Glow Up

1. Make solutions using the following recipes:
   • Solution A: Mix 0.1 g luminol and 20 mL 2.5 M NaOH (to help the luminol dissolve) in 1 L H₂O
   • Solution B: Mix 0.1 g fluorescein in 1 L H₂O
   • 20% bleach: Mix 200 mL bleach with 800 mL H₂O
2. Then prepare three sample cups to be tested:
   • Sample cup 1: Pour 30 mL solution A
   • Sample cup 2: Pour 30 mL solution B
   • Sample cup 3: Pour 30 mL solution A and 30 mL solution B

With the room lights on, observe the appearance of each sample.

Turn off the lights and use the pipette to add 10 drops of the 20% bleach solution to each cup. Notice what happens in each case.

Turn on the black light and take a look at all of the solutions again. If you have tonic water or liquid laundry detergent, look at those under the UV light as well.

You may have noticed that solution A had a blue glow with the addition of bleach and solution B did not glow at all. The mixture of solutions A and B should have exhibited a greenish glow when bleach was added. The types of light you see in this activity are examples of chemiluminescence and fluorescence. Detection of these two light sources serve as the basis for the majority of studies in molecular and cell biology.

Chemiluminescence is non-thermal, visible light produced as the result of a chemical reaction. A molecule in the reaction is excited to a higher energy state and then releases a photon as it returns to its ground state. This is distinct from thermal light generated by excess heat energy in a combustion reaction.

When bleach is added to a solution containing luminol, an oxidation reaction occurs, and electrons in the luminol are excited to a higher energy state. As they return to their ground state, they release the energy in the form of a photon. The wavelength of the photon corresponds to the blue light that you see. When all the electrons have returned to their ground state, the mixture should stop glowing. When chemiluminescence occurs in a living organism, it is called bioluminescence. A classic example of this is the light produced by fireflies when a luciferase enzyme catalyzes the oxidation of luciferin, another molecule produced by fireflies. The gene for luciferase can be engineered into other organisms so that they too can produce light in the presence of luciferin. Since the production of light is readily quantified by modern cameras, the luciferase system is commonly used by biologists as an indicator of gene activity.

Fluorescence is luminescence that occurs when a molecule is excited to a higher energy state through the absorption of a photon and then emits a different photon as it returns to its ground energy state. During this process, some energy is lost to heat or molecular motion, so the wavelength of the emission photon is longer than the wavelength of the excitation photon. Since this happens almost instantaneously, fluorescence usually is seen only in the presence of the excitation light. Fluorescein is a fluorescent molecule that absorbs UV light and emits green light. It is not chemiluminescent, so the addition of bleach to solution B does not result in any light generation. However, when solution A is present, the blue light emitted by the chemiluminescence of luminol is close enough to the excitation wavelength of fluorescein that the entire solution glows green, since the fluorescein absorbs the light from the luminol reaction and re-emits it in the green range. Other examples of fluorescent molecules are quinine in tonic water and the optical whiteners present in many laundry detergents. Both of these absorb UV light and emit in the blue range.

Shine a green laser through a bottle of olive oil. What color is the beam?

Fluorescent molecules can be excited by wavelengths other than UV light. Organic molecules present in olive oil fluoresce red when excited by green light. Green fluorescent protein (GFP) is a naturally occurring molecule in jellyfish that fluoresces green under UV light. Fluorescent molecules such as GFP and fluorescein are routinely attached (genetically or chemically) to biological molecules to assist in detection and monitoring in vitro and in vivo. Different lasers can supply light at specific wavelengths, so a large number of molecules can be simultaneously detected.

Instruments called fluorometers are used to measure water quality by detecting optical brighteners in water. Optical brighteners are frequently added to laundry detergents, and their presence can be an indication of a leak or spill from a waste water treatment facility, since household waste water typically contains laundry detergent.