Glow in the Flask
| Polish version is here |
The following article was originally published in the journal for educators Chemia w Szkole (eng. Chemistry in School) (3/2023):
I believe I do not need to convince the reader that chemistry is an incredibly fascinating scientific discipline that explores the composition, properties, and transformations of the matter around us. It encompasses a broad range of disciplines, from organic and inorganic chemistry to physical and analytical chemistry, offering numerous opportunities for knowledge acquisition. With its fundamental role in our daily lives, chemistry provides a unique perspective on the world around us.
One of the most intriguing aspects of chemistry is chemiluminescence, where light is emitted as a result of a chemical reaction. The emitted light can vary in color, intensity, and duration, creating visually striking displays that captivate students and can also be valuable for scientific purposes.
The use of chemiluminescent reactions has significant potential in education. By incorporating these reactions into educational activities, teachers can engage students in an exciting and interactive way. Spectacular light emissions can spark curiosity and thus promote a deeper understanding of chemical processes. It is worth noting that, due to their visual appeal, such reactions make even relatively complex and abstract concepts—such as energy transformations—more accessible and memorable. Furthermore, from an educational perspective, chemiluminescent reactions offer great versatility, from demonstrating reaction kinetics and energy transfer to studying the influence of various reagents and conditions on the reaction course.
Many substances exhibit chemiluminescent properties—one of the most well-known is luminol C8H7N3O2 and lophine C21H16N2 [1] [2]. However, today I would like to describe my work on the synthesis and observation of the chemiluminescence of lucigenin, a much less well-known compound with similar properties. I encourage the reader to conduct their own experiments in this area and attempt to reproduce the reactions described here.
The synthesis method outlined here is based on a protocol published in 1982, with some modifications [3].
Without further ado, let's move on to the first stage of synthesis.
Stage I – N-Phenylanthranilic Acid
To begin our work, we need to gather the following substances:
- 2-Chlorobenzoic acid (o-chlorobenzoic acid) ClC6H4CO2H,
- Aniline C6H7N,
- Anhydrous potassium carbonate K2CO3,
- Copper(I) oxide Cu2O,
- Activated carbon C,
- Hydrochloric acid HCl(aq).
2-Chlorobenzoic acid is one of the three isomers of chlorobenzoic acid and exhibits the strongest acidic properties among them. Under standard conditions, it is a white crystalline solid and serves as a precursor in the synthesis of various pharmaceuticals, food additives, and dyes [4]. This compound is irritating to the skin, eyes, and respiratory tract.
Aniline, on the other hand, is the simplest aromatic amine. It appears as a colorless liquid that gradually turns brown when exposed to air, with a characteristic odor reminiscent of spoiled fish. Its density is significantly greater than that of water, in which it is only slightly soluble. Aniline has widespread applications in the chemical, pharmaceutical, dye, and rubber industries, as well as in the production of explosives and as a component of certain rocket fuels.
I must emphasize that aniline is a toxic substance. It poses health risks through all routes of exposure, including inhalation, ingestion, and skin contact. Prolonged exposure, even to small amounts, can result in serious health hazards. Aniline particularly affects the blood and hematopoietic system, causing, among other effects, the destruction of red blood cells. It is also suspected of having mutagenic properties.
Copper(I) oxide naturally occurs as the red mineral cuprite and was one of the primary sources of copper for humans for centuries.
Under standard conditions, potassium carbonate is a white crystalline solid that is highly soluble in water. It has a relatively high melting point (reported as 891–899°C (1636–1650°F) in various sources). It has been known since antiquity as potash, traditionally obtained from wood ash through leaching.
To initiate the synthesis, I placed 20g (0.7 oz) of o-chlorobenzoic acid, 80g (2.8 oz) of freshly distilled aniline, 20g (0.7 oz) of anhydrous potassium carbonate, and 0.5g (0.02 oz) of copper(I) oxide in a round-bottom flask. The flask, fitted with a reflux condenser, was placed on a heating mantle, and the brown mixture inside was heated to boiling for 2.5 hours (Photo 1). After this time, heating was discontinued, and the reaction mixture was allowed to cool to room temperature before being diluted with 300 cm3 (10 fl oz) of water.
At this stage, any unreacted aniline must be removed from the reaction mixture. This can be done in several ways, including steam distillation. However, this process may be challenging for many experimenters, so an alternative approach is to perform multiple extractions using small portions of diethyl ether C4H10O (warning: highly flammable!) in a separatory funnel. Unfortunately, this procedure is quite tedious, as both the lower aqueous phase (containing the product) and the upper ether phase appear dark brown (almost black). As a result, distinguishing the boundary between them can be difficult. Strong side lighting can help in identifying this boundary more clearly (Photo 2).
Once most traces of aniline have been removed, the aqueous solution is heated to boiling with the addition of 10g (0.35 oz) of activated carbon C for several minutes, followed by hot filtration. After cooling, the filtrate is combined with 60 cm3 (2 fl oz) of hydrochloric acid at a concentration of approximately 14%, leading to the precipitation of a substantial amount of solid (Photo 3). This is N-phenylanthranilic acid, formed via a nucleophilic substitution reaction between o-chlorobenzoic acid and aniline (Figure 1).
The product should be filtered and dried in a desiccator. In its pure form, N-phenylanthranilic acid appears as white crystals, but when synthesized using this method, it often exhibits a slightly grayish tint (Photo 4). Its purity is sufficient for subsequent synthesis steps.
Approximately 23g (0.8 oz) of the acid was obtained using the given substrate proportions, corresponding to ~84% of the theoretical yield based on the o-chlorobenzoic acid used. The entire amount can be utilized for further synthesis, or a portion may be set aside for other applications.
Stage II – From N-Phenylanthranilic Acid to Acridone
This stage of the synthesis requires the following substances:
- N-phenylanthranilic acid C13H11NO2,
- Concentrated sulfuric acid (VI) H2SO4,
- Sodium carbonate Na2CO3.
N-phenylanthranilic acid is widely used as an intermediate in pharmaceutical synthesis and peptide chemistry. It exhibits irritating properties. Sulfuric acid (VI) is a strong corrosive agent capable of rapidly degrading organic tissues upon direct exposure.
To convert N-phenylanthranilic acid into the next intermediate, I dissolved 20g (0.7 oz) of the substance in 44 cm3 (1.5 fl oz) of concentrated sulfuric acid (VI) and heated it in a steam bath for 1.5 hours, obtaining a dark green solution. The next step is highly hazardous and requires maximum caution. The still-hot green solution must be carefully poured, in portions, into approximately 150 cm3 (5 fl oz) of near-boiling water, which can cause significant splashing of the highly corrosive mixture. Therefore, this reaction is best performed in a deep beaker of at least 1 liter (34 fl oz). Proper eye and face protection, such as a plastic shield, is essential. After adding the entire reaction mixture to the water, a dirty-yellow suspension forms, which should be heated to boiling for 5 minutes and then filtered. The precipitate should be transferred directly into 200 cm3 (6.8 fl oz) of an 8% sodium carbonate solution and heated again to boiling for several minutes.
Excess sodium carbonate may dissolve the precipitate. To prevent this, it is best to add the base solution in portions while monitoring the pH using universal indicator paper (author’s note).
After filtration, crude acridone is obtained as an amorphous solid (Photo 5), formed via an intramolecular electrophilic substitution reaction catalyzed by concentrated sulfuric acid (VI).
Unfortunately, practical experience shows that acridone obtained this way cannot be used in subsequent synthesis steps without a significant reduction in yield. To avoid this, the substance must be purified via recrystallization. However, acridone exhibits very low solubility in common solvents, making traditional recrystallization inefficient. A solution to this problem is the Soxhlet extractor, a laboratory apparatus designed for extracting poorly soluble compounds, invented in 1879 by Franz von Soxhlet [5].
The Soxhlet extractor consists of a system of glass tubes and a chamber positioned between the boiling flask and the reflux condenser (Figure 3). Boiling solvent vapors a travel from the lower flask through tube b to the condenser c (which has continuous cooling water circulation) above the extractor. The sample to be extracted is placed in the extraction chamber d in a special cellulose thimble e. At the bottom of the chamber, beneath its base where the thimble rests, there is an outlet siphon f. When the liquid level in the extraction chamber reaches a predetermined height set by the siphon’s design, the accumulated liquid automatically drains through the siphon tube back into the flask.
This system operates cyclically. The extraction chamber gradually fills with freshly distilled solvent, reaching the siphon level, at which point it empties itself and refills with fresh solvent.
Even if only a minimal amount of the substance in the thimble dissolves in a single cycle, the cyclic operation ensures that it is gradually transferred to the lower flask, leaving insoluble impurities behind. This allows for the repeated use of the same solvent volume, significantly reducing solvent consumption.
For purification, I placed crude acridone in a properly sized cellulose thimble (Photo 6).
Next, I assembled the Soxhlet extractor and inserted the thimble, positioning it on a small amount of cotton wool. A crucial detail is ensuring that the top edge of the thimble remains above the bend of the siphon (Photo 7).
In this case, ethanol with a concentration of at least 95% is the preferred solvent. The required volume should be determined experimentally; in my case, approximately 400 cm3 (13.5 fl oz) was used.
The Soxhlet extractor significantly accelerates and simplifies acridone purification, but it remains a relatively slow process. With the given quantities, the procedure took several hours, during which the mixture in the flask continuously boiled.
The solubility of acridone in ethanol is so low that crystallization from the boiling solvent became noticeable in the lower flask after only a few cycles.
The process was stopped once only insoluble residues remained in the thimble. After cooling, the mixture from the flask was filtered, and the collected precipitate was dried, yielding beautiful yellow crystals of purified acridone (Photo 8).
From the given amount of N-phenylanthranilic acid, approximately 16.3g (0.57 oz) of crude acridone was obtained, of which 14.8g (0.52 oz) remained after purification, corresponding to yields of 89% and 81%, respectively, relative to the starting material.
The purified acridone is now ready for the next stage.
Stage III – From Acridone to N-Methylacridone
For this stage, several additional reagents are required:
- Acridone C13H9NO,
- Potassium hydroxide KOH,
- Ethanol C2H5OH,
- Dimethylformamide (DMF) C3H7NO,
- Methyl iodide (iodomethane) CH3I.
Acridone is an organic compound with a structure based on the acridine skeleton (Figure 2). As previously observed, under normal conditions, it appears as a yellow crystalline solid. Some acridone derivatives are used as fluorescent markers in molecular biology. Additionally, acridone is an important precursor for the synthesis of various pharmaceutical compounds. It exhibits irritating properties.
Dimethylformamide (DMF) is an organic compound belonging to the amide group. Under normal conditions, it is a liquid that is miscible in any proportion with water and many organic solvents. Pure DMF is odorless, but the presence of dimethylamine C2H7N in technical-grade DMF imparts an unpleasant, fishy odor. DMF is widely used as a solvent due to its advantageous properties; its polar and hydrophilic nature makes it an effective medium for facilitating nucleophilic substitution reactions [6]. Special precautions must be taken when handling this substance, as it is suspected to be carcinogenic and teratogenic.
Methyl iodide (iodomethane) is an organic compound belonging to the alkyl halide group, specifically a monoiodinated derivative of methane. It is a colorless liquid that gradually turns brown upon exposure to light due to decomposition, with iodine as one of the byproducts responsible for the characteristic coloration. It has a melting point of −66°C (−87°F) and a boiling point of 42.4°C (108.3°F). The compound is only slightly soluble in water but readily dissolves in ethanol and diethyl ether. Methyl iodide is primarily used in organic synthesis and the pharmaceutical industry for methylation reactions, a process that will be employed in our synthesis. This compound is highly toxic and volatile. All manipulations involving it must be conducted under a well-functioning fume hood.
To initiate the synthesis, I dissolved 10g (0.35 oz) of acridone in 122 cm3 (4.1 fl oz) of ethanol with the addition of 3.15g (0.11 oz) of potassium hydroxide under heating. The ethanol was then evaporated, which is best done using a rotary evaporator under vacuum. However, I tested an alternative method that does not require this equipment. I found that simply evaporating the ethanol in an evaporating dish or crystallizer on a steam bath was sufficient, provided that care was taken to prevent the hygroscopic potassium hydroxide from absorbing moisture. This process yielded a yellow residue (Photo 9), which I subsequently dissolved in 122 cm3 (4.1 fl oz) of dimethylformamide, obtaining a dark green solution (Photo 10).
Next, I added 8.52g (0.3 oz) of methyl iodide dropwise to the solution, then heated it on a steam bath for 15 minutes, resulting in a slight change—the liquid no longer stained the walls of the vessel (Photo 11).
During this process, nucleophilic substitution of the methyl iodide occurs with the acridone anion. Upon adding the reaction mixture to water, a light yellow precipitate of crude N-methylacridone forms (Photo 12).
After filtration and drying, the product appears as a dull, cream-yellow amorphous solid (Photo 13).
The substance was then purified by classical recrystallization from hot ethanol. As the concentrated solution cooled, needle-like crystals of the derivative formed (Photo 14).
After filtration and drying, the derivative was ready for further processing (Photo 15).
During the synthesis, I obtained 9.32g (0.33 oz) of crude N-methylacridone and 8.46g (0.3 oz) after recrystallization, corresponding to yields of 87% and 79%, respectively, relative to the acridone used.
With this, the product is now ready for the next stage.
Stage IV – From N-Methylacridone to Lucigenin
For this step, the following reagents are required:
- N-Methylacridone C14H11NO,
- Ethanol C2H5OH (~95%),
- Concentrated hydrochloric acid HCl(aq),
- Finely powdered zinc Zn,
- Nitric acid (V) HNO3 (~6%).
N-Methylacridone is a derivative of acridone obtained through its methylation under the previously described conditions. It serves as an intermediate in organic synthesis.
The zinc used must be in the form of the finest powder available. It is important to note that highly powdered zinc has a strong staining effect and may even be pyrophoric, a property that must not be overlooked.
When handling acids, extra precautions must always be taken due to their corrosive properties.
In a round-bottom flask, I combined 9g (0.32 oz) of N-methylacridone, 450 cm3 (15 fl oz) of ethanol, and 90 cm3 (3 fl oz) of concentrated hydrochloric acid. Complete dissolution of the solids required heating under reflux (caution: irritating vapors). Then, I slowly added 28.8g (1 oz) of zinc powder over 40 minutes. The mixture was subsequently heated to boiling under reflux for another hour, producing a dark solution (Photo 16).
After cooling, the reaction mixture was slowly poured, with continuous stirring, into 1L (34 fl oz) of cold water, resulting in the precipitation of a green solid— the bis-acridine derivative—which was then filtered and dried (Photo 17, Figure 5).
The green precipitate was then dissolved in 540 cm3 (18.2 fl oz) of ~6% nitric acid (V) and heated in a steam bath for 30 minutes. The dark solution was filtered while hot and left to crystallize overnight at room temperature. The next day, well-formed orange-red crystals of lucigenin C28H22N4O6 were observed (Photo 18).
The lucigenin crystals were filtered and dried at a moderate temperature. When dry, their color appeared more orange than red (Photo 19).
Thus, N-methylacridone underwent reductive dimerization to form the bis-acridine derivative under the influence of zinc in an acidic medium. This compound was then oxidized with nitric acid, yielding the corresponding diazotate. From a chemical standpoint, lucigenin exists as a soluble diazotate of bis-N-methylacridine.
A total of 7.7g (0.27 oz) of lucigenin was obtained, corresponding to approximately 70% of the maximum theoretical yield based on the N-methylacridone used. The final lucigenin should be stored in a dark glass container.
Fluorescence of the Obtained Substances
Some of the synthesized compounds exhibit an interesting phenomenon—fluorescence, i.e., the emission of visible light when excited by ultraviolet radiation. This effect can serve as an additional verification that the expected compounds were obtained at each stage. To conduct this test, a small amount of each substance was dissolved in distilled water in separate test tubes. Since some compounds have low solubility, each test tube was shaken for one minute before exposure to UV light (Photo 20). The results showed that N-phenylanthranilic acid does not exhibit fluorescence (or the emission is too weak to observe), acridone and N-methylacridone fluoresce blue (the latter slightly weaker, but the exact cause of this effect is difficult to determine under such simple conditions), while the intermediate bis-acridine derivative and lucigenin fluoresce very strongly, emitting an intense green light.
Chemiluminescence of Lucigenin
After overcoming the challenges of synthesis and successfully completing each step, we finally arrive at the moment where we can test lucigenin’s ability to generate visible light during oxidation. To do this, two solutions must be prepared:
| A: | Dissolve a small pinch (0.05g / 0.002 oz) of lucigenin in 50 cm3 (1.7 fl oz) of water. |
| B: | Dissolve 15 cm3 (0.5 fl oz) of ethanol, 4g (0.14 oz) of sodium hydroxide, and 2.5 cm3 (0.085 fl oz) of 3% hydrogen peroxide (pharmaceutical-grade hydrogen peroxide) in 35 cm3 (1.2 fl oz) of water. |
Solution A appears orange-yellow, while solution B is completely colorless (Photo 21). Both solutions should be freshly prepared, although they can be stored briefly in a refrigerator.
To observe chemiluminescence, it is best to darken the room as much as possible before rapidly pouring the entire volume of solution B into the vessel containing solution A. Almost immediately, a green light emission begins, lasting for several minutes (Photo 22).
Interestingly, upon prolonged observation, the emission color gradually shifts from green to a more distinct blue hue as the reaction progresses (Photo 23).
Explanation
Lucigenin exhibits strong chemiluminescent properties when oxidized by hydrogen peroxide in an aqueous alkaline medium. The most probable reaction mechanism involves the oxidation of lucigenin to an unstable cyclic peroxide, which subsequently decomposes into N-methylacridone. Initially, N-methylacridone exists in a metastable excited state, spontaneously returning to the ground state while releasing excess energy. During the early stages of the reaction, when unreacted lucigenin is still present in large quantities, excitation energy is transferred to lucigenin molecules, causing them to emit green light. As the reaction progresses and lucigenin is depleted, the emission spectrum shifts toward blue, characteristic of excited acridone and N-methylacridone.
Beyond its educational applications, lucigenin and its derivatives are also utilized as molecular markers in biological research.
Summary
Lucigenin is a fascinating compound that exhibits both fluorescence and chemiluminescence. The synthesis described in this article enables its laboratory preparation using relatively accessible reagents. This process also provides an opportunity to explore a wide range of chemical reactions, including nucleophilic substitutions, oxidation-reduction transformations, and the fluorescent properties of organic compounds.
Through this work, we have gained insight into the fundamental principles of photochemistry and energy transfer at the molecular level. The ability to observe visible light emission from a chemical reaction not only enhances our understanding of these processes but also offers a visually striking demonstration of complex chemical phenomena.
In addition to its educational value, lucigenin and its derivatives play a significant role in scientific research, particularly as molecular probes and markers in biological studies. Their chemiluminescent properties are also valuable in analytical chemistry, where they are employed in highly sensitive detection techniques.
For those interested in further exploring chemiluminescence, I encourage replicating the synthesis and experimenting with various reaction conditions to observe their impact on emission intensity and color. I hope this article has inspired you to delve deeper into the world of photochemistry and its applications in both science and education.
References
- [1] Ples M., Widmowy blask - chemiluminescencja katalizowana związkiem miedzi (eng. Ghostly glow: copper-catalyzed chemiluminescence), Chemia w Szkole (eng. Chemistry in School), 2 (2016), Agencja AS Józef Szewczyk, pp. 13-17 back
- [2] Ples M., Synteza i chemiluminescencja lofiny - zimne światło, muzyka i migdały (eng. Synthesis and chemiluminescence of lophine - cold light, music, and almonds), Chemia w Szkole (eng. Chemistry in School), 5 (2020), Agencja AS Józef Szewczyk, pp. 44-47 back
- [3] Amiet R.G., The preparation of lucigenin: An experiment with charm, Journal of Chemical Education, 59(2), 1982, pp. 163-164 back
- [4] Maki T., Takeda K., Benzoic Acid and Derivatives, in: Ullmann's Encyclopedia of Industrial Chemistry, 2002, Wiley, Weinheim back
- [5] Harwood L. M., Moody Ch.J., Experimental organic chemistry: Principles and Practice, Wiley-Blackwell, 1989, pp. 122-125 back
- [6] Mohammadkhani L., Beyond a solvent: triple roles of dimethylformamide in organic chemistry, RSC Advances, 8 (49), 2018, pp. 27832-27862 back
All photographs and illustrations were created by the author.
Marek Ples