Lophine – The Great Synthesis
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The following article was originally published in the journal for educators Chemia w Szkole (eng. Chemistry in School) (6/2022):
Although chemistry, and science in general, is a fascinating part of our lives, certain families of chemical reactions consistently capture the imagination of both students and teachers. This is not only because of their clear educational and scientific value but also because of their striking visual effects. One such family comprises chemiluminescent reactions, processes that emit visible electromagnetic radiation through a nonthermal pathway. While these reactions may seem rare or limited to relatively expensive, hard-to-obtain, or synthetically challenging substances such as firefly luciferin Lampyris noctiluca (and other beetles, including the glowworm Phausis splendidula, author’s note) C11H8N2O3S2, lucigenin C28H22N4O6, and tetrakis(dimethylamino)ethylene C10H24N4, they can also be demonstrated with far more accessible chemicals. Cold light can be induced in the white allotrope of phosphorus P, metallic sodium Na and potassium K, the singlet form of oxygen 1O2, polyphenols found in green tea, certain Grignard compounds and organosilicon species (for example, Wöhler’s siloxene Si6O3H6, produced from ordinary sand here), and even readily available pharmacy-grade potassium permanganate KMnO4 [1][2][3][4][5]. Another chemiluminophore, and the focus of this article, is lophine C21H16N2.
The first synthesis of lophine is tied to fascinating episodes and remarkable historical figures. It was achieved in the second half of the nineteenth century by our compatriot, the distinguished organic chemist Bronisław Radziszewski. His initial method relied on tribenzylidenediimine, also known as hydrobenzamide, a compound first described by Alexander Borodin. This may come as a surprise, since Borodin is far better known for his music than for his chemistry. He was a composer who, together with Balakirev, Cui, Mussorgsky, and Rimsky-Korsakov, formed “The Mighty Handful,” a circle of renowned musicians drawing on Russian folk traditions. Borodin wrote symphonies, symphonic poems, fantasies, and string quartets. Strikingly, he considered music a secondary pursuit, though it is what he is most famous for today. His professional career and greatest passion, however, were always rooted in chemistry.
In an earlier issue of Chemia w Szkole, I described my own successful attempts to reproduce Radziszewski’s original lophine synthesis [6]. I chose this route because its main starting material, benzaldehyde, is inexpensive and readily available, whereas the newer and more efficient large-scale method requires benzil, which is harder to obtain. The drawback of the historical approach is its very low yield. After considering how to improve the efficiency of a process that begins with benzaldehyde and other easily obtained reagents, and after some experimentation, I became convinced that the outcome could indeed be improved. This involves a multistep sequence (benzaldehyde → benzoin → benzil → lophine) that also provides a straightforward and relatively safe introduction to hands-on laboratory work of this type. Let us proceed to the first stage, keeping in mind that every step must be performed using proper personal protective equipment.
Step I: from benzaldehyde to benzoin
To begin, gather the following substances:
- benzaldehyde C6H5CHO,
- ethanol C2H5OH 95% (rectified spirit),
- thiamine C12H17N4OS·HCl (vitamin B1),
- sodium hydroxide NaOH, 10% aqueous solution (based on [7], modified).
Benzaldehyde is a clear liquid with a strong almond-like aroma. In fact, the relationship is the reverse: almonds smell of benzaldehyde because they naturally contain small amounts of it. In principle, the experiment could be carried out using benzaldehyde extracted from the seeds of the almond tree Prunus dulcis, but such material is very costly. It is therefore far more practical to use synthetically produced benzaldehyde, which is what I employed.
Benzaldehyde oxidizes easily, especially when exposed to light, so it must be protected from both air and illumination. As oxidation proceeds, the liquid first turns yellowish and later brown. For our experiment, we need fresh benzaldehyde that is colorless or only slightly yellow. Strongly oxidized samples are unsuitable and should be redistilled to obtain material free of oxidation products. This compound may be harmful to health.
Thiamine C12H17N4OS (later named vitamin B1) is a heterocycle composed of a thiazole ring and a pyrimidine ring linked by a methylene bridge. It was first isolated in 1911 from rice bran by Kazimierz Funk, who introduced the term “vitamin” for this and other life-essential substances. The molecular structure is shown in Fig.1.
Under normal conditions thiamine is a fine white crystalline solid (Photo.1). It is water-soluble and plays a key role in cellular respiration, particularly in carbohydrate metabolism, as part of a carboxylase coenzyme. It enhances the activity of acetylcholine C7H16NO2, inhibits cholinesterase, acts synergistically with thyroxine C15H11I4NO4 and insulin, and stimulates the secretion of gonadotropic hormones. Thiamine promotes wound healing and has analgesic properties. Studies indicate that the daily requirement of vitamin B1 is 1.1 mg (0.000039 oz) for women and 1.2 mg (0.000042 oz) for men [8].
A deficiency of this compound can lead to central nervous system disorders such as weakness, fatigue, nystagmus, memory and concentration problems, and depression. It can also cause circulatory failure, including tachycardia, cardiomegaly, and peripheral edema. Digestive problems may also occur, including loss of appetite, nausea, vomiting, diarrhea, abdominal pain, and weight loss. In severe vitamin B1 deficiency, beriberi may develop, characterized by impaired function of neurons and muscle fibers, resulting in limb pain, muscle weakness, tremors, and ultimately circulatory failure and even death. Excess thiamine, however, is not harmful. Thiamine also exhibits catalytic properties, which we will take advantage of. Ethanol is toxic to humans, something to remember despite its long history as one of humanity’s favorite poisons. Sodium hydroxide is highly caustic and, like all hazardous laboratory substances, must be handled with great care.
To begin the synthesis, place 3.3 g (0.12 oz) of thiamine in a flask and dissolve it in 9.5 cm3 (0.32 fl oz) of distilled water. The vitamin dissolves easily, producing a solution with a distinctive odor that many find unpleasant. Once fully dissolved, add 25 cm3 (0.85 fl oz) of ethanol to obtain a clear, colorless solution (Photo.2).
Cool the solution in an ice-water bath, then add 9.5 cm3 (0.32 fl oz) of 10% aqueous NaOH dropwise with stirring. This liberates thiamine in its free-base form and the solution turns yellow (Photo.3). The bath prevents overheating that could degrade this delicate vitamin.
Allow the yellow solution to warm back to room temperature, then add 22 cm3 (0.74 fl oz) of benzaldehyde dropwise with continuous stirring. Because benzaldehyde is only sparingly soluble in water and ethanol does not ensure complete dissolution, the mixture remains turbid after the addition, as expected (Photo.4).
From this point the reaction may be run in one of two ways: for about two hours at 60°C (140°F), taking care not to exceed this temperature, or for several days at room temperature. I tested both approaches with comparable results. In the experiment described here, the reaction was carried out at room temperature for four days without continuous stirring; the flask was protected from light and kept in a fume hood. It is advisable to cap the vessel or wrap the opening with aluminum foil, as the combined odor of the vitamin and benzaldehyde can be overwhelming.
After the indicated time, abundant crystals were observed. This material is crude benzoin C14H12O2 (Photo.5).
Chill the mixture to about 0°C (32°F), filter off the benzoin, and wash the solid several times with distilled water. This removes residual thiamine, which is water-soluble, unlike benzoin. Perform a final rinse with 40–50% ethanol to remove any remaining unreacted benzaldehyde. The product on the filter is obtained as a white solid (Photo.6).
For subsequent steps it is worth purifying the product by recrystallization from ethanol. Benzoin is much more soluble in hot ethanol than at low temperature. In this case, several tens of cubic centimeters of 95% ethanol were sufficient to dissolve the crude benzoin at reflux, and upon cooling, much purer benzoin crystallized (Photo.7).
After filtration and rinsing with cold 50% ethanol, the benzoin crystals were dried and weighed (Photo.8).
The sequence above is an example of the benzoin condensation. Such reactions are commonly catalyzed by highly toxic cyanides CN-, but as early as the 1950s thiamine was proposed as a safer alternative, as in the present case (Fig.2).
The isolated mass of purified benzoin was 12.3 g (0.43 oz), which corresponds to about 50% of the theoretical yield based on the benzaldehyde used. Most of the benzoin obtained was carried forward to the next stage.
As a small reward for the effort, a portion of the benzoin can serve for a simple demonstration. Prepare a solution of 0.2 g (0.0071 oz) of benzoin in 25 cm3 (0.85 fl oz) of methanol CH3OH, then add 0.75 g (0.026 oz) of KOH dissolved in 1 cm3 (0.034 fl oz) of distilled water. The initially colorless solution (Photo.9A) soon turns pale violet (Photo.9B). Shaking the solution causes the color to disappear; it returns after a short rest. The cycle can be repeated many times [9].
B – return of color when left undisturbed
The color changes result from oxidation of benzoin by atmospheric oxygen in alkaline medium.
Step II: from benzoin to benzil
For this stage we need the following:
- benzoin C14H12O2,
- ammonium nitrate NH4NO3,
- copper(II) acetate (CH3COO)2Cu,
- glacial acetic acid CH3COOH.
Benzoin is an aromatic ketoalcohol used, among other applications, in fragrance compositions.
Ammonium nitrate, also called ammonium saltpeter, is a colorless crystalline solid. It is hygroscopic and highly water-soluble. It is used in explosives and in the production of mineral fertilizers.
When working, remember that concentrated acetic acid is corrosive to tissues and has a pungent, suffocating odor. Copper(II) acetate deserves a closer look. It can be purchased from a chemical supplier, but it is also easy to prepare in the laboratory. To do this, gather some metallic copper, such as clippings of uninsulated electrical wire or pieces of sheet metal (Photo.10).
Cover the copper fillings with about 100 cm3 (3.38 fl oz) of household vinegar (a 6–10% solution of acetic acid), then add 50 cm3 (1.69 fl oz) of 3% aqueous hydrogen peroxide H2O2 (drugstore grade). The proportions are not critical; even wide deviations will not prevent product formation. The solution is initially colorless (Photo.11). Protect the setup from evaporation and leave it at room temperature for several days, or warm gently to accelerate the reaction.
After a while the solution changes color visibly. The reaction can be considered complete once the liquid becomes distinctly blue (Photo.12). Metallic copper is typically used in large excess and will not dissolve completely, so it can be reused.
Decant the liquid and evaporate part of the water and residual acetic acid. Beautiful crystals of copper(II) acetate then begin to form according to the reaction:
After optional recrystallization and drying, the copper(II) acetate can be used in the benzil synthesis and appears as in Photo.13.
Returning to the benzil synthesis, charge a round-bottom flask with 11 g (0.39 oz) of the previously prepared benzoin, 31.4 cm3 (1.06 fl oz) of glacial acetic acid, 5 g (0.18 oz) of ammonium nitrate, and 6.3 cm3 (0.21 fl oz) of a copper(II) acetate solution prepared by dissolving 1.1 g (0.039 oz) of the salt in 50 cm3 (1.69 fl oz) of distilled water. The mixture is initially cloudy at room temperature because not all reagents dissolve. Place the flask in a heating mantle and attach a reflux condenser as shown in Photo.14.
Adjust heating and stirring so that the mixture boils gently for about 1.5 hours. During this time the reagents gradually dissolve and the reaction mixture turns green (Photo.15).
After the planned time, allow the mixture to cool, preferably overnight. It partially solidifies (Photo.16).
Filter the benzil C14H10O2, then wash the solid on the filter several times with distilled water and finish with a small portion of cold ethanol.
This transformation proceeds according to the scheme in Fig.3 [10].
After drying and verifying the melting point against literature values, 94–95°C (201–203°F), further purification by recrystallization was deemed unnecessary to avoid additional losses [11]. Under ambient conditions, benzil is a yellow solid (Photo.17).
The isolated mass of benzil suitable for further work was 10.45 g (0.37 oz), which corresponds to 96% of the theoretical yield based on the benzoin used.
Step III: from benzil to lophine
We now reach the final step toward the target compound. Prepare the following:
- benzil C14H10O2,
- benzaldehyde C6H5CHO,
- ammonium acetate CH3COONH4,
- glacial acetic acid CH3COOH 99%.
Benzil, also called dibenzoyl, is an aromatic diketone that can be viewed as a dimer of the benzoyl group. It is used as an intermediate in organic synthesis and as a photoinitiator for free-radical polymer curing. It may exert adverse effects on living organisms. Ammonium acetate is the ammonium salt of acetic acid and is used in the food industry as additive E264, one of the preservatives.
Technically this stage resembles the previous one [12] [13]. Charge a round-bottom flask with 10.4 g (0.37 oz) of benzil, 5.2 cm3 (0.18 fl oz) of benzaldehyde, 46.2 g (1.63 oz) of ammonium acetate, and 144 cm3 (4.87 fl oz) of glacial acetic acid. Place the setup in a heating mantle and heat with stirring at a gentle boil for three hours. The solution is initially distinctly yellow (Photo.18).
As the reaction proceeds, the color gradually fades (Photo.19).
Switch off the heat and allow the mixture to cool, then pour it in a thin stream into at least one liter (33.8 fl oz) of vigorously stirred cold distilled water. A large amount of white solid precipitates immediately. This is crude lophine C21H16N2 (Photo.20).
Filter the crude lophine, wash the solid several times with water, and dry it. The isolated mass of crude lophine was 13.6 g (0.48 oz) as a fluffy white powder (Photo.21).
For purification, recrystallize lophine from hot methanol. Ethanol or isopropanol can also be used, though larger volumes are required. Needle-like crystals often form on the vessel walls as the alcoholic solution cools slowly (Photo.22).
The purified lophine was filtered, dried, and stored for further experiments (Photo.23). The collected mother liquor still contains some lophine, so it is worth using it for initial chemiluminescence tests as described below.
The lophine synthesis follows the scheme shown in Fig.4.
The mass of recrystallized lophine was 10.6 g (0.37 oz), which corresponds to 72.3% of the theoretical yield based on the benzil used.
Chemiluminescence
To observe the chemiluminescence of lophine, we need:
- sodium hydroxide NaOH or potassium hydroxide KOH,
- hydrogen peroxide H2O2 3%,
- sodium hypochlorite NaClO.
Fortunately for the experimenter, sodium hypochlorite is easy to obtain and need not be purchased from a specialty supplier. It is the active ingredient in many household chlorine-based bleaches, especially the least expensive ones.
Here is a simplified procedure for preparing the lophine chemiluminescence reaction. Dissolve 0.1 g (0.0035 oz) of lophine in 50 cm3 (1.69 fl oz) of methanol or ethanol, then add a very small amount of sodium or potassium hydroxide, just a few pinhead-sized granules. The base will not dissolve completely but will raise the alkalinity sufficiently. Next, add 10 cm3 (0.34 fl oz) of 3% hydrogen peroxide. This solution is unstable and should be prepared immediately before use. Separately, prepare 40 cm3 (1.35 fl oz) of a bleach solution containing sodium hypochlorite by diluting it with an equal volume of distilled water.
For the demonstration, darken the room and pour the hypochlorite solution in a thin stream into the alcoholic lophine solution containing hydroxide and hydrogen peroxide (Photo.24A). A magnetic stirrer is helpful.
During the reaction, bright yellow-green light is emitted (Photo.24B). The mother liquor collected after lophine recrystallization can also be used to demonstrate chemiluminescence in the same way (Photo.25). At the end of the page, in the author’s supplements section, there is a video showing the chemiluminescence of lophine residues in the filtrate.
Explanation
Lophine is among the earliest artificially prepared substances known to exhibit chemiluminescence. As 2,4,5-triphenylimidazole, it belongs to the imidazole family. In alkaline solution and in the presence of hypochlorite ions ClO-, lophine is oxidized by hydrogen peroxide. One nitrogen atom bears a relatively acidic hydrogen, while the other is basic, so the molecule is amphoteric. During oxidation a peroxide bridge forms, giving a highly unstable cyclic peroxide that rapidly fragments. By energy conservation, the excess energy is released as light of wavelength λ = 525 nm, perceived as yellowish green.
The overall yield of a multistep sequence is the product of the individual step yields. In our case, the yield of the entire synthesis starting from benzaldehyde was about 35%, dominated by the lowest-yielding step (Step I). Even so, this exceeds the historical method by roughly an order of magnitude, in which lophine was obtained by cyclization of hydrobenzamide using atmospheric oxygen. Despite the effort, and with clear educational value, the approach proves worthwhile.
The chemiluminescence of lophine has practical value in chemical analysis and is more than a curiosity.
References:
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- [2] Ples M., Całkiem niezwykła herbatka (eng. A Rather Unusual Tea), Chemia w Szkole (eng. Chemistry in School), 4 (2015), Agencja AS Józef Szewczyk, pp. 6-9 back
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- [5] Ples M., Chemiluminescencja metalicznego sodu (eng. Chemiluminescence of Metallic Sodium), Chemia w Szkole (eng. Chemistry in School), 1 (2014), Wydawnictwo EduPress, pp. 5-7 back
- [6] 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
- [7] Dzieleńdziak A., Benzoina (kondensacja benzoinowa katalizowana tiaminą - witaminą B1), instrukcja do ćwiczeń, dostępne online: https://www.chem.umk.pl/panel/wp-content/uploads/Benzoina.pdf [dostęp 25.11.2022] back
- [8] Thiamin. Fact Sheet for Consumers, w: Dietary Supplement Fact Sheets, National Institutes of Health, 2015, dostępne online: https://ods.od.nih.gov/factsheets/Thiamin-Consumer/ [dostęp 25.11.2022] back
- [9] Pluciński T., Doświadczenia chemiczne, Wydawnictwo Adamantan, 1997, pp. 69 back
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- [11] Farmakopea Polska X, Polskie Towarzystwo Farmaceutyczne, Urząd Rejestracji Produktów Leczniczych, Wyrobów Medycznych i Produktów Biobójczych, Warszawa, 2014, s. 4276 back
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- [13] Radziszewski B. R., Untersuchungen über Hydrobenzamid, Amarin und Lophin, Berichte der deutschen chemischen Gesellschaft, 10 (1), 1877, pp. 70–75 back
All photographs and illustrations were created by the author.
Addendum
The video below shows the chemiluminescence of lophine residue in the filtrate left behind after recrystallization:
Marek Ples