Chem 197 - Spring 1998
Chemiluminescence: Synthesis of Luminol
Chemiluminescence is the process whereby light is produced by a chemical reaction with the evolution of little or no heat. Usually, a chemiluminescent reaction generates a product molecule in a highly energized state. In order to dispose of this excess energy, the excited molecule emits a photon of light. If this light is the product of a reaction that is biochemical in nature, the process is often referred to as bioluminescence. Numerous examples of biolumnescence exist in nature, most notably the flashes of light emitted by the male firefly. Other examples include various marine organisms such as sponges, corals, jellyfishes, clams, and a few types of fish. In these organisms, a compound called luciferin reacts with the enzyme luciferase, which has already formed a complex with ATP. The actual reaction may involve several steps but the end result is the production of an excited molecule that subsequently emits light. It is important to note that the names luciferin and luciferase are generic terms for the active agents in bioluminescent organisms and that the actual molecular structures may differ significantly from organism to organism. Of course, chemiluminescent reactions have been studied extensively and the chemical industry has developed these reactions for commercial purposes. For example, light sticks glow due to a reaction a Cyalume, created by the American Cyanamid Company, hydrogen peroxide.
In this experiment, you will synthesize a noncommercial chemilumnescent agent, luminol (1), and observe the reaction it undergoes to generate light. An alkaline solution of luminol (1) contains the doubly ionized anion (2) which is oxidized to the triplet excited state (two electrons with the same spin) by a combination of hydrogen peroxide and potassium ferricyanide. The triplet state undergoes a slow spin-flip (known as intersystem crossing) to form a singlet excited state (two unpaired electrons with opposite spin), which undergoes subsequent decay to the ground state (5) through the emission of a photon. The basic reactions are given below.
Luminol (1) is prepared by reduction of the nitro derivative (8) formed on thermal dehydration of a mixture of 3-nitrophthalic acid (6) and hydrazine (7). High-boiling triethylene glycol (bp 290 °) is added to an aqueous solution of the hydrazine salt, excess water is distilled, and the temperature is raised to a point where dehydration to (8) is complete within a few minutes. Nitrophthalhydrazide (8) is insoluble in dilute acid but soluble in alkali, by virtue of enolization. It is reduced to luminol (1) by sodium hydrosulfite (sodium dithionite) in alkaline solution. The synthesis scheme is shown below.
In dilute, weakly acidic or neutral solution luminol exists largly as the dipolar ion (9), which exhibits beautiful blue fluorescence.
Procedure:
1.Heat a flask containing 15 mL of water on the steam bath.
2.Then heat a mixture of 1g of 3-nitrophthalic acid and 2 mL of an 8% aqueous solution of hydrazine (caution) in a 20x150-mm test tube over a sand bath until the solid is dissolved.
3.Add 3 mL of triethylene glycol and clamp the tube in a vertical position in a hot sand bath.
4. Insert a thermometer in the sand bath, add a boiling chip, and an aspirator tube connected to an aspirator. Boil the solution vigorously to distill the excess water (110-130 °). Let the temperature rise rapidly until (3-4min) it reaches 215 °.
5. Remove heat, note the time, and by intermittent gentle heating maintain a temperature of 215-220 ° for 2 min.
6. Remove the tube, cool to about 100 °(crystals of the product often appear), add the 15 ml of hot water, coolunder the tap, and collect the light yellow granular nitro compound.
7. Transfer the nitro compound at once to the uncleaned test tube in which it was prepared. Add 5 mL of 3 M NaOH solution, stir with a rod, and to the resulting deep brown-red solution add 3g of fresh sodium hydrosulfite dihydrate. Wash the solid down the walls with a little water.
8. Heat to the boiling point, stir, and keep the mixture hot for 5 min, during which time some of the reduction product may separate.
9. Then add 2 mL of acetic acid, cool under the tap, and stir. Collect the resulting precipitate of light-yellow luminol. On standing, the filtrate usually deposits a further crop of luminol.
The Light Producing Reaction
1. Dissolve the first crop of moist luminol (dry weight about 40-60 mg) in 2mL of 3M NaOH solution and 18 mL of water; this is stock solution A.
2. Prepare a second stock solution, B, by mixing 4 ml of 3% aqueous potassium ferricyanide, 4 mL of 3% hydrogen peroxide, and 32 mL of water.
3. Now dilute 5mL of solution A with 35 mL of water, and in a dark place, pour this solution and solution B simultaneously into an Erlenmeyer flask. Swirl the flask and to increase the brilliance, gradually add further small quantities of alkali and ferricyanide crystals.