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Table-top spectrophotometer Beckman IR-1 Spectrophotometer, c. 1941 Beckman Model DB Spectrophotometer (a double beam model), 1960 Hand-held spectrophotometer used in graphic industry [1] Spectrophotometry is a branch of electromagnetic spectroscopy concerned with the quantitative measurement of the reflection or transmission properties of a ...
For spectroscopy, it is generally desirable for a source to cover a broad swath of wavelengths in order to measure a broad region of the absorption spectrum. Some sources inherently emit a broad spectrum. Examples of these include globars or other black body sources in the infrared, mercury lamps in the visible and ultraviolet, and X-ray tubes.
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.
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]
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.
Therefore, measurements at two wavelengths yields two equations in two unknowns and will suffice to determine the amount concentrations c 1 and c 2 as long as the molar attenuation coefficients of the two components, ε 1 and ε 2 are known at both wavelengths. This two system equation can be solved using Cramer's rule.
Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5 mL cuvettes or use a mobile smartphone camera (RGBradford method). [9] Wait 2 minutes and read the absorbance of each standard and sample at 595 nm. Plot the absorbance of the standards vs. their concentration.
In real atoms, there are sometimes more than two relevant transitions within the sample's Doppler profile (e.g. in alkali atoms with hyperfine interactions). This will generate the apparition of other dips in the absorption feature due to these new resonances in addition to crossover resonances.