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Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA
In this lab, you'll synthesize 3-aminophthalhydrazide, which is also called luminol, in a 2-step process. The first step is a condensation reaction between 3-nitrophthalic acid and hydrazine.
During this step, the carboxylic acid groups are substituted with NH groups to produce 3-nitrophthalhydrazide and two water molecules. You must work in a fume hood throughout this lab because the reactions produce toxic vapors and gases.
In the second step of the luminol synthesis, you'll reduce the nitro group of 3-nitrophthalhydrazide using sodium dithionite in a basic solution of NaOH. This will produce 3-aminophthalhydrazide dianion.
After the reduction, you'll add acetic acid to protonate the dianion, forming luminol. Sodium dithionite is highly reactive and degrades quickly, so you'll use a slight excess of it. After the reduction, you'll protonate the luminol dianion to decrease its solubility in water. Lastly, you'll precipitate and collect luminol as a yellow solid.
For the last part of the lab, you'll mix some of the luminol you made with dimethyl sulfoxide over solid potassium hydroxide. The hydroxyl ions deprotonate the two amine groups. Then, ambient oxygen oxidizes the luminol dianion to 3-aminophthalate, or 3-APA, in an excited state. This unstable complex will quickly relax to the ground state, releasing energy as visible light. Since the excited 3-APA was the product of a chemical reaction, the emitted light is called chemiluminescence.
Once you have seen the chemiluminescence from the oxidation reaction, you'll add fluorescein, a fluorescent molecule, to the mixture. Some excited 3-APA will transfer energy directly to fluorescein rather than emitting light, giving you a solution with two different light-emitting compounds.
Under your oxidation reaction conditions, excited 3-APA emits blue-green light in a broad range around 500 nm when it relaxes, and fluorescein is excited by absorbing light at 480 – 490 nm and 515 – 525 nm.
When you add fluorescein to the luminol oxidation reaction mixture, excited 3-APA can transfer energy that fluorescein would absorb as light directly to a nearby fluorescein molecule in a special interaction called nonradiative energy transfer. The resulting excited fluorescein emits yellow-green light when it relaxes.
Excited 3-APA is highly unstable, so if no fluorescein is close enough for nonradiative energy transfer, it will relax by emitting light as usual. Thus, a spectrum of your final glowing mixture would show contributions from both blue-green and yellow-green light.