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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.
Thus, an Absorbance (A) of 1 corresponds to a concentration of 50 μg/mL for double-stranded DNA. This method of calculation is valid for up to an A of at least 2. [2] A more accurate extinction coefficient may be needed for oligonucleotides; these can be predicted using the nearest-neighbor model. [3]
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 machine is controlled through a computer and, once it has been "blanked", automatically displays the absorbance plotted against wavelength. Getting the absorbance spectrum of a solution is useful for determining the concentration of that solution using the Beer–Lambert law and is used in HPLC.
An absorption spectrum can be quantitatively related to the amount of material present using the Beer–Lambert law. Determining the absolute concentration of a compound requires knowledge of the compound's absorption coefficient. The absorption coefficient for some compounds is available from reference sources, and it can also be determined by ...
σ λ is their absorption cross-section at wavelength λ (units: area) B λ (T) is the Planck function for temperature T and wavelength λ (units: power/area/solid angle/wavelength - e.g. watts/cm 2 /sr/cm) I λ is the spectral intensity of the radiation entering the increment ds with the same units as B λ (T)
[3]: 21–119 Because a spectrophotometer measures the wavelength of a compound through its color, a dye-binding substance can be added so that it can undergo a color change and be measured. [19] It is possible to know the concentrations of a two-component mixture using the absorption spectra of the standard solutions of each component.
The spectra of basic, acid and intermediate pH solutions are shown. The analytical concentration of the dye is the same in all solutions. In spectroscopy, an isosbestic point is a specific wavelength, wavenumber or frequency at which the total absorbance of a sample does not change during a chemical reaction or a physical change of the sample ...
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