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lose their excitation energy via nonradiactive pathways (e.g., heat). This type of luminescence provides interesting analytical applications thanks to its high sensitivity [1]. This entails considering its kinetic aspects in theoretical and applied terms.

For a reaction to produce detectable CL emission, it must fulfill the following conditions: (1) it should be exothermic so that sufficient energy for an electronically excited state to be formed (at least 180 kJ/mol for emission in the visible region) can be provided; (2) there should be a suitable reaction pathway for the excited state to be formed; and (3) a radiactive pathway (either direct or via energy transfer to a fluorophore) for the excited state to lose its excess energy should exist.

CL is observed in the liquid, gas, and solid phases. In the last decade, there has been growing interest in CL as a detection technique for quantitative analysis, particularly in the liquid (aqueous) phase [2, 3], which will be solely dealt with in this chapter, because of the excellent sensitivity and wide dynamic ranges that can be achieved by using relatively simple and inexpensive instrumentation.

This chapter focuses on analytical CL methodologies, with emphasis on the kinetic connotations of typical approaches such as the stopped-flow, the continuous-addition-of-reagent (a new kinetic methodology) and the pulse perturbation technique developed for oscillating reactions, among others. Recent contributions to kinetic simultaneous determinations of organic substances using CL detection (kinetometric approaches included) are also preferentially considered here.

2. KINETIC ASPECTS OF CHEMILUMINESCENCE

One key aspect of CL techniques is the transient signal that results from the underlying spectroscopic, chemical, and physical kinetics (see Fig. 1). This is primarily the result of the spectroscopic phenomenon (i.e., the emission of light by a molecule in an excited electronic state on return to its ground state) being intrinsically kinetic in nature. Because excited CL states are produced by a chemical reaction, chemical kinetics is also involved in the process. In fact, the intensity of the CL signal, lCL, is related to the reaction rate, v, via the CL quantum yield, φ. The way the ingredients of a CL reaction are mixed in an aqueous medium also influences the CL signal, particularly its initial portion. As a result, the physical kinetics inherent in the fluid dynamics must also be considered.

The typical profile of a CL transient signal (a plot of CL intensity vs. time) is a kinetic response curve that corresponds to a first-order sequence of two consecutive steps, namely: (1) generation of the light-emitting product by mixing of the chemical ingredients (the substrate and oxidant), and (2) formation of the end product (Fig. 2). The rate at which each step takes place depends on the formation

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Figure 1 Scheme of the spectroscopic, chemical, and physical kinetics involved in the CL signal (ICL, intensity of the CL signal; v, reaction rate; φ, CL quantum yield).

Figure 2 Schematic depiction of the kinetic foundation of the CL signal.

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and decay rate constants, k1 and k2, which correspond to the rising and falling portion, respectively, of the transient signal. Physical kinetics plays a role in the prior mixing process, whereas chemical and spectroscopic kinetics are involved in both steps.

The relationship between CL intensity and time is expressed by a kinetic equation including the reaction rate constants and the substrate concentration. Such is the case with the specific equation for the CL of the luminol reaction, which is one of the most widely studied in this context:

ICL k(substrate)	k1 k2	(e k 2 t e k 1 t)	(1)
	k1 k2

The CL phenomenon possesses three distinct analytical connotations, namely: (1) the chemical system, where the analyte may be directly or indirectly involved (for example, in immunoassay); (2) the way the process is implemented (i.e., whether a conventional or an innovative methodology is used); and (3) the measured parameters.

The formation of a light emitter (i.e., an excited product) can be accomplished by mixing the substrate and oxidant in the presence or absence of a catalyst or cofactor. The ingredients can also be electrochemically generated in situ, using so-called ‘‘electrogenerated CL’’ (ECL).

One must distinguish between two different alternatives: direct and indirect CL. In direct CL, the excited product returns to its ground state to give the starting product and emitted light. In indirect CL, also referred to as ‘‘energy-transfer process,’’ the excited product interacts with a fluorophore, which must be a fluorescent molecule, to form the product; simultaneously, the fluorophore is promoted to its excited state, from which it subsequently returns to its ground state with light emission.

In direct CL methods, the target analyte can be the substrate, the oxidant, or the catalyst. In some cases, the analyte can also be an inhibitor that decreases the intensity of the CL signal. This has helped expand the scope of direct methodologies, which was formerly restricted to the few available CL reactions. In indirect CL methods, the analyte is usually the fluorophore. These methods have a broader scope than their direct counterparts as a result of the wide variety of— mainly organic—substances that can act as fluorophore, either as such or following derivatization into fluorescent molecules.

The most widely used chemical systems in direct CL methods are based on classical substrates such as luminol, lucigenin, lophine, and pyrogallol, which are oxidized with hydrogen peroxide or dissolved oxygen [4, 5]. In these CL reactions, free and complexed metal ions (Cu, Co, Fe, Mn), and peroxidases, have prominent catalytic effects; also, some inorganic anions and organic ligands for metal catalysts have substantial inhibitory effects. Most of the target analytes

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used in this context are either the catalyst (a metal ion) or inhibitor (an organic compound); others are the oxidant or even the substrate of the CL reaction. In this context, it is worth mentioning immunoassay, which uses CL detection as an advantageous alternative to radioimmunoassay. A number of CL labels have been proposed for this purpose [6].

The most commonplace substrates in energy-transfer analytical CL methods are aryl oxalates such as bis(2,4,6-trichlorophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl) oxalate (DNPO), which are oxidized with hydrogen peroxide [7, 8]. In this process, which is known as the peroxyoxalate-CL (PO-CL) reaction, the fluorophore analyte is a native or derivatized fluorescent organic substance such as a polynuclear aromatic hydrocarbon, dansylamino acid, carboxylic acid, phenothiazine, or catecholamines, for example. The mechanism of the reaction between aryl oxalates and hydrogen peroxide is believed to generate dioxetane-1,2-dione, which may itself decompose to yield an excited-state species. Its interaction with a suitable fluorophore results in energy transfer to the fluorophore, and the subsequent emission can be exploited to develop analytical CL-based determinations.

Although the ECL phenomenon is associated with many compounds, only four major chemical systems have so far been used for analytical purposes [9, 10], i.e., (1) the ECL of polyaromatic hydrocarbons in aqueous and nonaqueous media; (2) methods based on the luminol reaction in an alkaline solution where the luminol can be electrochemically produced in the presence of the other ingredients of the CL reaction; (3) methods based on the ECL reactions of ruthenium(II) tris(2,2′-bipyridine) complex, which is used as an ECL label for other non-ECL compounds such as tertiary amines or for the quantitation of persulfates and oxalate (this is the most interesting type of chemical system of the four); and (4) systems based on analytical properties of cathodic luminescence at an oxide-coated aluminum electrode.

Before dealing with specific analytical CL methodologies, it is worth commenting on the parameters typically used to relate the transient signal to the analyte concentration in the sample. Such parameters can be of the classical type (e.g., peak height and peak area, which are kinetic in nature). It should be noted that the intrinsically kinetic nature of the CL signal requires using special systems to acquire and process data. In this context, CL chemometrics has opened up interesting avenues for development, some of which are discussed below.

In addition to conventional measured parameters (peak height or area under the CL response signal), one can use typically kinetic parameters such as CL formation and decay rates, both of which are directly related to the analyte concentration. These parameters can be easily determined from the straight segments of the rising and falling portions of the response curve, using a computer to acquire and process data. These alternative kinetic parameters result in improved selectivity and precision in CL analyses, as shown in Sec. 3.2.

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3. ANALYTICAL METHODOLOGIES

This section deals briefly with classical methods based on conventional mixing of the sample and reagents such as the batch mode and low-pressure flow mixing methods, as well as the use of CL detection in continuous separation techniques such as liquid chromatography and capillary electrophoresis for comparison with the unconventional mixing mode.

Analytical CL methodologies share a number of advantages, including: (1) low detection limits (in the nanogramor even subnanogram-per-milliliter region); (2) wide dynamic ranges (up to six orders of magnitude); (3) high signal- to-noise ratios resulting from the absence of a light source and the consequent absence of noise; (4) absence of Rayleigh and Raman scattering; (5) instrumental simplicity and affordability; and (6) absence of toxic effects from the usual CL reagents.

On the other hand, the most severe constraint of CL analyses is their relatively low selectivity. One major goal of CL methodologies is thus to improve selectivity, which can be accomplished in three main ways: (1) by coupling the CL reaction to a previous, highly selective biochemical process such as an immunochemical and/or enzymatic reaction; (2) by using a prior continuous separation technique such as liquid chromatography or capillary electrophoresis; or (3) by mathematical discrimination of the combined CL signals. This last approach is discussed in Sec. 4.

3.1 Conventional Mixing

Batch CL analyses are conducted by using commercially available ‘‘chemiluminometers.’’ The last ingredient of the CL reaction is injected, by means of a syringe, into a reaction vessel accommodated in a light-tight housing placed in front of a mirror. In many cases, a filter or monochromator is inserted between the sample light source and detector. The detector is usually a classical highvoltage photomultiplier tube (PMT) or, increasingly frequently, a low-power single photodiode or a linear two-dimensional photodiode array (referred to as a charge coupled device, CCD), which provides a CL spectrum [11, 12]. Chemiluminometers also include a signal processor, which can be connected to an analog-to-digital converter or a computer to obtain an oscilloscope-type fast recording or printout, respectively. The main drawbacks of the batch CL mode are that mixing is not efficient enough and the process is difficult to automate. On the other hand, its most salient advantage is simplicity.

Flow injection methodologies are highly suitable for implementing CL analyses using low-pressure continuous mixing. There are many reported applications of this type including immobilized reactants [13] or enzymes [14]. One recent example is the flow injection manifold used for the determination of poly-

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nuclear aromatic hydrocarbons [15], which act as fluorophores in an energy-trans- fer CL process. The sample is inserted into an acetonitrile stream that is merged with another resulting from the continuous mixing of aryl oxalate (DNPO) and hydrogen peroxide solutions. The merging point is near a planar coiled quartz flow cell, where the reaction takes place, which is located in front of a low-power photodiode. The detection limits thus achieved range from 0.6 to 70 ng/ml and the system is quite useful for screening purposes.

The flow injection/CL coupling provides a number of interesting advantages including: (1) automated mixing; (2) easy online coupling to automated sample treatment systems; (3) ready adaptation to the requirements of CL reactions thanks to the high flexibility of the flow injection technique; (4) low cost;

(5) easy assembling from available parts; and (6) commercial availability. However, this combined approach also has some drawbacks, such as: (1) medium to low mixing efficiency owing to the low pressure in the merging streams; (2) high dead volumes resulting from the mixing point being located outside the flow cell; and (3) no selectivity improvement relative to the conventional batch mode.

The lack of selectivity can be circumvented by coupling a postcolumn flow system to a liquid chromatograph. This has promoted the development of a number of efficient liquid chromatography-CL approaches [16, 17]. Eluted analytes are mixed with streams of the substrate and oxidant (in the presence or absence of a catalyst or inhibitor) and the mixed stream is driven to a planar coiled flow cell [18] or sandwich membrane cell [19] in an assembly similar to those of flow injection–CL systems. Many of these postcolumn flow systems are based on an energy-transfer CL process [20]. In others, the analytes are mixtures of metal ions and the luminol–hydrogen peroxide system is used to generate the luminescence [21].

In addition to its high sensitivity, automated mixing capability, and easy assembly, liquid chromatography–CL monitoring has the advantage of the high selectivity resulting from efficient discrimination on the chromatographic column relative to flow injection configurations. On the other hand, the medium-to-low mixing efficiency and high dead volumes of this coupled approach add up to compatibility problems of CL reactions with chromatographic mobile phases and to a high cost of the equipment required.

Capillary electrophoresis–CL detection [22] is an effective marriage of convenience where the main drawbacks of each partner are offset by the advantages of the other. In fact, the high discriminating capacity of capillary electrophoresis more than compensates for the low selectivity of CL reactions. Conversely, the high sensitivity of such reactions compensates for the low sensitivity of its partner. Despite its promising prospects, this coupling is not yet mature enough for use in routine analyses. In fact, the interfaces involved (mixing points, the CL cell) are rather complicated to implement and result in scarcely robust approaches. Also, commercially available equipment is scant. By contrast, a large

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number of capillary electrophoresis–CL systems have been reported [23], some of which include ECL detection [24]. By way of example, two recent applications, both of which use a metal-catalyzed luminol system, have been proposed. One is the resolution of a mixture of amino acids in an aspirated sample where the analytes act as CL inhibitors by complexing copper(II) ion dissolved in the buffer carrier, which is mixed in a capillary with a stream of luminol and hydrogen peroxide near the CL detection cell [25]. The other is the resolution of a mixture of metal ions in an aspirated sample [26]. A weak acid buffer carrier containing luminol and a ligand is used to form soluble complexes with the analytes to avoid precipitation. A stream of alkaline hydrogen peroxide is merged with the carrier in the capillary, near the CL detection cell.

3.2 Stopped-Flow Technique

The above-described drawbacks inherent in the conventional mixing mode are circumvented by using appropriate CL assemblies based on the high-pressure stopped-flow, the continuous-addition-of-reagent, or the analyte pulse perturbation technique. The first two have largely been used in kinetic methods involving fast reactions as they are particularly well suited to the monitoring of transient signals such as those produced by a CL reaction. These approaches meet the two principal requirements of CL analyzers, namely: (1) rapid, highly efficient mixing of sample and reagents; and (2) rapid detection of the light emitted after the CL reaction has started.

The combination of the high-pressure stopped-flow technique and a CL reaction is a new approach called ‘‘stopped-flow CL spectrometry’’ (SF-CLS) [27] that allows one to obtain entire CL profiles, which is impossible with continuous and flow injection systems owing to the fugacity of the CL signal. Figure 3 depicts a modular instrument for implementing high-pressure stopped-flow CL analyses. The sample and reagents are mixed at a high pressure from two propelling syringes actuated by manual or automatic pneumatic pulses in a flow cell or mixing chamber that also acts as the observation cell [28]. The cell is accommodated in the detector cell compartment of a commercially available spectrofluorimeter. When the flow is abruptly stopped by actuating the stopping syringe, the variation of the CL signal as a function of time is recorded and the data thus obtained are processed by a computer to deliver the analytical results [27]. Both the flow cell and the detector cell compartment are thermostated. The spectrofluorimeter’s light source is maintained off throughout the process.

This approach has several advantages with regard to the above-described conventional mixing mode including rapid, highly efficient mixing of sample and reagents; immediate detection of the CL signal; the ability to record the whole CL intensity-versus-time profile, even for extremely fast reactions; and the ability to use new measured parameters such as formation and decay rates. These param-

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Figure 3 Instrumental setup used to implement SF-CLS by using a modular stoppedflow device for mixing the sample and reagents.

eters are obtained from the slopes of the straight portions shown in Figure 4, both of which are proportional to the analyte concentration. The use of these kinetic parameters provides some advantages over conventional peak height and peak area measurements, specifically [27]:

1.They contain a greater amount of useful information for optimizing variables and performing mechanistic studies.

2.They provide a more accurate method for the simultaneous determination of formation and decay rate constants using curve-fitting software.

3.The use of formation and decay rates as measured parameters results in improved precision, selectivity, and throughout, which facilitates application to routine analyses.

By using the modular stopped-flow/CL approach and the above-mentioned kinetic parameters, analytes such as hydrogen peroxide [27], hypochlorite [29],

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Figure 4 CL profile provided by a modular stopped-flow device showing the portions in which formation and decay rates are measured.

periodate [30], and manganese(II) [31], which take part in the luminol reaction, can be advantageously determined in terms of sensitivity, throughput, and precision (see Table 1).

Luminol in a carbonate buffer solution at pH 10 is used to fill one of the drive syringes of the stopped-flow system, and the analyte in the sample to fill the other. The maximum emission wavelength is 425 nm and the operating temperature 20–25°C. Worth special note is the stopped-flow determination of manganese(II) with luminol, which was carried out in the absence of hydrogen peroxide, dissolved oxygen acting as the oxidant since the reactants were mixed at a high pressure so the activity of dissolved oxygen was very high and comparable to that of an oxidant such as hydrogen peroxide. Under these conditions, high concentrations of sodium chloride enhance the CL emission and provide a very sensitive method for the determination of manganese [31]. This enhancing effect can be ascribed to catalytic cleavage of a potential Mn(II)-O2-luminol-activated complex resulting in a chloride-manganese interaction in the presence of dissolved oxygen via the following reaction [31]:

MnCl42 O2 ↔ MnCl4 H2 O

This reaction is consistent with: (a) the observed enhanced reactivity of chloride ion toward manganese relative to other halide ions (e.g., bromide ion); and (b) the presence of H2 O species, which is indispensable for the subsequent formation

Table 1	Analytical Figures of Merit and Applications of Low-Pressure Stopped-Flow CL Spectrometry in Routine Analyses
							Linear dynamic									Detection		Sampling
Analyte		System							range, M							limit, M		frequency, h	1	RSD, %	Applications	Ref.
Direct determinations
Hydrogen peroxide		Luminol-Co(II)					10	8	–5 10				5			25 pmol		60		1.7		27
Hypochlorite		Luminol					7.5–5000 ng/mL											80		1.0	Tap water	29
Periodate		Luminol-Mn(II)-TEA					10	6	–2.5			10		4		1.2 10	7	100		0.7		30
Manganese(II)		Luminol-O		-TEA			2		10	7	–10		5			1.8 10	8	100		0.9	Biological materials	31
			2
Indirect determinations
Penicillins		Luminol-l	2				10	6	–1.2			10		4				120		2.0	Pharmaceuticals	32
Tartrate		Luminol-IO		4		-Mn(II)-TEA	5		10	6	–5			10	5	1.5 10	6	—		1.8	Pharmaceuticals	30
TEA, triethanolamine; RSD, relative standard deviation.

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