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oxidized, give rise directly to very weak or ultraweak CL, which, if not analytically useful in and of itself, may provide excited states of lifetime sufficiently long as to sensitize intense fluorescence in an acceptor fluorophore by energy transfer. In addition, a number of compounds, such as dioxetanes, organic oxalates, and oxamides, that are not necessarily chemiluminescent themselves can be thermally oxidized to electronically excited intermediates (aldehydes and ketones) whose lifetimes in the excited state are sufficient for transfer of their excitation energy to a suitable acceptor fluorophore to occur. These will be the subjects of this chapter.

Considerably more intense CL may be observed from a limited number of naturally occurring substances of biological origin (e.g., luciferin) when they are oxidized in vivo with the intermediacy of enzymes. These ‘‘bioluminescent’’ reactions are interesting and of considerable analytical utility as they can often be conducted under in vitro conditions. Gas-phase reactions also will be considered elsewhere in this book and do not have a wide range of applicability in organic analysis. For these reasons, they too will not be discussed in this chapter. Nor will electrogenerated CL, which entails electron transfer reactions occurring through the agency of electrode surfaces rather than oxidizing or reducing chemical species.

2.CHEMILUMINESCENT ORGANIC MOLECULES OF HISTORICAL SIGNIFICANCE

CL emissions can be characterized by four parameters, including color, intensity, rate of production, and decay of intensity. The properties of several organic, chemiluminescent reactions known to produce emissions of light are shown in Table 1.

2.1 Lophine and Other Indoles

Although bioluminescence had been observed in nature for centuries, that synthetic organic compounds could produce light was first established by Radziszewski [1, 2] in 1877 using lophine (2,4,5-triphenylimidazole) (Fig. 1).

Lophine emits yellow CL upon oxidation by molecular oxygen in alkaline solution. The oxidation is believed to produce a free radical [3], which is further oxidized to a hydroperoxide, which is the light-emitting species [4–6]. A number of chemiluminescent derivatives of lophine have been synthesized and have been shown to exhibit varying efficiencies of CL. Lophine has been used in the analysis of metal ions such as Co2 that catalyze the chemiluminescent reaction between it and hydrogen peroxide [7]. It has also been used as a chemiluminescent indicator in titrimetry [8].

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Table 1 Properties of Some Organic Chemiluminescent Reactions
Reaction	Color (λmax)	Quantum yield
Luminol oxidation in DMSO	Blue-green	0.05
	480–502 nm
Luminol oxidation in aqueous alkali	Blue	0.01
	425 nm
Lucigenin oxidation in alkaline H2 O2	Blue-green	0.016
	440 nm
Lophine oxidation in alcoholic NaOH	Yellow
	525 nm
Pyrogallol in alkaline H2 O2	Reddish-pink
ATP-dependent oxidation of D-luciferin
with firefly luciferase
pH 8.6	Yellow-green 560 nm	0.88
pH 7.0	Red 615 nm
Aliphatic aldehyde, reduced flavin mono-	Green
nucleotide oxidation with marine bac-	490 nm
teria
Peroxyoxalate [bis(2,4,6-trichlorophenyl) ox-	Blue	0.07–0.50
alate] oxidation using 9,10-dipheny-
lanthracene as the fluorophore

Figure 1 Mechanism for CL emission of lophine (2,4,5-triphenylimidazole).

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A number of other indole derivatives have also been observed to yield CL subsequent to oxidation. Among these is skatole (3-methylindole), which emits light after oxidation by O2 under strongly alkaline conditions [9].

2.2 Luminol and Related Acylhydrazides

Albrecht [10] was the first to report the CL of luminol (5-amino-2,3-dihydro- 1,4-phthalazinedione), in 1928. The chemiluminescent reaction involves the oxidation of luminol (usually by H2 O2) and often occurs in the presence of a catalyst (or co-oxidant) such as Fe(CN)6 3, Cu(II), or Co(II)) (Fig. 2). The light emission, which is blue in water and yellow-green in DMSO, is identical with the fluorescence of the 3-aminophthalate oxidation product [11].

The oxidation of luminol in basic solution is one of the best known and most efficient chemiluminescent reactions, having a quantum yield of CL of about 0.01 in water and 0.05 in DMSO.

White et al. [11, 12] showed that the production of CL from luminol follows from the formation of a dinegative ion of luminol, which reacts with oxygen or an alternative oxidizing agent to yield an excited singlet state of the aminophthalate ion that is responsible for the emission of light. Table 2 shows several oxidizing systems that react with luminol to generate light [13].

The luminol reaction occurs under a wide variety of conditions. Specific analysis using luminol requires that the chemistry be controlled so that the CL intensity is proportional to the concentration of the species of interest.

In the early studies on luminol and related hydrazides the systems used were composed of either sodium or potassium hydroxide, as base, hydrogen peroxide as the oxidizing agent (more recently molecular oxygen, hypochlorite, iodide, and permanganate have also been used), and some type of initiator or activator. This initiator was frequently hypochlorite, persulfate, a transition metal

Figure 2 Oxidation reaction of luminol to produce light.

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Table 2 Oxidizing Systems that React with Luminol to Emit Light

Oxidant Catalyst/(Co)oxidant

H2 O2 H2 S2 O8

Peroxidase

Ferricyanide

Heme compounds

Transition metal ions: (Co2 ,

Cu2 , Cr3 , Ni2 , Fe2 , VO2 )

Hypochlorite

O2 Ferricyanide

Fe2

OCl

I2

MnO4

NO2

complex such as ferricyanide or heme, or a metal ion such as Co(II), Cu(II), Cr(III), Ni(II), Fe(II), or VO2 . Lower alcohols as well as mixtures of water and other water-soluble organics [14] have been used as solvents in place of water. In place of the activating agent, pulse radiolysis [15, 16] and sonic waves have also been used [17]. No activating agent is necessary in some aprotic media, only oxygen and a strong base.

Drew et al. [18–20] showed that alterations to the heterocyclic part of the hydrazide system effectively blocked the chemiluminescent reaction. O- and N- methyl analogs, the N-amino imide, and the corresponding aminoquinazoline- 2,3-dione were not chemiluminescent under the reaction conditions used in the oxidation.

The unsubstituted phthalic acid hydrazide and several nonaromatic cyclic hydrazides such as maleic acid hydrazide or succinic acid hydrazide are either nonchemiluminescent or show extremely weak CL. However, the 6-amino isomer of luminol, which is called isoluminol, is chemiluminescent to about the same extent as is luminol. Isoluminol has been used in many chemiluminescent studies, and because the amino group is less sterically hindered than that of luminol, it is probably derivatized for chemiluminescent labeling far more often than is luminol (Fig. 3).

The amino group can be diazotized for coupling to various substrates, as is done in CL immunoassay, without loss of the chemiluminescent properties of the cyclic hydrazide.

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Figure 3 Structures of luminol and isoluminol.

Gundermann et al. [21, 22] have extended Drew’s work and showed that alkylation of the amino group enhances the efficiency, provided that the steric bulk of the alkyl groups does not interfere with the planarity of the nitrogen and the ring. Later work [23] showed that the chemiluminescent efficiency increased with the size of the aromatic ring.

The luminol reaction has been used for the determination of oxidizing agents such as hydrogen peroxide, for enzymes such as peroxidase and xanthine oxidase, and for metal ions such as copper or cobalt that catalyze this CL reaction [24].

2.3 Peroxyoxalate Chemiluminescence

The brilliant emissions resulting from the oxidation of certain oxalic acid derivatives, especially in the presence of a variety of fluorophores, are the bases of the most active area of current interest in CL. This group of chemiluminescent reactions has been classified as peroxyoxalate chemistry because it derives from the excited states formed by the decomposition of cyclic peroxides of oxalic acid derivatives called dioxetanes, dioxetanones, and dioxetanediones.

Peroxyoxalate chemistry has gained increasing importance in recent years, as it is the basis for a number of practical chemiluminescent devices that are marketed worldwide. The initial example of peroxyoxalate CL was reported in 1963 by Chandross [25]. He found that when oxalyl chloride, oxamide, or oxalate esters were treated with hydrogen peroxide in the presence of a fluorescent compound such as 9,10-diphenylanthracene (DPA), a bright, short-lived blue emission was produced corresponding to the fluorescence of the hydrocarbon. Thus, the energy produced in the chemical reaction was transferred to the fluorescer, sensitizing the formation of the lowest excited singlet state of the fluorescer that emits a typical fluorescence. The energy transfer step is common to all peroxyoxa-

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late chemiluminescent systems in that chemical reactions provide a key intermediate (an electronically excited ketone or aldehyde), which then transfers its energy to a fluorescent substance, thereby generating an excited state. Usually alkaline conditions are required to activate the reaction. The ‘‘color’’ of the emission is controlled by changing the energy acceptor fluorophore in the reaction mixture.

The reactions of certain esters of oxalic acid with hydrogen peroxide in the presence of a fluorescer give bright, long-lasting emission yielding the most efficient nonenzymatic chemiluminescent systems yet known with CL quantum yields in the range of 22–27% when the reactions are carried out under optimum conditions. Two of the most popular oxalyl derivatives in current use are bis (2,4-dinitrophenyl) oxalate (DNPO) and bis (2,4,6-trichlorophenyl) oxalate (TCPO) with TCPO being the more frequently used (Fig. 4, A and B).

The ‘‘leaving group’’ of the oxalic ester has a strong effect on the efficiency of the peroxyoxalate chemiluminescent system. The electron-attracting power of the substituents on the phenyl rings of the substituted diphenyl oxalates is important to the overall efficiency of the chemiluminescent reactions. Steric effects

Figure 4 Structures of TCPO (A) and DNPO (B).

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are also believed to play an important role. Concentration is also significant, as decreasing efficiency is usually observed with increasing concentration of the oxalic ester.

Leaving groups other than phenols were also found to be effective in producing moderately efficient chemiluminescent reactions [26, 27].

The structure of the fluorescent molecule can also contribute substantially to the overall efficiency of a chemiluminescent process. Excitation and fluorescence can be strongly influenced by the structure of the fluorescer.

A wide variety of different classes of fluorescent molecules has been investigated in the peroxyoxalate chemiluminescent systems. Among those screened were fluorescent dyes such as rhodamines and fluoresceins, heterocyclic compounds such as benzoxazoles and benzothiazoles, and a number of polycyclic aromatic hydrocarbons such as anthracenes, tetracenes, and perylenes. The polycyclic aromatic hydrocarbons and some of their amino derivatives appear to be the best acceptors as they combine high fluorescence efficiency with high excitation efficiency in the chemiluminescent reaction [28].

Quenching of the emissions of fluorophores sensitized by dioxetanes has also been used for chemical analysis [29, 30] but this is probably less specific and less sensitive than direct measurement of the sensitized emission of the analyte. Most analytical reactions of the dioxetanes require an organic solvent for optimal solubility and CL yields [31]. This has led to considerable interest in the development of water-soluble oxalate esters [32].

2.4 Lucigenin and Other Acridines

Blue-green CL arising from the oxidation of lucigenin (10,10′-dimethyl-9,9′-bisa- cridinium dinitrate) by hydrogen peroxide or oxygen in the presence of catalysts such as Co(II), Fe(II), Fe(III), Cu(II), Cr(III), or Ni(II), in alkaline solution, was first observed by Glue and Petsch [33]. The oxidation proceeds through a dioxetane intermediate and yields N-methylacridone as a final decomposition product (Fig. 5). The emission is identical with that of N-methylacridone (10-methyl- acridan-9-one) and it is the lowest excited singlet state of the latter that is believed to be the result of the lumigenic reactions [34].

Chemiluminescence from lucigenin is observed even without the catalytic transition metal ions but it is more intense when these ions are used. In aqueous or predominately aqueous solutions the CL yield is 0.01–0.02, which makes it a slightly better emitter than luminol [35]. The emission of lucigenin is also catalyzed by Pb(II), Bi(III), Tl(III), and Hg(I) ions, which do not catalyze the CL of luminol [36].

Lucigenin is often used to facilitate the measurement of reactive oxygen species in immunological studies, because it enhances the cellular CL intensity [37]. Both luminol and lucigenin can be used to measure reactive oxygen; how-

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Figure 5 Chemiluminescent reaction of lucigenin proceeding via a dioxetane intermediate.

ever, lucigenin is sometimes used in favor of luminol because lucigenin reacts only with O2 [38]. Lucigenin-enhanced cellular CL serves as a convenient and sensitive method for investigating factors influencing phagocyte biology and immunology, such as the creation of reactive oxygen species during phagocytosis and other immunopathic responses [37].

Several N-methyl-9-acridinecarboxylic acid derivatives (e.g., 10-methyl-9- acridinecarboxylic chloride and esters derived therefrom [39]) are chemiluminescent in alkaline aqueous solutions (but not in aprotic solvents). The emission is similar to that seen in the CL of lucigenin and the ultimate product of the reaction is N-methylacridone, leading to the conclusion that the lowest excited singlet state of N-methylacridone is the emitting species [40]. In the case of the N-methyl-9- acridinecarboxylates the critical intermediate is believed to be either a linear peroxide [41, 42] or a dioxetanone [43, 44]. Reduced acridines (acridanes) such as N-methyl-9-bis (alkoxy) methylacridan [45] also emit N-methylacridone-like CL when oxidized in alkaline, aqueous solutions. Presumably an early step in the oxidation process aromatizes the acridan ring.

The reaction in Figure 6 shows the CL-producing reaction of an acridinium ester and hydrogen peroxide in the presence of a base. This reaction makes acridinium suitable as a derivatizing agent (tag) for amino acids, peptides, and proteins in capillary electrophoresis. The positive charge provides greater mobility in an applied electric field, and it has high CL efficiency. Its rate can be adjusted for measurements in flowing systems, which require reaction completion

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Figure 6 Chemiluminescent reaction of an acridinium ester and hydrogen peroxide in the presence of a base.

in a few seconds to minimize band overlapping. In addition, the acridiniums have been modified to include functional groups suitable for the derivatization of other biomolecules [46].

2.5 Miscellaneous Organic Compounds

A number of organic compounds have demonstrated CL but have not been investigated, in this context, as extensively as the foregoing substances.

2.5.1Tetrakis (Dimethylamino) Ethylene

This compound chemiluminesces when exposed to air or oxygen. Its CL was first demonstrated by Fletcher and Heller [47, 48] and suggested to occur via formation of a dioxetane by addition of oxygen across the ethylenic double bond. Cleavage of the dioxetane to form excited tetramethylurea results in excitation of the tetrakis (dimethylamino) ethylene, whose CL is in good agreement with the fluorescence spectrum of the parent compound. The reaction has been used for the analysis of oxygen [49, 50].

2.5.2Diphenoyl Peroxide

Diphenoyl peroxide is the cyclic peroxide of diphenic acid (2,2′-biphenyldicar- boxylic acid) (Fig. 7). It undergoes thermal decomposition to form 3,4-benzocou-

Figure 7 Structure of diphenoyl peroxide.

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marin with no emission of light [51, 52]. However, in the presence of some of the larger aromatic hydrocarbons (e.g., 9,10-diphenylanthracene and rubrene) emission corresponding to the fluorescence of the hydrocarbons is observed. In this respect, not surprisingly, diphenoyl peroxide behaves very much like the cyclic peroxides formed by the oxidation of oxalic acid derivatives. The reaction is believed to proceed through a charge-transfer complex formed between the aromatic hydrocarbon and the diphenoyl peroxide, followed by electron transfer from the former to the latter with subsequent decomposition of the complex to benzocoumarin and the excited hydrocarbon. This is called ‘‘chemically initiated electron exchange luminescence’’ (CIEEL).

The oxidation of naphthalic acid (1,8-naphthalenedicarboxylic acid) by peroxide, rather surprisingly, does not proceed by formation of a cyclic peroxide but rather via a dioxirane [53] (a three-membered ring containing a carbon atom and a peroxide group). CL is observed from this reaction.

2.5.3Schiff Bases

9-Aminoanthracene forms a Schiff base with dimethylacetaldehyde (isobutyraldehyde). This compound can be oxidized by peroxide under basic conditions to form 9-formamidoanthracene and acetone in dimethylformamide as a solvent [54, 55]. CL from this system can be observed in other aprotic solvents as well. A limited amount of work has been done with the CLs of Schiff bases or anthracene derivatives. Presumably, this will change in the future.

3.RECENT DEVELOPMENTS IN ORGANIC CHEMILUMINESCENCE

(E)-2-(phenylsulfonyl)-3-phenyloxaziridine, commonly known as Davis’ oxaziridine, and its analogs have been shown to luminesce in the presence of strong bases. Davis’oxaziridine has been widely used in organic syntheses, such as in the hydroxylation of enolates to produce α-hydroxy ketones. Stojanovic and Kishi observed in 1995 that when using more than two equivalents of a strong base, such as lithium diisopropylamide (LDA), Davis’ oxaziridine instantly decomposed and no α-hydroxy ketone was produced. However, this reaction was accompanied by an emission of intense yellow light (about 520 nm). They found that not only would LDA produce CL, but tert-butyllithium, n-butyllithium, and potassium hexamethyldisilazanide also cause emission of visible light [56]. In addition to Stojanovic and Kishi’s work, other studies suggest that Davis’ oxaziridine reacts with LDA through protonand oxygen-transfer processes [57].

Akhavan-Tafti et al. have developed a new class of peroxidase substrates that produce CL upon enzymatic oxidation. Horseradish peroxidase (HRP) is