Luminol Synthesis and Chemiluminescence

Written by Lena


In this experiment, we synthesized luminol and used the product to observe how chemiluminescence works. Our starting material was 5-nitro-2,3-dihydrophthalazine-1,4-dione, which was, after addition of reaction agents, refluxed and vacuum filtered to retrieve luminol. Using two stock solutions, we missed our precipitated luminol with sodium hydroxide, potassium ferricyanide, and hydrogen peroxide, in their respective solutions, in a dark room, to observe the blue light emission.


Anyone who has watched a CSI show on the television has probably seen the wonders of chemiluminescence. There is hardly an episode where we do not see one member of the CSI team spraying an unknown substance onto a surface, and using a black-light to show that all too familiar blue glow that indicates the presence of blood or body fluids. The unknown substance in the spray bottle is, in fact, luminol; and although its immediate effect is exaggerated on the television screen, it is effective and chemiluminescence does occur. Iron in hemoglobin serves as the ‘active ingredient’ in blood that causes the familiar glow.

In chemiluminescence, light is released without the heat from a chemical reaction; light is produced in this reaction through the energy released by the breaking, formation, or restructuring of chemical bonds. In a fluorescence reaction, the absorbance of light at a higher frequency, and consequent release at a lower frequency visible to the human eye, is the cause for the release of light.

The process of refluxing, which we use in this experiment, involves boiling a solution while continually condensing its vapor by cooling and returning the liquid to the reaction flask. Due to the fact that most organic reactions do not occur too quickly, chemists use this method to heat a reaction mixture for a long time without losing reagents. The reflux apparatus includes a jacketed condenser, where water flows into the bottom outlet and out of the top outlet. The apparatus is clamped to a stand, and a round-bottomed flask, or conical vial, containing a solution is attached before refluxing begins.

Also utilized in this experiment is the process of vacuum filtration, which is used for quick and complete separation of a solid from a liquid in a mixture. Filtration can be done using either a water aspirator line or a compressor-driven vacuum system. In this lab, we use a water aspirator line. In vacuum filtration, a Hirsch funnel, fitted with filter paper, is inserted into a filter flask which is attached to the vacuum trap. As mixture is poured into the funnel, the vacuum draws out liquid; and, leaving the aspirator running, the solid is allowed to dry.


Mohrig, J.R.; Hammond, C.N.; Schatz, P.F. Techniques in Organic Chemistry, 2010, 59-60, 109.



To begin our experiment, we weighed out 5-nitro-2,3-dihydrophthalazine-1,4-dione (0.15g, 0.72 mmol), and added it to a 5mL conical vial with a spin vane. Into this same vial, we added sodium hydroxide (2mL, 3M), sodium hydrosulfite (0.25g, 1.4 mmol), and stirred. We washed solid residue from the sides of the conical vial using water (1mL). We then assembled the reflux apparatus using the jacketed condenser and water lines and attached the conical vial. This was followed by 5 minutes of reflux and stirring simultaneously, after which the solution was cooled to room temperature.

When the solution was sufficiently cooled, we added acetic acid (1mL, 17mmol, 1 equiv.) to the conical vial and stirred it for 5 minutes. The solution was then cooled on ice for 10 minutes. Using the vacuum filtration system, we filtered the precipitate and left it to dry with the aspirator running for 10 minutes. The precipitate recovered was luminol (0.24g, 1.4 mmol).

Moving on to the chemiluminescence experiment, we made four solutions: stock solution A, solution A, stock solution B, and solution B. Stock solution A was prepared using luminol (0.24g, 1.4 mmol) dissolved in sodium hydroxide solution (2mL, 3M) in a 25mL Erlenmeyer flask. Taking stock A (1mL) diluted in water (9mL) in a 50mL beaker, we made solution A. Stock solution B was prepared using potassium ferricyanide (4mL) and hydrogen peroxide solution (4mL) in a 25mL Erlenmeyer flask. Taking stock B (4mL), and diluting it with water (16mL), in a 50mL beaker, we got solution B. Finally, diluting solution A (3mL) with water (16mL) in a 150mL beaker, and pouring solution B (20mL) into this beaker, in a dark room, we were able to see the light emission as our solution turned blue.


The initial stirring of 5-nitro-2,3-dihydrophthalazine-1,4-dione (0.15g, 0.73 mmol), sodium hydrosulfite (0.25g, 1.4 mmol), and sodium hydroxide (2mL, 3M)  made a deep red/brown solution. After reflux and continuous stirring, a yellow coagulation/precipitate appeared on top of the solution. After the addition of acetic acid, yellow lumps of precipitate formed within the solution. Upon reflux and stirring of the solution, it turned orange and opaque, with visible floating flakes of precipitate. After cooling on ice and running through vacuum filtration, a mustard-colored, pasty luminol precipitate was recovered. For our experiment, we were able to recover 0.2436g (1.375 moles) of luminol. At first we thought the luminol would dry completely, but soon realized that it maintained a pasty consistency throughout the drying process. Stock solution A was a translucent red color, and stock solution B was a clear yellow, with frothy consistency on top. Our initial attempts at mixing the solutions say not emission light, for reasons we were unable to determine; but we mixed the solutions from stock again, and, fortunately, were able to see the blue luminescence in the beaker which lasted for about one minute, before fading away.


The experiments in today’s lab allowed us to see how luminol is instrumental in chemiluminescence. We see the outcome of chemiluminescence in contemporary media, but, in a laboratory setting, we are better able to be involved in the process. We can now understand that it is not blood itself, but the iron in its hemoglobin that causes this chemiluminescence. With this knowledge, we see the relevance of using potassium ferricyanide ( as a reactive agent. By investigating this multistep process, we have the opportunity to see the chemical roots of well-known phenomena.