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JoVE Lab Manual
Lab: Chemistry

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Education
Synthesis of Luminol
 

Learning Objectives

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

What is the lowest energy state that an electron can occupy?

The lowest energy state that an electron can occupy is the ground state.

How is an electron elevated to a higher energy level?

An electron is elevated to a higher energy level when it absorbs energy equal to the difference between the higher energy level and the lower energy level. This correlates to specific energy intervals as electrons cannot exist between energy levels.

What happens when an excited electron relaxes back down to the ground state?

When an excited electron relaxes back down to the ground state, it releases energy in the form of a photon of light equal to the energy difference between the higher and lower level.

How does fluorescence differ from chemiluminescence?

In fluorescence, an electron is excited to a higher energy level by absorbing energy in the form of light. However, in chemiluminescence, an electron is excited to a higher energy level by absorbing energy that is released during a chemical reaction.

What is the chemiluminescent chemical luminol used for?

Luminol emits a blue/white glow when oxidized and reacts with the iron in hemoglobin. This enables forensic scientists to identify trace amounts of blood.

List of Materials

  • Stirring hotplate
    5
  • Bunsen burner with tubing and striker
    5
  • Lab Stand
    10
  • 3-prong clamp
    15
  • Insulated gloves or flask tongs
    5
  • 10-mL glass graduated cylinder
    20
  • 50-mL glass graduated cylinder
    5
  • 25-mL Erlenmeyer flask
    15
  • 250-mL glass filter flask
    5
  • 50-mL glass beaker
    5
  • 250-mL glass beaker
    10
  • 400-mL glass beaker
    5
  • 600-mL glass beaker
    5
  • 25-mL glass test tube
    10
  • Büchner funnel
    10
  • Long glass stirring rod
    5
  • Medium stir bar
    5
  • Thermometers
    10
  • Spatulas
    10
  • Boiling chips
    30
  • Medium filter adapter
    5
  • Test tube stopper
    5
  • Stopper for 25-mL Erlenmeyer flask
    5
  • Tweezers
    5
  • Laboratory tape
    5 rolls
  • Labeling pen or marker
    5
  • Pipette bulbs
    10
  • Circular filter paper (9-mm)
    10
  • Hydrazine hydrate (64% hydrazine)
    5 mL
  • Sodium hydroxide
    5 g
  • 3-nitrophthalic acid
    5 g
  • Triethylene glycol
    25 mL
  • Anhydrous sodium dithionite
    20 g
  • Glacial acetic acid
    15 mL
  • Potassium hydroxide
    5 g
  • Dimethyl sulfoxide
    20 mL
  • Fluorescein sodium salt
    2 g
  • 10 wt% sodium hypochlorite solution
    300 mL
  • 1 M sodium carbonate
    100 mL
  • 1-mL graduated pipette and pipette controller
    1
  • 10-mL glass graduated cylinder
    2
  • 50-mL glass graduated cylinder
    1
  • 30-mL glass beaker
    1
  • 50-mL glass beaker
    1
  • 20-mL glass bottle with cap
    2
  • Test tube brush
    2
  • 250-mL wash bottle filled with DI water
    Dependent on lab size
  • Crushed ice
    Dependent on lab size
  • Ice scoop
    Dependent on lab size
  • Insulated ice cooler
    Dependent on lab size
  • Analytical balance (at least 1)
    Dependent on lab size
  • Weigh boats (at least 5 per group)
    Dependent on lab size
  • Spatulas
    Dependent on lab size
  • Lab wipes
    Dependent on lab size
  • Deionized water
    Dependent on lab size
  • Pasteur pipettes
    Dependent on lab size
  • Paraffin film
    Dependent on lab size
  • Scissors
    Dependent on lab size

Lab Prep

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

  1. Preparation of Solutions

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

    • Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. The chemicals that you will use are hazardous, so both solutions must be prepared and handled in a fume hood.
    • Prepare 10 mL of a 10% by volume solution of hydrazine in water. Measure 8.4 mL of deionized water and add it to a 30-mL beaker.
    • Measure 1.6 mL of hydrazine hydrate and pour it into the beaker for a total volume of 10 mL. Stir the hydrazine solution with a glass rod until it is well mixed. Note: Hydrazine is flammable, toxic, and explosive.
    • Pour the solution into a 20-mL glass bottle. Cap it and label it with the solution's name and concentration. Store the hydrazine hydrate and the hydrazine solution in a cabinet for flammables.
    • Prepare 20 mL of 2.8 to 3 M aqueous NaOH. Note: NaOH is highly corrosive. Avoid skin contact.
    • Measure 2.2 to 2.4 g of solid NaOH and add it to a 50-mL beaker. Add 20 mL of deionized water and stir the mixture on a stir plate until the NaOH has completely dissolved.
    • Pour the solution into a 20-mL glass bottle and close it with a lid. Label the bottle with the solution's name and approximate concentration. Store the NaOH pellets and solution in the appropriate cabinets.
    • Lastly, clean your glassware following your usual procedures.
  2. Preparation of the Laboratory
    • Before you set up the main lab, reserve a separate room that you can make as dark as possible.
    • Prepare a container for fluorescein waste and ensure that a glass waste container is available.
    • Set out a 10% sodium hypochlorite (bleach) solution and a 1 M sodium carbonate solution in the waste hood.
    • Fill a wash bottle with deionized water and place it in the same hood. Confirm that there are paper towels and a test tube brush by each sink in the main lab.
    • Bring containers of 3-nitrophthalic acid, anhydrous sodium dithionite, solid potassium hydroxide, and fluorescein to the balance area.
    • Confirm that there are enough laboratory wipes, spatulas, and weighing boats for the class.
    • Set triethylene glycol, the hydrazine solution, the NaOH solution, glacial acetic acid, and dimethyl sulfoxide in a central hood. Place a 1-mL graduated pipette and a pipette controller designated for hydrazine in the same hood.
    • Put Pasteur pipettes, filter papers, scissors, and plastic paraffin film in a central area.
    • Set out the following glassware and equipment at each student lab station (we suggest that students work in pairs):
       1    Stirring hotplate
       1    Bunsen burner with tubing
       2    Lab stands
       3    Clamps
       1    Pair of flask tongs
       4    10-mL graduated cylinders
       1    50-mL graduated cylinder
       3    25-mL Erlenmeyer flasks
       1    250-mL filter flask
       1    50-mL beaker
       2    250-mL beakers
       1    400-mL beaker
       1    600-mL beaker
       2    25-mL test tubes
       2    Büchner funnels
       1    Long glass stirring rod
       1    Medium stir bar
       2    Thermometers
       2    Spatulas
       6    Boiling chips
       1    Medium filter adapter
       1    Test tube stopper
       1    Small flask stopper
       1    Pair of tweezers
       1    Fire striker or lighter
       1    Roll of laboratory tape or labels
       2    Pipette bulbs
       1    Pen

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