Lab: Chemistry
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Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA
In the first part of the lab, you'll use ultraviolet and visible light absorption spectroscopy, or UV-Vis absorption spectroscopy, to analyze the absorption characteristics of fluorescein, β-carotene, and indigo dye. To perform UV-Vis spectroscopy, you'll place a solution between a light source and a photodetector. The molecules will absorb light at wavelengths that correspond to the energies needed to excite their electrons and scatter or transmit the rest.
An absorption spectrum represents the variation in the number of photons of each wavelength that reach the detector. A higher absorbance corresponds to fewer detected photons of that wavelength. You'll take a spectrum of the pure solvent first, which the instrument will subtract from the spectrum of the dye solution to show you just the absorbance data from the dye. This reference is called a solvent blank.
Absorbance range (nm) | λmax (nm) | |
Fluorescein | ||
β-carotene | ||
Indigo |
In the second half of the lab, you'll use UV-Vis spectroscopy to relate absorbance intensity and concentration for β-carotene by measuring the absorbances of 5 β-carotene solutions with different concentrations. This will let you make a calibration curve of absorbance versus β-carotene concentration and derive the equation describing that relationship.
Test | Concentration (µM) | Absorbance at λmax (450 nm) |
1 | 1.9 | |
2 | 3.7 | |
3 | 7.5 | |
4 | 11 | |
5 | 15 |
First, look at the spectra of the three dyes. Fluorescein in water absorbs blue and purple light, with the maximum absorbance at 490 nm. It does not absorb red light, and it only absorbs some yellow and green light. Consistent with this, solid fluorescein is red, and fluorescein solutions are usually yellow to green.
β-carotene also absorbs blue and purple light. The maximum absorbance of β-carotene in hexane is 450 nm, and you'll see another large peak at 478 nm. The strong absorption of purple light is part of why β-carotene appears orange.
Indigo in DMF absorbs UV light and red, orange, and yellow light, with a distinct peak at 611 nm. Thus, indigo dye reflects primarily blue and purple light, giving it its characteristic color.