The extent to which a sample absorbs light depends strongly upon the wavelength (λ) of light. For this reason, spectrophotometry is performed using monochromatic light. Monochromatic light is light in which all photons have the same wavelength.
In analyzing a new sample, a chemist first determines the sample's absorbance spectrum. The absorbance spectrum shows how the absorbance of light depends upon the wavelength of the light. The spectrum itself is a plot of absorbance vs wavelength (A vs λ) or molar absorptivity vs wavelength (ε vs λ). The spectrum is characterized by the wavelength (λmax) at which the absorbance (or molar absorptivity) is the greatest.
The value of λmax is important for several reasons. Each compound has a characteristic λmax that provides information on the electronic structure of the compound. Also, spectrophotometry has the highest sensitivity and most closely obeys Beer's Law when using light with a wavelength of λmax.
Indigo Carmine is a common food dye known as Blue No 2 or E132. The absorbance spectrum of Blue No 2 is shown below. Observe that there is a peak in the spectrum where the light is most strongly absorbed. Measurements at this wavelength provide the greatest sensitivity, that is, the highest absorbance for a given concentration and cell path length. There are also regions (above 720 nm, for example) where there is little or no absorbance of light.
The experiment below uses a 2.00 cm cell containing a sample solution of an unknown concentration of Blue No 2.
Perform the experiment using light of various wavelengths between 400 and 780 nm. Because the output from the light source varies with wavelength, it will be necessary to perform a blank measurement to determine I0 at each wavelength. Use the intensity data for the sample and blank to determine the absorbance at various wavelengths. For each measurement, plot the point on the graph. For comparison, the spectrum of Indigo Carmine is plotted.
Operation of the Spectrophotometer: Select the desired cell path length and tartrazine concentration. Then start the simulation. Once photons begin reaching the detector, start the Data Acquisition. The intensity of light (photons per second) reaching the detector will be displayed. Note that the simulation employs more photons than are shown on the screen.
- Do the measured absorbances lie along the graphed absorbance spectrum?
- What is the value for λmax ?
- What is the value of εmax ?
Absorbance-Spectrum.html version 3.0
© 2000, 2014, 2020 David N. Blauch