Chemiluminescence in Analytical Chemistry

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Some of the investigations carried out in the first half of the twentieth century were related to CL associated with thermal decomposition of aromatic cyclic peroxides [75, 76] and the extremely low-level ultraviolet emission produced in different reaction systems such as neutralization and redox reactions involving oxidants (permanganate, halogens, and chromic acid in combination with oxalates, glucose, or bisulfite) [77]. In this period some papers appeared in which the bright luminescence emitted when alkali metals were exposed to oxygen was reported. The phenomenon was described for derivatives of zinc [78], boron [79], and sodium, potassium, and aluminum [80].

In 1950, Pruett et al. synthesized tetrakis(dimethylamino)ethylene, a clear, slightly yellow and mobile liquid. The authors observed a prolonged bright bluegreen luminescence when the compound was exposed to oxygen or air in protic solvents [81]. The mechanism for this CL reaction, as proposed by Fletcher and Heller in 1967 [82], is shown in Figure 7.

In the sixties, with the development of instrumentation and the use of more sensitive photomultiplier tubes (PMT), the range of CL reactions studied was extended. Vasil’ev’s group studied intensively the low emission produced in the autoxidation of a variety of hydrocarbons and noted that the addition of certain fluorescent molecules considerably enhanced the luminescence intensity [83, 84]. These mechanisms refer to the term ‘‘sensitized’’ CL. Chandross in 1963 [85] and McKeown and Waters in 1964 [86] observed the visible light emitted during the reaction of hydrogen peroxide with oxalyl chloride or certain nitriles. Hercules [87] and Visco and Chandross [88] independently reported the visible production of light generated in the vicinity of the cathode when a series of highly condensed aromatic hydrocarbons were electrolyzed in acetonitrile or dimethylformamide with tetraethylammonium salts employed as supporting electrolytes. This was the first time that the phenomenon of electrogenerated CL (ECL) was investigated in detail. Chandross and Sonntag [89] found a similar behavior when chemically produced aromatic radical anions were reacted with electron acceptors such as 9,10-dichloro-9,10-diphenyl-9,10-dihydroan- thracene, benzoyl peroxide, oxalyl chloride, mercuric chloride, and aluminum chloride. Throughout the 1960s and 1970s there was much interest in these phenomena for studying new compounds, the different mechanisms, and the nature of the emitting state. In particular, polyaromatic hydrocarbons and their derivatives, ruthenium, osmium, platinum, palladium, and other transition metal complexes, and molybdenum and tungsten clusters have been studied in relation to their photochemical and electrochemical properties. However, only in the 1990s was application of ECL in analytical chemistry fully exploited (Chapter 9).

In 1961, Ashby reported the weak light emission produced from several polymers such as nylon, when heated [90]. The phenomenon was termed oxyluminescence because it was caused by oxidative processes and required the presence

Chemiluminescence of Evolution Historical

Figure 7

Chemiluminescent reaction of tetrakis(dimethylamino) ethylene (TDE).

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of oxygen. This property was employed for determining the stability of a polymer toward oxidative degradation for estimating the ability of several compounds to act as antioxidants.

The CL behavior of 1,2-dioxetanes, bearing a four-membered ring, was studied by McCapra [91] in 1968. He showed that these compounds were easily converted to an excited product by heat and produced light due to compound cleavage to form two carbonyl compounds, one of them simultaneously being electronically excited and producing emission of light (Fig. 8A). This explanation supported the assumption that the CL reaction of lophine or indole includes a 1,2-dioxetane as an active intermediate. Kopecky et al. synthesized 3,3,4-tri- methyl-1,2-dioxetane for the first time, and observed that the CL emission was strongly enhanced by the addition of a fluorophore [92]. On the other hand, Richardson et al. proposed another mechanism by which dioxetane is decomposed by heat to an excited carbonyl compound via a biradical form (Fig. 8B) [93, 94]. Because of the weak emission by these compounds, analytical applications were not so common although Hummelen et al. synthesized stable 1,2-dioxetanes as a label for thermoluminescence immunoassay of proteins [95, 96]. Some of these derivatives are shown in Figure 9.

Indole derivatives were studied by Philbrook et al. in 1965, showing the CL emission produced in the presence of oxygen and under strong alkaline conditions [97], following the reaction scheme depicted in Figure 10 for skatole (3- methylindole).

In 1965, Rauhut et al. [73] reviewed the oxalyl chloride CL system and showed that oxalyl esters could be used for this system instead of oxalyl chloride. Since then, they synthesized a number of oxalates including oxamides and established a new, potent luminescent system, namely the peroxyoxalate CL (PO CL) system. Much work has been carried out to synthesize suitable oxalic compounds. The first study dealing with different reagents was published in 1967 by Rauhut et al. [98] for the American Cyanamid Company with the purpose of developing

Figure 8 Chemiluminescent mechanism for 1,2-dioxetanes: (A) a concerted decomposition process; (B) a two-step biradical process.

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Figure 9 Some dioxetane derivatives.

reagents suitable for different kinds of emergency lights, where a high quantum yield in combination with a long duration of emission were considered optimal properties. They found that phenyl esters with strongly electron-withdrawing substituents were most efficient, whereas aryl oxalates with electron-donating or weak electron-withdrawing groups were capable of producing only low CL emission. The most efficient oxalate ester appeared to be bis(2,4-dinitrophenyl)oxalate (DNPO), being one of the most widely used esters together with bis(2,4,6-trichlo- rophenyl)oxalate (TCPO). Maulding et al. compared the reactivity of 19 oxamides in the peroxyoxalate reaction and found that the strongest intensity of all oxamides tested was obtained from 1,1′-oxalylbisbenzimidazole [99]. The PO CL system is subject to catalysis by weak bases (e.g., amines) and to inhibition by organic acids. Sherman et al. [100] used the TCPO system to determine hydrogen peroxide and aromatic hydrocarbon fluorescers in a static system; when metal

Figure 10 Chemiluminescent mechanism for 3-methylindole (skatole).

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Figure 11 Proposed mechanism for the PO CL reaction.

chelates are employed as fluorescers, trace metals can be determined. In 1977, Curtis and Seitz [101, 102] applied the PO CL reaction to the detection of fluorescers separated by thin-layer chromatography (TLC). Dansyl derivatives separated by TLC could be detected by successive spraying with solutions of TCPO and hydrogen peroxide in dioxane. The suggested method, comparable to conventional fluorescence detection, had the advantages that it did not require excitation source and could be used to excite the plate uniformly. However, in our opinion, further TLC applications of analytical CL reactions were rarely published thereafter.

From the first papers produced by Rauhut et al. [98, 103] numerous mechanisms for these reactions have been reported to explain the suitability of peroxyoxalate reactions to easily excite oxidizable fluorescers down to 280 nm, although efficiency decreases markedly in the UV region. A widely accepted mechanism

Figure 12 Proposed chemiluminescent mechanism for Schiff bases.

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is termed ‘‘chemically initiated electron exchange luminescence’’ (CIEEL) [104], which is based on the formation of an intermediate of the reaction of 1,2-dioxe- tanedione, which forms a charge-transfer complex with a fluorophore that donates one electron to the intermediate. This electron is transferred back to the fluorophore at a higher energy level, resulting in an excited fluorophore (Fig. 11). The energy content of the intermediates has been determined to be 105 kcal/mol 1, which corresponds to an excitation wavelength around 280 nm [105]. The light emitted corresponds to the first singlet excited state of the fluorophore. Different ‘‘key intermediates’’ for this reaction have been proposed. For example, Catherall et al. proposed substituted 1,2-dioxetanedione as a key intermediate alternative to 1,2-dioxetane [106, 107]. This assumption was supported by the results of analysis by computer simulation by Givens’s group, who studied the complex role of some base catalysts in this mechanism [108, 109]. The mechanism and application of the PO CL system are discussed in Chapter 7.

In 1976, McCapra and Burford [110] studied CL reactions of Schiff bases, proposing the mechanism shown in Figure 12. Although the efficiency of the CL reaction is high, a strong base is needed; furthermore, the reaction takes place

Figure 13 Chemiluminescent reaction of diphenoyl peroxide based on CIEEL mechanism.

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only in aprotic or anhydrous solvents, which restricts analytical applications of this system.

In 1977, Koo and Schuster studied the CL emission produced when diphenoyl peroxide was decomposed at 24°C in dichloromethane in the dark producing benzocoumarin and polymeric peroxide [111, 112]. No CL emission was observed directly as benzocoumarin is nonfluorescent; however, in the presence of aromatic hydrocarbons light was produced because of the fluorescence of these hydrocarbons. The explanation of this phenomenon was based on the abovementioned CIEEL: the aromatic hydrocarbons, which have a low oxidation potential, transfer one electron to diphenoyl peroxide to form a charge-transfer complex, from which benzocoumarin and the corresponding hydrocarbon in the excited state are produced (Fig. 13).

4.THE FIRST ANALYTICAL USES OF BIOLUMINESCENCE AND CHEMILUMINESCENCE

Investigations of CL for analytical use began around 1970 [113]. In 1974 Isacsson and Wettermark presented an extensive review covering the general field of analytical methods based on the recording of CL [114]. The applications included gas-phase, solid-state, and liquid-phase analysis as well as special applications (identification of bloodstains in forensic chemistry, the analysis of microorganisms, and the CL of organic compounds induced by ozone). In another review article, Seitz and Neary reported in the same year on the advantages of CL and BL for chemical analysis, in relation to the extreme sensitivity and simple instrumentation. They described a small number of CL and BL analytical uses because of lack of available reactions [115].

Burdo and Seitz reported in 1975 the mechanism of the formation of a cobalt peroxide complex as the important intermediate leading to luminescence in the cobalt catalysis of the luminol CL reaction [116]. Delumyea and Hartkopf reported metal catalysis of the luminol reaction in chromatographic solvent systems in 1976 [117], while Yurow and Sass [118] reported on the structure-CL correlation for various organic compounds in the luminol-peroxide reaction.

Routine application of CL as an analytical tool dates from around the 1980s for the liquid phase. In 1978, Paul focused attention on recent advances in CL analysis in solution, stressing the high sensitivities that are possible and the use of rather inexpensive equipment [119].

4.1 Chemiluminescence in the Gas Phase

The development of CL methods for determining components of a gas largely originated from the need to determine atmospheric pollutants. In 1965, a hydro-

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gen-rich flame photometric detector was developed, in which a strong emission was produced in the presence of volatile compounds of phosphorus or sulfur because of a reduction reaction in the flame [120, 121]. The detector was sensitive down to ppb levels of phosphorus compounds and less than ppm levels for sulfur compounds. In the case of sulfur dioxide and other sulfur compounds the emission was due to the electronically excited sulfur produced [122,123]:

SO2 2H2 → S 2H2 O

S S → S2*

S2* → S2 hν (300–425 nm)

Also, a CL reaction between sulfur dioxide and oxygen atoms was studied by Mulcahy and Williams [124] and proposed for the analysis of sulfur dioxide with a sensitivity of 0.001 ppm. However, a disadvantage of this analysis lies in the difficulty for finding a stable source of oxygen atoms.

O• O• SO2 → O2 SO2*

SO2* → SO2 hν (280 nm)

The oxidation of phosphorus by molecular oxygen occurs with vapor just above the solid to give a green emission. The mechanism of the reaction is not known [125] though the emitting species have been identified as (PO)2 and HPO [126]. In 1965 Nederbragt et al. proposed a method for analysis of ozone based on a CL reaction in the presence of ethylene. Light intensity was proportional to the concentration of ozone and was emitted between 300 and 600 nm [127]. Using this method, monitors for field use as well as ozone detectors were constructed [128].

Some methods were proposed for the determination of nitrogen oxides based on the reaction with ozone [128, 129]:

NO O3 → NO2* O2

NO2* → NO2 hν (600–2800 nm),

with atomic oxygen [128]:

NO O• → NO2* → NO2 hν (450–1800 nm)

NO2 O• → NO O2

or with hydrogen atoms [130]:

NO2 H• → NO •OH

H• NO M → HNO* M

HNO* → HNO hν (650–760 nm)

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Applying these processes, several commercial instruments were developed for pollutant monitoring with sensitivities at the ppb level.

Bowman and Alexander described the CL emission induced by ozone from a variety of organic compounds when absorbed on a silica gel surface and dissolved in an organic solvent [131]. The intensity of emission was proportional to the quantity of analyte in the ng range, offering a sensitivity comparable to fluorescence-based methods. In this sense, Regener proposed a very sensitive and selective method for the determination of ozone based on the CL induced when rhodamine B, adsorbed on silica gel, was exposed to ozone [132]. The system was improved by Hodgeson et al. eliminating the humidity by treating the gel surface with a hydrophobic agent [133]. This procedure has been used for the study of vertical distribution of ozone in the atmosphere using a rocket-borne probe.

The characteristics of this kind of CL emission, design of reactors, CL reactions in gas phase, and applications as detection technique in gas chromatography (GC) and atmospheric research are extensively described in Chapter 13.

4.2 Chemiluminescent Systems as Indicators in Titrations

For analysis in solutions, the most frequently used CL reaction is alkaline oxidation of luminol and lucigenin in the presence of hydrogen peroxide as oxidant, although sodium hypochlorite, sodium perborate, or potassium ferricyanide may also be used. CL reactions involving alkaline oxidation have been used to indicate acid-base, precipitation, redox, or complexometric titration endpoints either by the appearance or the quenching of CL when an excess of titrant is present [114, 134]. An example of these mechanisms is shown in Figure 14.

Figure 14 Some examples of endpoint determination in titrations using chemiluminescent indicators. (A) Acid-base titration: the endpoint is detected by the emission of light;

(B) complexometric titration: the endpoint is detected by disappearance of light. M, metal acting as a catalyst; X*, excited state from the CL precursor acting as indicator.

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The systems luminol-H2 O2-catalyst [135, 136], lucigenin-H2 O2 [137, 138], lophine-H2 O2-catalyst [137, 139], and pyrogallol [140, 141] were used as acidbase indicators based on the fact that these substances emit light only in an alkaline medium, allowing the titration of bases as well as acids when including hydrogen peroxide in the indicator system. Lucigenin can be considered the most satisfying indicator because no catalyst is required. These systems find useful application in the determination of acidity of dark-colored or turbid solutions such as red wine, fruit juices, milk, mustard, etc. and in the determination of the acid and saponification numbers of fats and oils.

Luminol, lucigenin [142–145], and siloxene were found suitable for redox titrations. In these systems, an oxidizing agent is used as titrant, and the titration is carried out until light emission begins. In some cases a reducing agent is used as titrant, the titration being carried out until light emission decreases. The use of luminol as redox indicator involves the application of hypochlorite or hypobromite as titrant in alkaline medium. The endpoint is observed when oxidization of luminol starts and light emission appears. In the case of lucigenin, the CL emission is produced when this reagent is oxidized by hydrogen peroxide in alkaline solution, which allows its use as indicator. Titrations with hydrogen peroxide can be carried out, and light is produced only when hydrogen peroxide is in excess. Siloxene can also be used as redox indicator in the direct titration of several inorganic species employing different oxidizing agents such as MnO4 , Ce(IV), Cr2 O72 , and VO3 [142, 146–149]. Kenny and Kurtz found that a potential of about 1.17 V had to be reached before the indicator emits sufficient light to be detected [147]. The reaction is instantaneous in the presence of a small excess of oxidant and no catalyst is required. Indirect methods using siloxene as indicator are based on the reduction of I2 or IO3 with zinc to iodide, on the precipitation of Ag(I) as AgI by adding an excess of iodine, which is then titrated in the presence of the precipitate, or on the addition of an excess of Fe(II) for the analysis of VO3 . Some applications of these systems are shown in Table 1.

Precipitation titrations were developed using lucigenin in the presence of hydrogen peroxide as an adsorption indicator for the argentometric determination of I in the presence of Cl and Br [150]. The indicator, as a positive species, was absorbed onto the precipitated silver iodide, which is negatively charged with I and the luminescence disappears. At the endpoint, I is desorbed from silver iodine and the solution emits light. Siloxene was also employed for the determination of Pb(II) by formation of a precipitate with potassium chromate solution [151]. The endpoint was observed by the emission of siloxene when a small excess of oxidant is present. The method was also used for indirect determination of sulfate by precipitating lead sulfate and titrating the excess of Pb(II) [152] and for determination of Cd(II) by precipitation with hexacyanoferrate(III) [153]. Using this methodology, potassium was determined by precipitation with a known quantity of a standard sodium tetraphenylborate solution, of which the