most commonly expressed in units of moles of solute per litre of solution. [1] For use in broader applications, it is defined as amount of substance of solute per unit volume of solution, or per unit volume available to the species, represented by lowercase c {\displaystyle c} : [ 2 ]
where λ is a wavelength, is the optical path length of the cuvette which contains the solution of the N compounds (chromophores), , is the molar absorbance (also known as the extinction coefficient) of the ith chemical species at the wavelength λ, c i is its concentration. When the concentrations have been calculated as above and absorbance ...
When an isosbestic plot is constructed by the superposition of the absorption spectra of two species (whether by using molar absorptivity for the representation, or by using absorbance and keeping the same molar concentration for both species), the isosbestic point corresponds to a wavelength at which these spectra cross each other.
The two parameters, K or ε are determined by using the Solver module a spreadsheet, by minimizing a sum of squared differences between observed and calculated quantities with respect to the equilibrium constant and molar absorbance or chemical shift values of the individual chemical species involved.