Assignments - Chemiluminescence Lab Report

Luminol Synthesis and Chemiluminescence Abstract In this experiment, luminol was synthesized and used to investigate its characteristic chemiluminescence. We synthesized luminol by reducing 5-nitro-2,3-dihydrophthalazine-1,4dione with sodium hydrosulfite and sodium hydroxide, and then refluxing and vacuum filtering the resulting solution. We then oxidized the retrieved luminol by mixing stock solutions containing sodium hydroxide, potassium ferricyanide, and hydrogen peroxide in a dark fume hood. The overall reaction was successful because we were able to see the luminol’s characteristic blue light emission when the lights were turned off in the fume hood. Introduction Chemiluminescence reactions are well-known because they are the reactions that cause glow sticks to glow in the dark and blood to glow in crime scene investigation. Chemiluminescence is a specific type of luminescence in which the release of light is secondary to chemical bonds breaking, forming, and being restructured as part of a chemical reaction. No heat is released as part of this reaction. During a chemiluminescence reaction, an atom gains energy from chemical reactions, causing excitation of an electron from its ground state to a higher energy level. When the electron falls back to its ground state, a photon of light is released at a wavelength that is within the range of visible light. This wavelength is detected by the human eye as a glow of a particular color that corresponds to this wavelength (Welsh 2011). In this particular procedure, students used sodium hydrosulfite in sodium hydroxide to reduce 5-nitro-2,3-dihydrophthalazine-1,4-dione. This reaction reduces the nitro group down to an amino group, thus synthesizing 5-amino-2,3-dihydrophthalazine-1,4-dione, or luminol. The resulting luminol is then oxidized by a solution of sodium hydroxide, hydrogen peroxide, and potassium ferricyanide. This solution cleaves the ring and converts the amine groups into carbonyl groups, thus forming 5-aminophthalate salt. The hydrogen peroxide and potassium ferricyanide are used in this reaction to add a triplet oxygen to the luminol. This triplet oxygen serves to excite the 5-aminophthalate salt to the S1 excited state. The blue light is consequently released when the molecule converts back down to the S0 ground state. This is chemiluminescence. Experimental Procedure Scheme 1: Reduction of 5-nitro-2,3-dihydrophthalazine-1,4-dione Scheme 2: Oxidation of 5-amino-2,3-dihydrophthalazine-1,4-dione to the corresponding sodium phthalate salt with the release of light 5-nitro-2,3-dihydrophthalazine-1,4-dione (0.153 g, 0.739 mmol) and NaOH solution (2 mL, 3 M) were added to a 5 mL conical vial. The solution was then stirred using the magnetic spin vane on the magnetic stirrer until the color changed to a dark red. Sodium hydrosulfite (0.253g, 1.43 mmol) was then added to the dark red solution. All solid was washed from the sides of the vial using a maximum of 1 mL of water. The reflux apparatus was assembled by attaching the jacketed condenser to the 5 mL conical vial containing the dark red solution. The water in line was placed on the bottom inlet and the water out hose was attached to the top outlet, with the end placed into the drain at the back of the fume hood. The water was turned on and allowed to run through the condenser. The system was placed on the aluminum block and hot plate until it began to condense inside of the condenser. At this point, reflux had begun and was timed for 5 minutes. At the end of 5 minutes, the system was allowed to cool until the student could no longer feel heat coming off of the vial. The reflux apparatus was then disassembled. At this point, acetic acid (1.00 mL) was added to the conical vial and the solution was allowed to stir on the magnetic hot plate for 5 minutes. No heat was applied to the system. The conical vial was then placed in a large beaker containing an ice bath for the next 10 minutes. The vacuum filtration apparatus was then assembled by placing a neoprene adapter on a Hirsch funnel and placing this system into a 25 mL filter flask and attaching the vacuum hose from the fume hood. Filter paper was placed over the perforations in the Hirsh funnel and was wet using ice cold water. The conical vial was rinsed using ice cold water and the aspirator was run for 10 minutes to allow the solid to sufficiently dry. The resulting solid luminol (0.187g, 1.38 mmol) was then weighed. We then began the chemiluminescence part of the experiment by creating four different solutions. The first, located in a 25 mL flask labeled Stock Solution A, consisted of the crude luminol (0.187g, 1.38 mmol) synthesized in the previous portion of the experiment dissolved into and NaOH solution (2.00 mL, 3 M). The second solution, located in a 50 mL beaker labeled Solution A, consisted of Stock Solution A (1.00 mL) diluted in water (9.00 mL). The third solution, located in a 25 mL flask labeled Stock Solution B consisted of potassium ferricyanide solution (4.00 mL, 3% by weight in water) added to a hydrogen peroxide solution (4.00 mL, 3% by weight in water). The fourth solution, located in a 50 mL beaker labeled Solution B, consisted of Stock Solution B (4.00 mL) diluted with water (16.0 mL). After all solutions were mixed and covered to prevent evaporation or interaction with air, we diluted Solution A (3.00 mL) with water (16.0 mL) in a 150 mL beaker. We then turned the lights off in our fume hood and poured Solution B into the 150 mL beaker that held the diluted Solution A. Chemiluminescence was observed. All observations were noted in the lab manual at each step of this experiment. Results/Discussion The following paragraph include observations and results for the luminol synthesis part of the experiment. When I added NaOH (2 mL, 3 M) and sodium hydrosulfite (0.253g, 1.43 mmol) to the 5-nitro-2,3-dihydrophthalazine-1,4-dione (0.153 g, 0.739 mmol), the solution changed color from clear to dark red/brown. This color change corresponds to reduction of 5nitro-2,3-dihydrophthalazine-1,4-dione to 5-amino-2,3-dihydrophthalazine-1,4-dione, or luminol. When I added acetic acid to this solution, the solution blood orange solid began to form in the solution. This color change coordinates to the sodium hydrosulfite being neutralized in the reaction and the luminol precipitating out of solution. When I cooled the solution on ice and ran the solution through vacuum filtration, a blood orange precipitate resulted (luminol). We recovered 0.187g (1.38 mmol) of luminol. This solid was still pasty and wet after 10 minutes of drying. This gave us a percent yield of 143%. A yield greater than 100% may be due to the filtered solid still having water in it. The solid would have needed to be dried overnight or placed in an oven in order to extract all of the water, but that was not an option for this lab. The following paragraph includes the observations and results from the chemiluminescence part of the experiment. When I dissolved luminol in the NaOH solution to make Stock Solution A, the solution change from clear to a dark orange. When yellow potassium ferricyanide was mixed with clear hydrogen peroxide to make Stock Solution B, the color changed to bright yellow. When Solution B was added to this diluted Solution A, I observed the expected emission of blue light, which lasted about 3 seconds. We also mixed Stock Solution A with Stock Solution B and observed a more intense emission of blue light, which lasted about 5 seconds. Conclusion Overall, in this lab we were successful in demonstrating why glow sticks and blood glow. This glowing is not the result of the entire solution itself glowing. Instead, this chemiluminescence is caused by an electron being excited from its ground state to a higher energy level, and consequently returning to its ground state. From the oxidation of luminol with sodium hydroxide, hydrogen peroxide, and potassium ferricyanide, we can conclude that these compounds are the source of the triplet oxygen that excites the electrons. This is because the blue light was only emitted after the luminol and sodium hydroxide in Solution A came into contact with Solution B containing the hydrogen peroxide and potassium ferricyanide. Further evidence that these two compounds are the sources of the triplet oxygen come from the fact that when we combined the two stock solutions the blue emission was more intense and longer lasting than when we combined the diluted Solution A and diluted Solution B. From this reaction, we concluded that chemiluminescence will occur whenever a solution of luminol comes into contact with a solution of iron, base, and an oxidizing agent. In this reaction, the source of iron was potassium ferricyanide, the base was sodium hydroxide, and the oxidizing agent was hydrogen peroxide. A similar reaction will occur when crime scene investigators combine luminol, a base, and an oxidizing reagent and spray the resulting solution onto an area that may contain blood. If the area contains blood, it will glow blue secondary to reaction between the solution and the iron contained in the hemoglobin of the blood. The iron in both reactions is a catalyst and only needs to be present in very small amount in order for the reaction to result in chemiluminescence (Welsh 2011). Discussion Questions 1. What is the compound being oxidized in the luminol synthesis reaction? The compound being oxidized is sodium hydrosulfite. 2. What is the purpose of acetic acid in the luminol synthesis reaction? How would the amount of product obtained be affected if the number of equivalents for the two reagents were reversed? The acetic acid was used to cause the luminol to precipitate out of the solution. More specifically, it is used to neutralize the remaining sodium hydrosulfite in the solution. If the equivalents of the two reagents were reversed, it would have altered the ratio of acetic acid to NaOH, and would have caused there to be more NaOH in solution. This would have resulted in a more basic solution and a smaller yield of luminol. Bases tend to deprotonate solutions, whereas we wanted the 5-nitro-2,3-dihydrophthalazine-1,4-dione to be reduced. These two processes would have acted in opposition to one another and we would not have had favorable yields of luminol (Feld 2014). 3. In the luminol synthesis reaction, why was COLD water used for rinsing the filtration step? How would the efficiency of the filtration be affected if HOT water were used instead? We use cold water for this step to prevent any of the solid from re-dissolving in the solution. If the solid was rinsed with hot water, it might cause the recently re-crystallized luminol to dissolve into solution again, which would be counter-intuitive for filtering out the solid. (Columbia University 2014). 4. When isolating the solid through crystallization and filtration, why was the solid not rinsed with a substance like ethyl acetate or dichloromethane? According to Sigma Aldrich, luminol is not soluble in ice cold water (Sigma 1996). Therefore, we rinsed the solid luminol with ice cold water to prevent any of the solid from redissolving and being vacuumed filtered into the filter flask. On the other hand, luminol is soluble in substances such as ethyl acetate or dichloromethane, which would have caused the luminol to be dissolved into the solution we rinsed it with. This would have resulted in smaller yields of solid luminol after vacuum filtration. 5. What is the difference between chemiluminescence and fluorescence? Where does the ENERGY for light emission come from in each case? Fluorescence is the release of light secondary to excitation of electrons by light, whereas chemiluminescence is the release of light secondary to the excitation of electrons by a chemical reaction (Welsh 2011). The energy for light emission for fluorescence comes from a substance absorbing light at a high frequency, then subsequently releasing the light at a frequency that is within the range of visible light (Welsh 2011). The energy for light emission during chemiluminescence comes from bonds being broken, formed, and restructured during chemical reactions. This causes electrons to go from their ground states to an excited state, and then subsequently fall back to the ground state and release light. 6. In the chemiluminescence reaction, a diene is generated on the luminol molecule. Why does the reaction not progress from that point forward as a Diels-Alder reaction with molecular oxygen (O2) acting as a dienophile? The reaction does not progress as a Diels-Alder reactions because the oxygen (O2) does not contain a double bond, and therefore does not act as a dienophile. Instead, the oxygen (O2) is added to the solution in the triplet form (see Lewis Structure below). The two oxygen atoms are bonded together with a single bond, and each oxygen atom a total of 5 lone electrons.