Chemiluminescence

At its core, chemiluminescence follows the same fundamental principles as all luminescent phenomena: an electron is excited to a higher-energy orbital through an energy input and, upon returning to its ground state, emits that energy in the form of a photon. While this process is relatively well understood and predictable for photoluminescence—where photon absorption is governed by clear quantum mechanical rules—it becomes significantly more complex in chemiluminescence. Here, the required excitation energy must originate from a preceding chemical reaction, adding several layers of mechanistic and energetic intricacy.

To illustrate, consider the emission of teal-green light at a wavelength of arround 500 nm. It corresponds to approximately 228 kJ/mol—an amount of energy that many chemical reactions are theoretically capable of releasing. Yet chemiluminescent reactions remain rare. Why?

Additional constraints make such reactions exceptionally difficult to achieve. As early as 1964, Chandross and Sonntag [1] proposed that, for chemiluminescence to occur efficiently, the reaction enthalpy must be released in a single, rapid, and spatially confined reaction step. This concentrated energy release increases the likelihood of directly exciting an electron. However, even when these conditions are met, chemiluminescence is by no means guaranteed. The process is highly sensitive to the precise arrangement of molecular orbitals, the dynamics of the reaction pathway, and the coupling between electronic and nuclear motion—all of which contribute to the rarity and complexity of efficient chemiluminescent systems.

International activities related in particular to bioluminescence and chemiluminescence research are coordinated by the "International Society of Bioluminescence and Chemiluminescence", which organizes conferences every two years to facilitate collaboration and knowledge exchange in this exciting and interdisciplinary field.

Reaction Mechanism & Potential Energy Surfaces:

The conceptual framework of a chemiluminescent reaction involves a transition from a chemical reactant (A) to a product (P), with light emission resulting from the formation of an electronically excited state. A key challenge is aligning the potential energy surfaces of the ground state (S₀) and the excited state (S₁) closely enough—typically at or near the transition state of an exergonic decomposition reaction, such as that of an organic peroxide—so that an efficient transition to the excited state can occur [2].

The diagram on the right illustrates this concept through a cross-sectional view of both energy surfaces along the reaction coordinate. Modern quantum chemical methods allow for detailed investigation of these processes, offering valuable insights into the factors governing efficient excitation.

In principle, such a transition is always possible, but the probability decreases significantly as the energy gap between the two surfaces widens. As a result, even subtle changes to the molecular structure can dramatically reduce—or entirely suppress—the emission of light. This is one reason why classic compounds like luminol or lucigenin, discovered over a century ago, are still widely used: they represent an optimized balance within their molecular class. Despite many efforts, significantly increasing their quantum yields has proven difficult.

Today, highly sensitive detection techniques allow us to observe even the emission of individual photons. When chemiluminescence becomes too weak to be seen by the dark-adapted human eye, it is referred to as ultraweak luminescence. Such low-intensity emissions can be readily measured with instruments like luminometers. In biological systems, these faint emissions—part of the field of biophotonics—occur naturally during normal metabolic processes. In contrast, ultraweak chemiluminescence remains relatively understudied in chemistry, primarily because a wide range of more efficient, high-intensity systems are already available, despite their limitations.

General reaction scheme of a chemiluminescence reaction producing excited state molecules via the decompositon of organic peroxides

Cyclic Peroxides in Chemiluminescent Reactions

Chemiluminescence is observed in a wide range of oxidation reactions, but one particular class of compounds consistently plays a central role in producing intense and efficient light emission: peroxides.

All known organic chemiluminescence reactions involve a high-energy intermediate containing a peroxy moiety. The chemical decomposition of these peroxide species generates electronically excited-state molecules, which subsequently relax to the ground state by emitting light. Organic peroxides are therefore fundamental to all organic chemiluminescent reactions. Their molecular structure, stability, and decomposition mechanisms are key factors in determining both the efficiency and intensity of light emission.

In many highly luminous bioluminescent systems, cyclic peroxides—especially 1,2-dioxetanes—play a critical role. The parent compound, 1,2-dioxetan, serves as the structural basis for a wide variety of derivatives. These include substituted 1,2-dioxetanes, dioxetanones, and dioxetane dianions, typically generated via oxidation. Although dioxetanes are metastable, meaning they are synthetically accessible but decompose readily, their stability can be significantly enhanced with suitable substituents. This has enabled the development of commercially viable chemiluminescent reagents, such as AMPPD.

Dioxetanones, while even less stable, are believed to act as key intermediates in numerous chemiluminescent and bioluminescent reactions [3]. Due to their transient nature, detecting and studying these intermediates poses a considerable challenge. Nevertheless, their existence and behavior can be probed through advanced kinetic analyses, the synthesis of stabilized model compounds, and a multitude spectroscopic methods.

The general and computational aspects of cyclic peroxide chemiluminescence are very well summarized in our 2018 ChemRev Review:
M. Vacher, I. Fdez. Galván, B.-W. Ding, S. Schramm, R. Berraud-Pache, P. Naumov, N. Ferré, Y.-J. Liu, I. Navizet, D. Roca-Sanjuán, W. J. Baader, R. Lindh, Chemical Reviews 2018, 118, 6927-6974. Link

Selected Chemiluminescence Systems & Demonstration Experiments:

White Phosphorus:

The first recorded observation of chemiluminescence dates back to 1669, when the Hamburg alchemist Heinrich Hennig Brand accidentally discovered it during his attempts to isolate the Philosopher’s Stone. By distilling large quantities of urine to dryness and gently heating the residue, he observed a faint yellow-green glow emanating from the flask.

Today, we understand that this glow was the result of the oxidation of white phosphorus, a reaction that produces visible chemiluminescence. Remarkably, this discovery predates modern chemistry and marks one of the earliest documented instances of light emission from a chemical reaction—long before the phenomenon was scientifically understood.

Following the demystification of phosphorus chemiluminescence, the reaction became known as the Mitscherlich Test, named after the chemist Eilhard Mitscherlich (1794–1863), and found its way into forensic chemistry. The glowing oxidation of white phosphorus was used as a detection method and captivated observers with its striking light emission. Even today, for those willing and skilled to handle white phosphorus under controlled conditions, the Mitscherlich Test remains a fascinating demonstration—not only of chemiluminescence but also as a testament for ingenuity of early scientists, who investigated complex natural phenomena with the simplest of means.

Mitscherlich Test, chemiluminescence reaction during the oxidation of white phosphorus

Experimental Procedure Mitscherlich Test: A 500 mL round-bottom flask with a standard ground joint (NS 29) is filled with 250 mL of water, and a piece of white phosphorus about the size of a cherry pit is carefully added. A riser tube approximately 50 cm in length is then attached to the flask.The room is darkened, and the water is brought to a gentle boil. As the water heats, phosphorus atoms are carried upward by the rising steam. Upon contact with atmospheric oxygen, these phosphorus atoms undergo oxidation, producing a bluish-white chemiluminescent glow. The glowing vapor ascends through the tube, forming a luminous column, and eventually creates a small chemiluminescent flame at the top of the tube—an impressive visual demonstration of phosphorus chemiluminescence.

Oxidation of Lophine:

The first chemiluminescence of an organic molecule was observed in 1877 by Radziewski [4]. He wrote, “It is sufficient to pour a concentrated alcoholic solution of either potassium or soda over a few centigrams of lophine in a test tube to observe the emission of light.” The synthesis of lophin requires only benzaldehyde and ammonia. An easy procedure is available at the german synthesis webpage Illumina. Benzaldehyde and ammonia first form hydrobenzamide, which then converts into Amarine upon heating, which at higher temperatures, transforms into lophine.

Synthesis and chemiluminescence mechanism of Lophine

The chemiluminescence mechanism of lophine is ultimately, a somewhat elaborate oxidation of benzaldehyde to benzoic acid. In alkaline solution, lophine reacts with atmospheric oxygen and emits chemiluminescence light. The high energy intermediate of this reaction is a cyclic peroxide, a typical chemiluminescent intermediate found in many chemiluminescent reactions. The reaction can be also be triggered when lophine is treated with hydrogen peroxide and hypochlorite is added. The resulting singlet oxygen reacts directly with lophin to form the high energy intermediate, and the final product is benzoic acid, which arises from the hydrolysis reaction product.

Bronislaus Radziewski is not only the discoverer of lophine chemiluminescence but also identified other chemiluminescent reactions that have since fallen into obscurity [8].

In 2019, we revisited the classical lophine chemiluminescence system and successfully transferred the solution-based reaction into a solid-state thermochemiluminescent process. Specifically, we focused on lophine hydroperoxide, a derivative that can be photochemically synthesized from lophine. This compound readily crystallizes into large, centimeter-sized crystals that remain stable up to approximately 110 °C. Upon heating beyond this threshold, the crystals decompose, releasing visible chemiluminescent light.

For a detailed exploration of this system, please refer to our publication:
S. Schramm, et al. Nature Communications 2019, 10, 997. Link

A centermeter size crystal of lophine hydroperoxide on a 120°C heating plate

Trautz-Schorgin Reaction:

The discovery of lophine chemiluminescence by Radziewski in 1877 sparked interest in exploring other light-emitting chemical reactions. Researchers soon observed chemiluminescent behavior in compounds associated with analog photographic processes. During the early development of photographic plates and gels, extensive empirical experimentation revealed that solutions containing formaldehyde and aromatic phenols emitted a faint glow upon exposure to air [5].

In 1905, P. Schorgin published a paper [6] dedicated to luminescence, describing the oxidation of formaldehyde in the presence of phenols—a reaction later termed the Trautz-Schorgin Reaction. The mechanism behind this chemiluminescence is complex and not yet fully understood. Interestingly, formaldehyde can be substituted with glutaraldehyde, methanol, or even glycerin. However, in the case of methanol and glycerin, the reaction only proceeds when the mixture is heated to at least 70°C. Phenolic compounds can also be sourced from natural substances, such as tannins or the polyphenols found in black tea [7].

Video of the two-color chemiluminescence during the Trautz-Schorgin Reaction usung the reaction precedure as above

The Trautz-Schorgin Reaction can relatively easily be recreated in visually appealing way with simple chemicals for demonstration experiments:

Experimental Procedure of the Trautz-Schorgin Reaction:

Solution 1: 1 g of pyrogallol in 10 ml of water
Solution 2: 5 g of potassium carbonate in 10 ml of water ( + few mg of Luminol)
Solution 3: 10 ml of 35% formaldehyde
Solution 4: 15 ml of 30% hydrogen peroxide

In a darkened room, the solutions are poured sequentially into a tall beaker or Erlenmeyer flask and stirred briefly. The reaction is highly exothermic, heats up rapidly, produces foam and an orange-red chemiluminescence. One can exploit the intense heat development to produce a two-color emission by adding a small amount of luminol. At approximately 80°C, luminol is oxidized by hydrogen peroxide even without a catalyst and glows blue, producing a sequential red then blue emission. The video below show the typical emission of the Trautz-Schorgin reaction with additional Luminol. This way a two-color chemiluminescence reaction can be achived.

Luminol:

Perhaps the most famous chemiluminescent compound, luminol was first synthesized in 1902, although its light-emitting properties were not described until 1928 [9]. Since then, it has become the textbook example of chemiluminescence, widely known for its brilliant blue glow.

In an alkaline solution, luminol is oxidized—typically using hydrogen peroxide in the presence of a metal catalyst such as iron, copper, or even the iron-containing hemoglobin in blood. This oxidation produces an electronically excited 3-aminophthalate ion, which emits blue light as it returns to the ground state.

The luminol reaction is remarkably sensitive, capable of detecting trace amounts of blood or metal ions, and is therefore a staple in forensic science for crime scene investigations. Its vivid glow and simple setup have also made it a popular demonstration experiment in classrooms and science shows, where it illustrates key chemical concepts such as oxidation, catalysis, and excited-state emission.

Due to its importance and widespread use, we’ve dedicated a separate section to this iconic chemiluminescent molecule:

Luminol

Hemine catalysized chemiluminescence of Luminol
everyone's idle from berlin, germany, CC BY-SA 2.5, via Wikimedia Commons

Synthesis and chemiluminescence mechanism of Lucigenin

Lucigenin:

Lucigenin was first synthesized and described in 1936 [10]. It is prepared via the reductive dimerization of N-methylacridone, followed by ion exchange. Well-documented and illustrated synthesis protocols are available through online sources such as Lambdasyn.

In chemiluminescent reactions, lucigenin reacts with the peroxide dianion, forming a dioxetane intermediate that decomposes with the emission of light, yielding two molecules of acridone. The actual light emitter in this process is acridone, which emits at 401 nm. Interestingly, lucigenin itself fluoresces green, and this fluorescence plays a role in the reaction’s color dynamics.

At the start of the reaction, the high concentration of lucigenin allows energy transfer from the excited acridone to unreacted lucigenin, resulting in green emission. As the reaction progresses and lucigenin is consumed, the emission gradually shifts to blue, corresponding to direct acridone emission.

Thanks to its water solubility and relatively high quantum yield, lucigenin is particularly well-suited for detecting hydrogen peroxide in biological systems, and is frequently used in studies involving cells and tissues [11].

Peroxyoxalate Chemiluminescence:

In the early 1960s, Chandross discovered that oxalyl chloride emits light when reacted with hydrogen peroxide in the presence of a fluorescent dye [12]. This marked the beginning of peroxyoxalate chemiluminescence research. Today, instead of using the reactive acid chloride, more stable oxalic acid esters with electron-deficient aromatic groups are employed—offering safer handling and improved performance.

Thanks to the vast array of available dyes, virtually any emission color can be achieved, and the reaction mechanism is now largely understood [13]. The chemiluminescent process begins with the reaction of the oxalate ester (e.g., bis(2,4,6-trichlorophenyl) oxalate, TCPO) with hydrogen peroxide, producing a high-energy intermediate, likely 1,2-dioxetanedione. This intermediate transfers its energy to a fluorescent dye, which then emits light as it relaxes to the ground state.

Peroxyoxalate chemiluminescence is also one of the most efficient non-biological light-emitting reactions, with quantum yields exceeding 90% in optimized systems.Because of its brightness, simplicity, and safety, this system is widely used in glow sticks,

Due to its importance and widespread use, we have dedicated a separate page to this fascinating class of chemiluminescence.

Peroxyoxalate

Reaction mechanism of TCPO chemiluminescence and demonstration of peroxyoxalate chemiluminescence with different fluorescent dyes

Chemiluminescence mechanism of AMPPD

1,2-Dioxetanes:

1,2-Dioxetanes are a key class of cyclic peroxides known for their ability to emit light upon thermal or enzymatic decomposition. These four-membered ring structures are metastable [14], and when triggered—either by heat, pH change, or enzyme activity—they decompose to form carbonyl compounds, depending on the mechanism involved in either an electronically excited state singlet or triplet state that emits light as it relaxes [14, 15].

A particularly important derivative is AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane), a stabilized dioxetane widely used in biochemical assays. AMPPD remains stable until it is cleaved by alkaline phosphatase, at which point it undergoes chemiluminescent decomposition, producing blue light. This reaction is highly specific and sensitive, making AMPPD a powerful tool in enzyme-linked immunoassays (ELISAs), nucleic acid hybridization assays, and clinical diagnostics with limits of detection in the single molecule range.

Due to its importance and widespread use in bioanalytical assays we’ve dedicated a separate section on 1,2-dioxetanes:

1,2-Dioxetanes

2-Coumaranones:

2-Coumaranones represent one of the most recently discovered classes of chemiluminescent substances. The first compounds in this group were synthesized and described in 1979 [16], but due to the relatively weak light emission, research interest quickly faded. The system was briefly revisited in 1996 [17], but limited success again led to its decline into obscurity.

Driven by pure curiosity—and a desire to observe their chemiluminescence firsthand—we re-synthesized the original 2-coumaranones, developed an improved synthetic route, unraveled the chemiluminescence mechanism and expanded the scope compound class to a around 100 derivatives. This effort led to a decisive breakthrough: certain 2-coumaranones now rank among the brightest known chemiluminescent compounds [18, 19].

The chemiluminescence is oxygen-dependent and is initiated by dissolving the coumaranone in acetonitrile or acetone, followed by the addition of a non-nucleophilic base (e.g., DBU). If the coumaranone concentration is too high, the light emission diminishes due to local oxygen depletion. However, simply swirling the solution allows oxygen to re-dissolve, restarting the chemiluminescent reaction.

The synthesis of these compounds is relatively straightforward and is described in detail (in German) on the synthesis page Illumina.

In 2025, we compiled all available scientific data on chemiluminescent 2-coumaranones published to date and authored a comprehensive review article. Published under an open-access license, this work covers the historical discovery, synthesis, luminescence mechanisms, and emerging applications of this fascinating chemiluminescent system.

For more information, please refer to our review:
S. Schramm, T. Lippold, I. Navizet, Molecules 2025, 30, 1459. Link

Singlet Oxygen:

Molecular oxygen is one of the few naturally occurring substances that exists in a triplet ground state, meaning its two unpaired electrons occupy separate molecular orbitals with parallel spins. When energy is supplied—either chemically or photochemically—oxygen can be converted into its singlet excited state, in which the electrons pair up.

Singlet oxygen is highly reactive and short-lived, with a typical lifetime of just a few milliseconds. As it returns to its stable triplet form, it releases the excess energy as faint red light, a form of chemiluminescence.

A simple and illustrative way to generate singlet oxygen is through the oxidation of hydrogen peroxide by elemental chlorine. In this demonstration, chlorine gas is produced by reacting potassium permanganate or sodium hypochlorite with hydrochloric acid. The gas is then bubbled into an alkaline hydrogen peroxide solution, typically set up in a fermentation tube or gas-washing bottle. As the chlorine reacts with the hydrogen peroxide, singlet oxygen is generated, resulting in visible red light emission.

The overall reaction is as follows:

2NaOH + Cl₂ + H₂O₂ → O₂(¹Δg) + H₂O + 2NaCl

The reaction can also be carried out entirely with household chemicals. Acidic toilet cleaner (which contains hydrochloric acid) and alkaline toilet cleaner or bleach (which contains hypochlorite) are mixed to produce chlorine gas—a very effective method. The alkaline hydrogen peroxide solution can be prepared from a percarbonate bleach products.

When chlorine gas bubbles through the hydrogen peroxide solution, each bubble produces a red glow—the emission of singlet oxygen.

References:

E. A. Chandross, F. J. Sonntag, J. Am. Chem. Soc. 86 (1964) 3179
D. Wöhrle, M. Tausch, W.-D. Stohrer, Photochemie, Wiley-VCh, 1998
W. J. Baader, C. V. Stevani, E. Bastos, Chemiluminescence of Organic Peroxids, in: The Chemistry of Peroxids Vol 2., John Wiley & sons 2006
Radziszewski, B.; Ber. Chem. Ges. 1877, 10, 70
M. Trautz, Studien über Chemilumineszenz, Z. Phys. Chem. 53 (1905) 1-111
Schorgin, P.; Die Lichterscheinung während der Kristallisation und die temporäre Tribolumineszenz, über die chemische Lumineszenz; Inaugural-Dissertation, Universität Freiburg i. B. 1905
H. Brandl, PdN-Ch. 42/1 (1993) 24-26
B. Radziszewski, Über die Phosphoreszenz der organischen, organisierten Körper, Liebigs Analen der Chemie, 203 (1880) 305-336
H. O. Albrecht, Z. Phys. Chem. 136 (1928) 321
Gleu, K.; Petsch, W.; Angew. Chem. (1935), 48, 57
M. A. Baker, R. J. Aitken, Reproductive Biology and Endocrinology (2005) 3:67
E. A. Chandross, Tetrahedron Lett., 761 (1963)
Mechanismus Peroxioxalat
E. L. Bastos, W. J. Baader, Theoretical studies on the thermal stability of alkyl-substituted 1,2-dioxetanes; ARKIVOK 2007 (viii) 257-272
S. Wildey, F. bernardi, M. Olivucci, M. A. Robb, S. Murphy, W. Adam, The Thermal Decomposition of 1,2-Dioxetane Revisited, J. Phys. Chem. A 103/11 (1999) 1669 - 1677
G. J. Lofthouse, H. Suschitzky , B. J. Wakefield , R. A. Whitetaker ,B. Tuck, J. Chem. Soc. Perkin Trans. I, 1979, 1634
B. Matuszczak, Monatsh. Chem., 1996, 127, 1291
S. Schramm, D. Weiß, I. Navizet, D. Roca-Sanjuán, H. Brandl, R. Beckert, H. Görls, ARKIVOC 2013 (iii) 174-188
L. F. M. L. Ciscato, F. H. Bartoloni, A. S. Colavite, D. Weiss, R. Beckert, S. Schramm, Photochem. Photobiol. Sci., 2014, 13, 32

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