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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.
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.
An example batch calculation may be demonstrated here. The desired glass composition in wt% is: 67 SiO 2, 12 Na 2 O, 10 CaO, 5 Al 2 O 3, 1 K 2 O, 2 MgO, 3 B 2 O 3, and as raw materials are used sand, trona, lime, albite, orthoclase, dolomite, and borax. The formulas and molar masses of the glass and batch components are listed in the following ...
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.
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.
The equation displayed on the chart gives a means for calculating the absorbance and therefore concentration of the unknown samples. In Graph 1, x is concentration and y is absorbance, so one must rearrange the equation to solve for x and enter the absorbance of the measured unknown. [25]
In this method, the sum of the molar concentrations of the two binding partners (e.g. a protein and ligand or a metal and a ligand) is held constant, but their mole fractions are varied. An observable that is proportional to complex formation (such as absorption signal or enzymatic activity) is plotted against the mole fractions of these two ...
The attenuation coefficient of a volume, denoted μ, is defined as [6] =, where Φ e is the radiant flux;; z is the path length of the beam.; Note that for an attenuation coefficient which does not vary with z, this equation is solved along a line from =0 to as: