Chemiluminescence in Analytical Chemistry

146	Stigbrand et al.

119], and urea [120], which have been quantified with the POCL reaction. The high sensitivity attainable, combined with the wide scope of the reaction through the choice of the limiting or catalytic/quenching component, has resulted in a considerable number of analytical applications of peroxyoxalate chemiluminescence. The relative absence of interference is also a contributing factor to the wide variety of applications associated this chemistry [6–8].

Numerous fluorophores have been tested for their suitability as energy acceptors in the POCL reaction. Detection limits are generally in the low femtomole range and improvements of 10–100 times are possible compared to conventional fluorescence [75, 94, 121]. Another parameter that shows the contrast between POCL and fluorescence detection is the selectivity, where POCL, owing to its inability to excite all fluorescent compounds, is more selective than fluorescence and can provide simpler chromatograms, as has been demonstrated by Sigvardson and Birks [100]. Shale oil extracts were separated and detected with either CL or fluorescence detection. The chromatogram obtained with fluorescence detection was dominated by overlapping peaks, whereas when POCL chemistry was used, only well-resolved amino-polyaromatic hydrocarbons (amino-PAHs) were detected. This property can also be utilized in derivatization, wherein the 5-di- methylaminonaphthalene-1-sulfonyl (dansyl) group is the fluorescent moiety most commonly introduced. Dansylating derivatization can be directed toward many different kinds of analytes since this fluorophore has been synthesized with several functional groups, e.g., chloride [61], hydrazine [83], hydroxyl [122], ethanol amine [123], and aziridine [124].

It should also be noted that optimization of POCL detection is a relatively complex process due to the number of variable parameters in the reaction. Most papers dealing with practical applications of the POCL reaction also feature some degree of optimization studies. In addition, a number of studies have focused specifically on the optimization process [125–134], and Hadd and Birks [135] have recently summarized the most important aspects in a comprehensive overview.

2. REACTION MECHANISM

The mechanism of the POCL reaction is a complex multistep process and it has proved to be difficult to elucidate. Side reactions as well as light-generating reactions are fast and overlapping in time, and many of the intermediates are unstable. Because of this complexity, the complete reaction mechanism has still not been fully resolved, despite numerous investigations since its discovery.

Rauhut et al. [2, 3] conducted a number of mechanistic experiments aimed at elucidating the reaction mechanism of the POCL reaction. It was noted that fluorophores were not consumed in the reaction, and that the CL emission spec-

Peroxyoxalate Chemiluminescence

147

trum is essentially identical to the fluorescence emission spectrum of the fluorophore compound, demonstrating that the deexcitation of the fluorophore occurs via the first singlet excited state. A 1:1 stoichiometry between oxalyl chloride and hydrogen peroxide was observed, and the gaseous products of the reaction were CO2 and CO [2]. However, the ratio between these two gases varied substantially and difficulty was encountered in obtaining reproducible data. The changes in the infrared absorption of carbonyl, acid chloride, and ester groups were studied during the course of the reaction. In the case of both oxalyl chloride [2] and diaryloxalate esters [3], the absorbances from acid chloride and ester groups decreased faster than the total carbonyl absorption, leading to the suggestion that at least one intermediate contains a carbonyl group(s). Furthermore, the effect of adding certain compounds was studied. Water increased the initial intensity and the reaction rates, without affecting the quantum yield. Addition of ethanol caused a side reaction in which no CL was produced, and the radical inhibitor 2,6-di-tert-butyl-4-methylphenol decreased the quantum yield, but did not affect the reaction rates to a great extent. Some differences in behavior between oxalyl chloride and the oxalate esters were noted. The reaction between oxalyl chloride and hydrogen peroxide proceeded under acidic conditions [2], whereas the oxalate esters generally required neutral or basic conditions [3]. The quantum yield increased with increasing hydrogen peroxide concentration in the oxalyl chloride reaction, whereas a plateau was reached in the ester reaction. This led Rauhut et al. to the conclusion that intermediates formed in the oxalyl chloride reaction were short-lived, whereas in the ester reaction they were long-lived. These differences do not rule out a common intermediate; if there is one, it can be formed through different reaction paths. Furthermore, competing side reactions, especially in the presence of certain additives, can affect the reaction paths significantly.

Recently, Orosz et al. [136] reviewed and critically reevaluated some of the known mechanistic studies. Detailed mathematical expressions for rate constants were presented, and these are used to derive relationships, which can then be used as guidelines in the optimization procedure of the POCL response. A model based on the ‘‘time-window concept,’’ which assumes that only a fraction of the exponential light emission curve is captured and integrated by the detector, was presented. Existing data were used to simulate the detector response for different reagent concentrations and flow rates.

2.1 Reactive Intermediates

Early research suggested that the ‘‘key intermediate’’ in the POCL reaction was 1,2-dioxetandione (structure II in Fig. 5) [2, 3], which is formed following the multistage nucleophilic attack by the hydroperoxide ion on one of the carbonyl carbons in the oxalate compound. This series of reactions is proposed to occur

148	Stigbrand et al.

Figure 5 Proposed intermediates in the POCL reaction.

as follows: deprotonation of the newly formed hydroperoxyoxalate ester group on the other carbonyl carbon is followed by a nucleophilic attack, which in turn triggers an intramolecular cyclization. This would explain why the nature of the aryl groups has a major influence on the efficiency in the POCL reaction. 1,2- Dioxetandione was once claimed to have been positively identified using mass spectrometry [137], but in a later study [138], it was shown that the peak corresponding to the dioxetandione (m/z 88; C2 O4 ) was formed in the ion source itself.

Catherall et al. [139, 140] continued to study the mechanism and claimed that there was no experimental evidence to support the existence of a common intermediate. Instead they suggested another ‘‘key intermediate,’’ one that still has a side chain (R) attached to it (structure III in Fig. 5). These workers also claimed that based upon their research, the key intermediate exhibited a lifetime of approximately 5 10 7 s at ambient temperatures.

Rauhut and Semsel [141] and Steinfatt [142] have suggested an unusual mechanism whereby the proposed intermediate 1,2-dioxetandione forms a structural (isoelectronic) isomer as outlined in route B in Figure 6. Route A represents a dark reaction. The isomerized molecule is then thought to decompose into singlet oxygen and carbon trioxide [143].

A kinetic investigation of photoinduced peroxyoxalate chemiluminescence led to the postulation of a new series of high-energy intermediates thought to be capable of inducing the chemiexcitation of fluorophore compounds [143]. Photoinitiation of the POCL reaction was performed by irradiation with a laser pulse to generate hydrogen peroxide in situ from oxygen in the presence of a hydrogen donor, 2-propanol. The kinetic behavior of the ensuing reaction(s) between TCPO, hydrogen peroxide, and a fluorophore was monitored. Complex reaction

Peroxyoxalate Chemiluminescence

149

Figure 6 Proposed formation of an alternative C2 O4 structure in the excitation mechanism for peroxyoxalate chemiluminescence.

schemes were proposed, giving rise to a number of reactive intermediates whose existence was considered by the authors to be speculative.

When the POCL reaction is initiated using diaryloxalate ester reagents in the absence of a fluorophore molecule, a weak emission may be observed. This emission, subsequently referred to as background emission, was found by an early study [100] to be a fundamental characteristic of the POCL reaction. Mann and Grayeski [144] studied the background emission from three different diaryloxalate esters and one oxamide compound. The results indicated that there were two emission bands, one with a maximum centered at 450 nm with a weaker band at a longer wavelength, which appeared to be reagent dependent. The two emission bands were attributed to two separate key intermediates of the POCL reaction, formed and decomposed at different rates. Recently, Barnett et al. [145] demonstrated that the background emission associated with oxalate esters was absent in the case of certain oxamides, but if the oxamides were mixed with chlorinated phenols, emission spectra exhibiting the same spectral characteristics as those of the corresponding bis(chlorinated phenyl) oxalate esters were seen. The intensity of the background emission arising from diaryloxalate ester reagents was also found to be significantly enhanced upon further addition of the corresponding phenol to the reaction. Accordingly, the background emission observed with diaryloxalate ester reagents was postulated to originate from phenoxyl radical intermediates, produced in situ as a result of the oxidizing conditions, and not from key intermediates in the POCL reaction.

The formation of stable intermediate(s) during the storage of POCL reagents under cold, acidic conditions has been observed [146]. Although the exact nature of the intermediate(s) is not yet understood, it is known that following prolonged cold storage ( 20°C) of either TCPO or bis(4-nitro-2-(3,6,9-trioxa- decyloxycarbonyl)phenyl) oxalate (TDPO) with hydrogen peroxide in the presence of a strong acid such as trifluoroacetic acid, a stable compound(s) of unknown structure was formed. When either of these aged solutions was reacted with a fluorophore under suitable conditions, chemiluminescence was observed.

150	Stigbrand et al.

The POCL mechanism has also been studied by NMR spectroscopy by Chokshi and co-workers [147], who used bis(2,6-difluorophenyl) oxalate as reagent and measured the 19F signals. Only one intermediate was found at the concentration level detectable with NMR, and the authors attributed it to a hydroperoxyoxalate ester (structure I in Fig. 5). They proposed that this was the key intermediate in the POCL reaction. In a later paper, Stevani et al. [148] were able to synthesize an analogous structure, 4-chlorophenyl-O,O-hydrogen monoperoxyoxalate. This compound did not transfer its excess energy to fluorophores without the addition of a base catalyst to deprotonate the molecule. This result indicated that this type of molecule was not a ‘‘key intermediate’’ but instead an important precursor that was capable of generating CL following deprotonation by a base. In a similar study, Hohman et al. [149] synthesized two new oxalate esters, triisopropylsilylperoxy 2,6-difluorophenyl oxalate and triisobutylsilylperoxy 2,6-difluorophenyl oxalate. These oxalate derivatives were found to be stable precursors of the half-ester peroxy acid and were intended for mechanistic studies, in pursuit of the ‘‘key intermediate.’’ Following the addition of fluoride ion in the presence of a fluorescer, a bright, short-lived chemiluminescence was observed.

In the case of the experiments performed by Hohman and co-workers [149], the fluoride anion would readily displace the silicon-leaving group. The peroxide anion could then further react via an intramolecular nucleophilic attack, resulting in cyclization to form the reactive intermediate responsible for the chemiluminescence that was observed. A recent kinetic study by Stevani and Baader [150] of the reaction of 4-chlorophenyl-O,O-hydrogen monoperoxyoxalate with various oxygen and nitrogen bases suggested that the intermediate formed must be 1,2- dioxetandione.

2.2 General-Base and Nucleophilic Catalysis

Over the years, there has also been considerable confusion regarding the interaction mechanisms of compounds that appear to catalyze the POCL reaction. The complexity of the reaction and its apparent dependency upon a significant number of parameters has resulted in slow progress in the understanding of the role of these catalysts. Recent work in this area [151–157] has considerably extended our knowledge of the catalyst reaction mechanisms in the POCL reaction, widening the possibilities for development of new and more carefully designed catalysts.

In the first systematic study of the reaction between several different diaryloxalates, hydrogen peroxide, and fluorophores [3], it was recognised that the chemiluminescence reaction was highly sensitive to base catalysis by potassium hydroxide or benzyltrimethylammonium hydroxide, and that acidic conditions markedly diminished the light production. The addition of bases was noted to

Peroxyoxalate Chemiluminescence

151

both accelerate the reactions and, when less reactive oxalates were used, increase the quantum efficiency [4]. A reaction rate increase of about 100 times was observed when 0.1 molar percent of 2,6-lutidine was present in the reaction with DNPO [158]. Almost two decades later, a comprehensive report on POCL kinetics [139] stated that reproducible kinetics could not be obtained in the reaction between bis-(pentachlorophenyl) oxalate (PCPO) and hydrogen peroxide, unless the base catalyst sodium salicylate was present. It was also concluded that the reaction was general-base-catalyzed, and that the attack of the hydroperoxide anion was the step limiting the rate of the light production. Moreover, the quantum yield was found to decrease with increasing base concentrations, and the rate of light decay declined when the base concentration exceeded the oxalate concentration. The authors ascribed this to the formation of a complex between the diaryloxalate and the base catalyst, and noted that the quantum yield decrease was larger than what could be explained by a 1:1 stoichiometry in the assumed complex. The studies above supported the impression of general-base catalysis, but it should be emphasised that none of the bases employed are known to be capable of engaging in modes of reaction initiation other than simple deprotonation.

Following the successful introduction [159, 160] of imidazole as a buffer component in a postcolumn POCL reaction detection in liquid chromatography, the catalytic ability of several bases was compared in static systems [125, 161]. The reaction intensity versus time profiles were recorded under semiaqueous conditions with catalyst buffers containing either imidazole, pyrazine, pyridine, aniline, diethylamine, mono-, di-, and triethylamine, tris(hydroxymethyl)aminomethane (TRIS), or salts of phthalate, phosphate, and tetramethylammonium ions. The use of imidazole was found to generate light intensities approximately 10 times higher than any other catalyst buffer. The kinetics of the light production was also highly dependent upon the concentration of imidazole [125]. Under dry conditions in aprotic media, 4-dimethylaminopyridine has been found to be a superior catalyst compared to sodium salicylate and tetrabutylammonium perchlorate [162]. As a result of these observations, the authors suggested that the pH of the medium was of prime importance to the reaction rate.

Following the disclosure of the outstanding catalytic ability of imidazole compared to other bases, the catalysis of the POCL reaction by imidazole was studied in more detail [163], and it was concluded that the POCL reaction mechanism included the concurrent catalysis by two imidazole molecules, by what was described as ‘‘general-base’’ and ‘‘nucleophilic’’ pathways, respectively. The mechanism for this was suggested to be a ‘‘base catalysis of imidazole catalysis by imidazole itself’’ as previously reported for imidazole-catalyzed reaction of esters [164, 165]. Despite this, it was not until the introduction [151] of 1,1′- oxalyldiimidazole (ODI) as a chemiluminescence reagent, and the postulation of its intermediate appearance in the imidazole-catalysed POCL reaction, that the

152	Stigbrand et al.

true catalytic action of imidazole was revealed. The identity of this transient intermediate formed in the nucleophilic replacement reaction between TCPO and imidazole was later confirmed by UV spectroscopy to be ODI [153, 154]. Moreover, the 13C NMR results [154] of the proceeding reaction could also be explained by ODI appearing as an intermediate. From these studies it was thus evident that the POCL reaction was subject to nucleophilic catalysis by imidazole, which is also apparent by an observed autocatalytic breakdown of ODI caused by the presence of imidazole [152]. The use of pure reagents and meticulously dry solvents was thus the key to obtaining these results. The subsequent reaction of ODI with hydrogen peroxide has recently been confirmed [155] to be the prime reaction pathway in imidazole-catalyzed POCL.

The nucleophilic catalytic reaction route was found to be second-order with respect to imidazole with moderately reactive diaryloxalates [153–155] whereas only one imidazole molecule was involved in the reaction with more electronegatively substituted diaryloxalates [154]. Analogous to the imidazole-catalyzed hydrolysis of esters [164, 165], this was rationalized [153, 154] by general-base catalysis of one imidazole molecule on another imidazole molecule attacking the diaryloxalate, and by a difference in the leaving-group ability of the substituted phenol on these types of reagents [157]. The rate-limiting step of ODI formation appeared to depend upon the types of leaving group on the reagent [157]. With TCPO [154, 156] the first replacement was slowest, whereas with 4-nitrophenyl oxalate and DNPO [153, 154] the second replacement was the rate-limiting step of the reaction. Recently, substituted imidazoles and other azoles have been shown to catalyze the POCL reaction of TCPO via the formation of intermediates [166], although the intensity of the chemiluminescence was lower than that observed with imidazole.

In attempting to summarize these extensive investigations, it is clear that the POCL reaction is subject to both nucleophilic catalysis and general-base catalysis by concurrent mechanisms, and that the nucleophilic catalysis can itself be influenced by general-base catalysis. From the POCL reaction mechanism (see Fig. 7), the general-base catalysis can readily be accounted for. The nucleophilic attack of hydrogen peroxide on the diaryloxalate substrate is subject to catalysis by a base that can assist in the removal of a proton from the peroxide. The hydroperoxide anion is a stronger nucleophile than hydrogen peroxide and thus reacts with the diaryloxalate and forms the arylperoxyacid at a far faster rate. Likewise, the suspected and postulated cyclization of the arylperoxyacid is accelerated by proton acceptors. Both the intermolecular and intramolecular peroxide attacks proceed at very slow rates, or not at all, with less reactive oxalates. The nucleophilic catalysis path involves the stepwise in situ formation of different oxalic acid derivatives. This may be either a monosubstituted or disubstituted reagent, depending on the relative reactivity of the leaving group from the oxalate and

Peroxyoxalate Chemiluminescence

153

Figure 7 Schematic diagram of the POCL reaction with general-base catalysis according to the left route and nucleophilic (Nu) catalysis following the right route.

the nucleophilic catalyst at a certain pH. The in situ–generated intermediate then reacts with hydrogen peroxide in the same manner as the original reagent would have done, but at a faster rate.

General-base catalysis can, as the name suggests, be accomplished by any adequately strong base, whereas very special demands are placed upon compounds acting as nucleophilic catalysts. The efficiency of these catalysts depends on three factors: basicity, nucleophilicity, and leaving-group ability [166]. Each of these characteristics is in turn the combined result of several attributes.

At low base concentrations, very complex reaction kinetics have been observed by several authors [143, 156, 167, 168]. Emission profiles containing dual maxima has been reported with triethylamine [167] and sodium salicylate [168], which are known to act as general-base catalysts, and also with the nucleophilic catalyst imidazole [156, 168]. From these observations, reaction mechanisms containing two or more speculative intermediates suggestively capable of exciting the fluorophore, plus some metastable ‘‘storage intermediate,’’ have been proposed [143, 154, 167, 168]. Another, and equally likely, explanation is that at low catalyst concentrations there is simply not enough catalyst present to maintain constant reaction conditions throughout the light-generating reaction. The

154	Stigbrand et al.

variable reaction conditions cause changes in the reaction kinetics and thus induce an apparent pulse in the intensity-time profile.

2.3Chemically Initiated Electron Exchange Luminescence (CIEEL)

The POCL reaction is thought to follow the CIEEL mechanism proposed by Schuster [169], later modified by McCapra et al. to fit the POCL reaction [170]. According to this mechanism, the intermediate forms a charge complex with the fluorophore, which donates one electron to the intermediate. The radical anion of 1,2-dioxetandione is believed to decompose into carbon dioxide and carbon dioxide radical anion, and the latter transfers its extra electron back to the fluorophore at a higher energy level, resulting in an excited fluorophore. The energy of the intermediates has been determined to be 105 kcal/mol, which corresponds to an excitation wavelength of 270 nm [171].

The logarithm of the excitation efficiency is a linear function of the oxidation potential of the fluorophore, as experimentally verified in several studies [67, 171]. Among the most efficient energy acceptors are polyaromatic hydrocarbons (PAHs) [100] and amino-PAHs [102]. Kang and Kim [172] studied the effect of substitution on PAHs. The results showed that CL intensity was increased with substitution by phenyl, phenylethenyl, and amino groups. In contrast, cyano and carboxyl substituents lowered the CL efficiency. The higher efficiency of aminoPAHs compared to other PAHs is not only explained by a lowering of the oxidation potential; it is also believed to be due to differences in solvation energies between the compounds and the radical ions formed in the charge-transfer complex.

In conclusion, the intermediate(s) responsible for excitation and the process of the energy transfer step(s) in POCL appear to be extremely difficult to both observe and characterize owing to their complex and inherently unstable nature. The final definitive elucidation of the mechanism and the positive identification of the key intermediates of this enigmatic reaction will continue to provide a significant challenge to researchers.

3. ANALYTICAL APPLICATIONS

3.1 Determination of Hydrogen Peroxide

Hydrogen peroxide plays an important role in many processes in the atmosphere and in natural aqueous systems. It affects numerous redox reactions, which in turn influence the stability and transport of other chemical substances, e.g., pollutants. In the atmosphere, hydrogen peroxide is believed to be involved in several important oxidation reactions, e.g., conversion of sulfur dioxide to sulfuric acid

Peroxyoxalate Chemiluminescence

155

and nitrous acid to nitric acid [173, 174]. In the atmosphere, the concentrations of hydrogen peroxide can be as high as 0.65 parts per million volume (ppmv). Measured concentration levels range from 5 to 500 nM in seawater [175], 0 to 66 nM in groundwater [176], and up to as much as 80 M in rainwater [175]. There is one significant problem when working with detection of low levels of hydrogen peroxide, and that is its presence as a trace component in purified water. Several methods are available to reduce this background level of hydrogen peroxide. Lazrus et al. [177] used catalase, which decomposes hydrogen peroxide to water and oxygen. Dasgupta and Hwang [178] reported the use of granular manganese dioxide packed in a column and incorporated into a flow system. This latter method was used with some modifications [29] to enable protection of hydrogen peroxide ‘‘free’’ water without incorporating a manganese dioxide reactor in the system. Helium-degassed distilled water was circulated through a column filled with manganese dioxide. This water contained approximately 5–10 nM hydrogen peroxide, which corresponded to a 75% decrease in blank signals compared to untreated Milli-Q water. Low concentration standards were prepared by diluting stock solutions of hydrogen peroxide with this water, and to prevent light-induced formation of hydrogen peroxide, these standards were kept in dark, high-density polyethene bottles. It should, however, be emphasized that it is very difficult to prepare reliable standards with a concentration lower than 25 nM.

The POCL reaction has been used for trace determinations of hydrogen peroxide, most commonly in environmental and clinical analysis [26–52]. The latter applications often include various enzyme systems [34–52], where a number of substrates can be indirectly determined by measuring the hydrogen peroxide that is produced as a by-product in the enzymatic reaction.

3.1.1Direct Determination of Hydrogen Peroxide

Klockow and Jacob have used POCL chemistry to study the concentration levels of hydrogen peroxide in different kinds of precipitates and air. In their first study [26], determinations of hydrogen peroxide in rainwater were carried out. The instrumental configuration consisted of a dual-line flow injection (FIA) manifold, where one aqueous carrier delivered the analyte and the other contained TCPO and perylene dissolved in acetone. Extensive studies of potential interferents, including metal ions, nitrite, sulfite, formaldehyde, hydrogen sulfide, organic hydroperoxide, and ozone, were made. The results demonstrated that none of these had any influence when they were added in 100-fold excess over hydrogen peroxide. At higher concentration levels, nitrite, sulfite, and formaldehyde caused signal depression, whereas hydrogen sulfide and organic hydroperoxides gave a positive signal. Furthermore, they concluded that ozone present at normal atmospheric levels (30 pptv) corresponds to a signal equivalent to 0.7 pM hydrogen peroxide, i.e., several orders of magnitude lower than the detection limit