UV-Vis Spectroscopy of Dyes

Source: Lara Al Hariri at the University of Massachusetts Amherst, MA, USA

1. Preparation of Dye SolutionsExpand

   Here, we show the laboratory preparation for 10 students working in pairs, with some excess. Please adjust quantities as needed.

   • First, put on a lab coat, safety glasses, and nitrile gloves. Some solutions use hexane or dimethylformamide, so prepare those solutions in a fume hood.
   • Prepare a 1 mM stock solution of fluorescein in water. Measure 0.0166 g of fluorescein and place it in a 50-mL volumetric flask.
   • Add about 30 mL of deionized water to the flask, stopper it, and swirl it until as much solid has dissolved as possible. Then, fill the flask to the line with deionized water.
   • Stopper the flask, wrap the top in plastic paraffin film, and invert it several times while firmly holding the stopper in place to thoroughly mix the solution. Label the flask with the solution's name and concentration and wrap it in aluminum foil to protect the dye from light. Label the outside of the foil as well.
   • Prepare a 3 µM fluorescein test solution from the stock solution. Use a micropipette to transfer 150 µL of the stock solution to a clean 50-mL volumetric flask. Add about 30 mL of deionized water and swirl the flask to mix them.
   • Fill the flask to the line with deionized water, stopper and seal it, and invert the flask several times until the solution is well mixed. Label the flask with the solution's name and concentration, wrap it in foil, and label the outside of the foil as well.
   • Prepare 1 mM stock solutions and 3 µM test solutions of indigo in dimethylformamide and β-carotene in hexane in the same way. The masses of solid β-carotene and indigo that you will need to make the solutions are listed in the tables.

     All stock solutions in 50 mL solvent
     Dye	Stock concentration	Mass	Solvent
     β-carotene	1 x 10-3 (1 mM)	0.027 g	Hexane
     Indigo	1 x 10-3 (1 mM)	0.013 g	DMF

     All test solutions diluted to 50 mL
     Dye	Test concentration	Stock volume	Solvent
     β-carotene	3 x 10-6 (3 µM)	150 µL	Hexane
     Indigo	3 x 10-6 (3 µM)	150 µL	DMF

   • Use the stock solution of β-carotene to make five more solutions of the concentrations listed in this table. Wrap the flasks in foil afterward.

     All solutions diluted to 50 mL with hexane
     Dye	Concentration	Stock volume
     β-carotene	1.9 x 10-6 (1.9 µM)	95 µL
     β-carotene	3.7 x 10-6 (3.7 µM)	185 µL
     β-carotene	7.5 x 10-6 (7.5 µM)	375 µL
     β-carotene	11 x 10-6 (11 µM)	550 µL
     β-carotene	15 x 10-6 (15 µM)	750 µL

   • When you're finished, you'll have three micromolar solutions of fluorescein, β-carotene, and indigo dye for the first part of the lab and five β-carotene solutions with different concentrations for the second part of the lab. Store the solutions in a refrigerator for now if you have one. If not, store them at room temperature for up to 24 hours.
2. Preparation of the LaboratoryExpand
   • Put organic and aqueous waste containers in a fume hood with a glass waste container nearby.
   • Fill a 250-mL wash bottle with acetone and place it with the waste containers. Next, place the dye solutions and containers of hexane and DMF in the hood.
   • Fill another wash bottle with deionized water and place it with the solvents.
   • Set Pasteur pipettes, several pipette bulbs, and lab wipes nearby. Then, place quartz cuvettes in a central area for the students. Place another box of lab wipes by the spectrometer.
   • About 10 to 20 min before the lab, turn on the UV-Vis spectrometer and the instrument computer.
   • Once the spectrometer is ready, configure it so the default scan is absorbance with a range of 200 – 800 nm.
   • Create a folder where students can save their spectra and set the default file path to that folder. When the students arrive, explain how to use the software and save their data.