Synthesis of Luminol

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

1. Synthesis of 3-NitrophthalhydrazideExpand

   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.

   • Before you get started, put on a lab coat, splash-proof safety glasses, and nitrile gloves. Note: Hydrazine is highly toxic and flammable, so avoid touching it and keep anything containing hydrazine closed or covered when you transport it between fume hoods.
   • Now measure 10 mL of deionized water and pour it into a 25-mL Erlenmeyer flask. Clamp the flask on a hot plate and place a thermometer in it.
   • Then, start heating the water. You'll want the temperature of the water to be 80 °C, so set the hot plate setting to slightly higher.
   • While it heats, set up a Bunsen burner under a clamp fixed on a lab stand.
   • Obtain a 25-mL test tube and adjust the clamp so that it will hold the test tube about 5 – 7 cm above the flame. Then, remove the test tube and place it in a 400-mL beaker.
   • Once the water reaches 80 °C, adjust the heat setting to keep the temperature stable.
   • Then, weigh 0.6 g of 3-nitrophthalic acid in a tared weighing boat. Pour the 3-nitrophthalic acid into your test tube. Then, bring the test tube and a test tube stopper to the solvent hood.
   • Use the provided volumetric pipette to carefully measure 0.9 mL of the 10% v/v hydrazine solution and transfer it to the test tube. Remember to cap the bottle of hydrazine solution and stopper your test tube before you return to your hood.
   • Clamp the test tube over the Bunsen burner and remove the stopper. Then, pour ~2 mL of triethylene glycol into a graduated cylinder and set it out of the way in your hood.
   • Now, add 2 or 3 boiling chips to the test tube. Place a high-temperature thermometer in the test tube and ignite the Bunsen burner. Heat the mixture with a medium flame until the 3-nitrophthalic acid dissolves.
   • Then, add the triethylene glycol and increase the heat of the flame. Note: Once the water from the hydrazine solution has boiled off, the temperature will continue increasing.
   • When the reaction mixture reaches 210 °C, turn down the heat and keep the mixture between 210 °C and 220 °C for 3 – 4 min.
   • Then, extinguish the flame and wait for the mixture to cool to 100 °C.
   • Turn off the hotplate, remove the thermometer, and unclamp the flask. Use tongs to hold the flask and measure 8 mL of hot water with the graduated cylinder. Pour this hot water into the test tube and remove the thermometer.
   • Obtain a piece of plastic paraffin film and cover the mouth of the test tube. Then, use tongs to transfer the test tube to the holding beaker.
   • At a lab sink, hold the test tube under running tap water to cool it, being careful not to let water into it.
   • Then, dry the outside of the tube and bring it back to your hood. Clamp the tube upright and remove the paraffin film. Let the yellow 3-nitrophthalhydrazide precipitate in the tube for 15 min.
   • While you wait, add ~10 mL of deionized water to a 25-mL Erlenmeyer flask. Prepare an ice bath in a 250-mL beaker and start chilling the water in it.
   • Then, set up for vacuum filtration using a 250-mL filter flask, a Büchner funnel, and a piece of circular filter paper.
   • Once the product has precipitated for at least 15 min, obtain a Pasteur pipette and wet the filter paper with a few drops of cold water.
   • Then, turn on the vacuum and pour the contents of the test tube into the Büchner funnel. Rinse the remaining solid into the funnel with 1 – 2 mL of cold water.
   • Remove the boiling chips with tweezers and rinse them with a few drops of cold water to finish collecting the product.
   • Turn off the vacuum pump once no more liquid is dripping into the flask from the funnel.
2. Synthesis of 3-AminopthalhydrazideExpand

   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.

   • Place a clean 25-mL test tube in a beaker and use a clean spatula to transfer your 3-nitrophthalhydrazide to the test tube.
   • Set the Büchner funnel aside and clamp the test tube over the Bunsen burner. Leave the filter flask in place for later.
   • Now, use a 10-mL graduated cylinder to obtain 3 mL of approximately 3 M NaOH. Pour the sodium hydroxide solution into the test tube and stir the mixture with a glass rod until the 3-nitrophthalhydrazide dissolves.
   • Now, weigh 2 g of sodium dithionite in a tared weighing boat and add it to the test tube. Obtain 3 mL of deionized water and pour it into the tube.
   • Stir the mixture well to dissolve as much of the solid as you can. Then, add a few clean boiling chips to the test tube and ignite the Bunsen burner.
   • Heat the mixture to boiling over a medium flame and then let it boil for 5 min.
   • While you wait, measure 1.5 mL of glacial acetic acid and bring it to your hood.
   • Once the reaction mixture has boiled for 5 min, shut off the Bunsen burner and add the acetic acid to the tube. Let the mixture cool to room temperature, which usually takes 10 –15 minutes. Meanwhile, prepare an ice bath in a 600-mL beaker.
   • Once the mixture has cooled, place it in the ice bath and wait 15 min for the luminol to precipitate.
   • Reassemble the vacuum filtration setup using a clean Büchner funnel and a new piece of filter paper, and start cooling another 10 mL of deionized water in an ice bath.
   • Once the luminol has been in the ice bath for at least 15 min, wet the filter paper with cold water. Then, collect the solid luminol by vacuum filtration.
   • Retrieve the boiling chips and rinse the luminol with more cold water. Once liquid stops dripping from the funnel, turn off the vacuum and transfer the luminol to a 50-mL beaker.
   • Now, prepare the combined filtrate for disposal. First, label a 250-mL beaker ‘hydrazine and dithionite waste’. Transfer the filtrate to the beaker and rinse the flask with deionized water to remove residual hydrazine and dithionite.
   • Next, dilute the filtrate with 10 mL of deionized water. Then, measure 10 mL of 1 M sodium carbonate and add it to the combined filtrates.
   • Bring a 50-mL graduated cylinder to the waste hood. Measure 40 mL of 10% sodium hypochlorite and pour it into the filtrate mixture. Place the beaker on the hot plate, add a stir bar, and secure a thermometer in the beaker.
   • Heat the mixture to 50 °C while stirring. The mixture must stay at 50 °C for 1 h to oxidize leftover hydrazine and dithionite, so make sure that the temperature is stable and set a timer for 1 h before starting the last part of the lab.
3. Chemiluminescence and FluorescenceExpand

   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.

   • Now, measure about 0.2 g of the luminol you made and place it in a 15-mL Erlenmeyer flask. It's OK if the solid is still damp.
   • Obtain 5 – 7 pellets of potassium hydroxide and pour them into the flask. Then, bring the flask, a stopper, and a 10-mL graduated cylinder to the solvent hood.
   • Measure 2 mL of DMSO, add it to the flask, and stopper it. Put the graduated cylinder in your fume hood and bring the flask to a dark room.
   • Shake the stoppered flask vigorously until you see chemiluminescence and then put the flask down. The light fades quickly because both the oxidation and relaxation are fast processes.
   • Shake the flask and let the light fade 6 – 10 more times without opening the flask. The light will be progressively dimmer as the oxygen in the flask is consumed. Note: To make it brighter, leave the flask open in your fume hood for a minute to replenish the oxygen.
   • Close the flask, return to the dark room, and shake the flask again to see the effect.
   • Now, measure 0.2 g of fluorescein dye and add it to your flask. Stopper the flask and return to the dark room. Shake the flask vigorously until the mixture is glowing brightly and then set it down to observe the difference in color from luminol alone.
   • When you're finished, check on the filtrate. Once it has been at 50 °C for at least 1 h, turn off the hotplate and wait for the beaker to cool to room temperature.
   • Then, add about 10 mL of tap water to the filtrate and flush it down the drain.
   • Dispose of the remaining chemical and glass waste in the appropriate containers, clean your glassware and tools, and put away your lab equipment.
4. 結果Expand

   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.