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JoVE Lab Manual
Lab: Chemistry

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Education
UV-Vis Spectroscopy of Dyes
 

Learning Objectives

At the end of this lab, students should know...

What happens to light when it interacts with a substance?

Light is either absorbed, reflected, or transmitted. The color of a substance depends on the wavelengths that it reflects. A substance that appears blue absorbs orange and reflects blue light.

What energy must light have in order to excite an electron from the HOMO to LUMO?

The energy of light must be equal to the energy gap between the HOMO and LUMO.

What property do dyes typically have that make them able to absorb and reflect visible light?

Dyes typically contain conjugated double and single bonds. This results in a lower energy gap between the HOMO and LUMO, and therefore, a lower required energy of light is needed to excite an electron. Lower energy light has a longer wavelength, making most dyes able to absorb light in the visible range.

What does a spectrophotometer measure, and how does it report a result?

A spectrophotometer measures transmittance, or the amount of light that passes through a substance. This is related to the absorbance, or the amount of light absorbed by the substance. The spectrophotometer produces a plot of absorbance versus wavelength called an absorbance spectrum.

How is absorbance related to the concentration of a sample?

The concentration of a sample affects the absorbance through the Beer-Lambert law. Absorbance is dependent on the concentration times the molar attenuation coefficient and the path length. Therefore, as concentration increases, so does absorbance. A standard curve of a sample’s absorbance versus known concentrations can derive a linear equation that can be used to find the concentration of an unknown sample based on the absorbance.

List of Materials

  • β-Carotene
    40 mg
  • Fluorescein
    20 mg
  • Indigo dye
    20 mg
  • Hexane
    1000 mL
  • Dimethylformamide
    500 mL
  • Acetone
    250 mL
  • Pasteur pipette bulb
    15
  • Quartz cuvette with cap (1 cm x 1 cm)
    10
  • UV-Vis single-beam spectrophotometer OR Handheld Data Acquistion Device with UV spectrophotometer attachment
    Dependent on lab size
  • 50-mL glass volumetric flask with stopper
    11
  • 50-mL glass graduated cylinder
    3
  • Small glass funnel
    3
  • 200-µL micropipette with tips
    1
  • 1000-µL micropipette with tips
    1
  • Pasteur pipettes
    Dependent on lab size
  • 250-mL plastic wash bottle
    Dependent on lab size
  • Analytical balance
    1
  • Weighing boats
    3
  • Spatulas
    3
  • Lab wipes
    Dependent on lab size
  • Labeling tape
    1 roll
  • Labeling pen
    1
  • Paraffin film
    1 box
  • Deionized water
    Dependent on lab size
  • Aluminum foil
    1 roll

Lab Prep

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

  1. Preparation of Dye Solutions

    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 Laboratory
    • 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.

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