science experiments with luminol

How to Make Luminol Glow: Glowing Reaction Activity

Luminol is a chemical that produces a beautiful blue fluorescence when oxidized by hydrogen peroxide. In addition to providing one of the best-known examples of chemiluminescence, it is also a valuable crime scene investigation tool whose blue glow reveals the presence of blood.

For teachers, demonstrating the luminol reaction can add to discussions of oxidation-reduction reactions, conservation of energy, and electron energy levels. The following demonstration is ideal for middle and high school students.

• 1 g Luminol
• 20 mL Sodium Hydroxide Solution (1 M)
• 10 mL Hydrogen Peroxide (3%)
• 0.2 g Potassium Ferricyanide
• 4-ft Piece of Rubber Tubing
• Support Ring

Preparation

You will need a separate beaker for each of the 2 stock solutions you’ll prepare. Prepare the solutions immediately before use. Don lab coat or apron, goggles, and gloves before preparing solutions.  Caution:   Hydrogen peroxide is a strong oxidizer. Avoid skin contact. Sodium hydroxide and its solutions are caustic and can irritate skin. Avoid skin contact.

• To prepare stock solution A, fill a beaker with 100 mL of water. Add 0.18 g of luminol and 3.0 mL of sodium hydroxide solution (1 M).
• To prepare stock solution B, fill another beaker with 100 mL of water. Add 1 mL of hydrogen peroxide (3%) and 0.03 g of potassium ferricyanide.

To set up the apparatus, follow the steps in the figures below.

• Dim the lights.
• Simultaneously pour an equal amount of solution A and solution B into the funnel.
• As the 2 solutions mix, a blue light is emitted that is relatively bright and should last for several minutes.

Reactions that produce light without heat are called  chemiluminescent reactions . Perhaps the most familiar chemiluminescent reactions are those that occur in living organisms and are referred to as  bioluminescence . A classic example of this is the light produced by fireflies.

The reaction in this demonstration is an oxidation-reduction reaction in which a photon of light is released from an excited molecule. In the reaction, luminol is oxidized and its electrons elevated to an excited state. When the electrons return to the ground state, visible light is emitted.

Light’s wavelength determines its color. Light at a wavelength of 680 nm is red; at 500 nm, green; and at 425 nm, blue. The energy of one quantum (one photon, one particle) of light is inversely proportional to its wavelength. Thus: E = hc/l

where  E  is the energy of one quantum of light of wavelength (l),  h  is Planck’s constant and  c  is the speed of light.

In the reaction, hydrogen peroxide oxidizes luminol to produce aminophthalic acid, nitrogen gas, water, and light.

Whether from fireflies or luminol, visible light is produced by the release of light energy from energized atoms. Our chemistry kits below include material along with complete instructions and background information for this interactive activity.

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Hello, I followed the experiment exactly and was not able to get any light. What could be some issues? Is there a difference between regnant grade vs lab grade ?

Thank you for reaching out! After consulting with the product team specific to the Carolina Chemonstrations Luminol Light Up Kit, their suggestions were to check your hydrogen peroxide. The hydrogen peroxide should be 3% and check expiration dates since it can degrade. Additionally the hydroxide solution cannot be old, too weak or too strong—the 1M solution should be made up fresh. Hopefully this helps!

How bright is it suppose to glow? I tried it out in a dark room and it wasn’t really visible.

Hi! Make sure to check your hydrogen peroxide. The hydrogen peroxide should be 3% and check expiration dates since it can degrade. Additionally the hydroxide solution cannot be old, too weak or too strong—the 1M solution should be made up fresh. Hopefully this helps!

Hello, I was wondering on how you are supposed to dispose of the chemicals after use.

Hi! Please follow your state’s guidelines for chemical disposal, or also the instructions listed on the chemical’s mSDS sheet. You can find all of Carolina’s mSDS sheets at the link below: https://www.carolina.com/teacher-resources/msds-material-safety-data-sheets/10857.co?intid=srchredir_msds

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• American Chemical Society
• Students & Educators
• Chemistry Outreach Activities

Chemistry That Glows

Ages 5 - 18 years

Activity Time Prep: 20 mins Activity: 5-10 mins

Group Size 25-250 observers

ACS Student Chapter at the University of Pittsburgh at Johnstown Presents: Chemistry That Glows

Youtube ID: KktL6JFaTFg

• Concepts to Explore
• Chemiluminescence: Reaction of luminol with oxidants emits light
• Forensics: The chemiluminescence of luminol can be used to detect certain biological compounds, even after cleaning
• Other applications: Chemiluminescent reactions are used in glow sticks and certain plants and animals
• Safety & Other Considerations
• Present in a laboratory setting or venue in which presenters and spectators can be separated by >10 ft. 
• Best viewed in a dark setting.
• All cloth used in this activity may become permanently bleached. 
• Potential hazards include:  - Acids and bases - Broken glassware - Inhalation hazards - Oxidizers - Spills and splashes
• Conduct your own RAMP assessment prior to presenting the activity.
• Learn more about Safety in Outreach Settings and review a sample RAMP worksheet.
• Materials Required
• 100 mL of commercial bleach, or 5-6% NaOCl solution
• 4 g NaOH (lye)
• 0.46 g luminol (5-amino-2,3- dihydrophthalazine-1,4-dione)
• 2 L distilled, deionized water, plus extra for rinsing
• One (1 L) storage container, tinted or opaque
• One (1 L) storage container, plastic, preferably HDPE
• Two (100 mL) graduated cylinders
• 500 mL beaker or Erlenmeyer flask
• Two spray bottles
• Cloth towel or shirt
• Additional materials you identified in your RAMP analysis
• Optional: - Iron ring stand - Iron ring - Large funnel - 3 ft of clear, colorless plastic tubing that fits snuggly around the funnel’s stem - Clamps

• Preparation

Prior to Activity

Customize Activity to Venue

• Work in a well-ventilated area.
• Revise procedure to adapt to your specific venue and participants.
• List appropriate procedures for accidents, emergencies.

Identify Safety Practices

• Wear appropriate personal protective equipment (e.g., goggles, gloves, etc.).
• Secure loose hair, clothing.
• Prohibit eating, drinking.
• Clean work area, wash hands after activity.
• Ensure a minimum of 10 ft between presenters and audience

Prepare Materials

• Dilute 100 mL of commercial bleach (usually 5-6% NaOCl) to a 1 L solution with water. Store in a tinted or opaque bottle for up to 1 month.
• Dissolve 4 g NaOH in water and dilute to 1 L. Add 0.46 g luminol and stir until dissolved. Store in plastic bottle.
• Label one graduated cylinder and one spray bottle, “bleach solution.”
• Label one graduated cylinder and one spray bottle, “luminol solution.”
• Prepare a space in which: a. The lights can be dimmed b. The audience is at least 10 ft away from the activity c. Spills can be easily contained 
• Fill graduated cylinders with 100 mL each of indicated solution.
• Set out beaker/flask.
• (Optional) Position the iron ring at the top of the ring stand. Set a funnel in the ring with the stem facing down. Affix one end of tubing to the stem of the funnel. Use the clamps to secure the tubing in a loose spiral around the ring stand. Set the loose end of the tubing in the bottom of the beaker/flask.
• Fill each of two spray bottles about 1/3 full with the solution indicated on its label.
• Position towel or t-shirt so that it can be easily seen by the audience.
• Instructions & Talking Points

Introduce Concept of Chemiluminescence

Instructions

• Explain that some reactions emit light.

Talking Points

• Have you ever seen fireflies?
• Where else do you see examples of glowing in nature, also known as bioluminescence?

Demonstrate Luminol Reaction

• Turn off lights.
• Simultaneously pour 100 mL each of bleach solution and luminol solution into the beaker/flask - Optional: simultaneously pour the bleach and luminol solutions into the funnel so the audience can see the reaction as it moves down the tube.
• Turn lights back on.
• What do the solutions look like before they are mixed? 
• What happens when the solutions are mixed?
• Why is bleach used?

Demonstrate Use of Luminol in Forensics

• Spray towel/shirt with luminol solution.
• Spray towel or shirt with bleach solution.
• How might this reaction be used outside of the lab?
• Why do you think luminol might not be the first analysis used at crime scenes?
• Dispose of all solids from this activity in the trash.
• Dispose of all liquids down the drain with lots of water.
• Clean all work surfaces with water or a damp cloth.
• Wash hands thoroughly.

• Explore the Chemistry

Here are some key themes to explore with the audience once they've completed the activity. Adjust the details to match the level of your audience.

What makes luminol glow?

When the chemical energy of a reaction is converted to visible light energy, the resulting glow is called “chemiluminescence.” Chemiluminescent reactions are what make glow-sticks and some road safety lights glow.

When luminol is dissolved in a base, such as NaOH, the H + on its nitrogens are stripped off, leaving a dianion (i.e., a molecule with two negative charges). The dianion forms resonance structures, which stablizes it just long enough to be oxidized by the bleach, which removes the nitrogens to form a dicarboxylate ion and N 2 .

The oxidized luminol is left with a lot of energy, which it releases as light. Similarly, glow sticks rely on the oxidation of a trichlorosalicylate oxalate ester in a basic solution by hydrogen peroxide.

Luminol and forensics

Hemoglobin, an oxygen-carrying protein in blood, catalyzes the luminol oxidation reaction for a strong glow. Luminol is very sensitive and able to detect trace amounts of blood, even latent blood that has been cleaned or removed.

Luminol also reacts with other oxidizing compounds, such as those found in urine or saliva (even horseradish), so a positive luminol test is usually followed by one that is more specific for blood. Because the application of luminol can dilute any blood that may be present or damage other evidence, other non-destructive techniques are generally used.

Chemiluminescence in nature

When a chemiluminescent reaction occurs in an living organism, the phenomenon is called “bioluminescence.” Bioluminescent organisms include fireflies, some fungi, and certain jellyfish, bacteria, algae, and saltwater fish. These organisms either produce or absorb luciferin, a light-emitting compound.

• American Chemical Society, 2023
• ACS Student Chapter at University of Pittsburgh at Johnstown
• Procedure developed by Dr. Marsha Grimminger, University of Pittsburgh at Johnstown, based on resources from Science in Motion, Juniata College

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