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One of the most severe shortcomings of PO-CL reactions is their relatively low selectivity; this problem can be overcome in two ways: by physical discrimination (i.e., by sequentially separating the analytes prior to their determination), or by mathematical discrimination, the usefulness of which is restricted to only a few components. The former approach is the more interesting; in fact, PO-CL is widely used as very sensitive detection method for HPLC [46, 47]. The latter approach is a novel alternative in CL analysis; it is discussed in detail in Sec. 4.

However, an essential condition to ensure high sensitivity with PO-CL detection in HPLC is that the reactants (aryl oxalate and hydrogen peroxide) should be mixed with the column eluate at a point as close to the PMT as possible. In other words, a zero-dead-volume detector is required to avoid losses of emitted light and hence of sensitivity. Reported devices for PO-CL detection based on flow systems have non-zero-dead volumes, so some compromises must be made in their optimization. Based on the principles and technical aspects of the CAR technique, our group has developed a zero-dead-volume integrated derivatization CL detection system coupled to a liquid chromatograph [48]. This combined system circumvents the shortcomings of conventional postcolumn flow CL detection systems. As can be seen in Figure 9, one such system consists of a standard 1.0-cm spectrofluorimetric quartz cell in which the two reactant solutions (a) aryl oxalate in ethyl acetate and b) hydrogen peroxide, Tris buffer in an isopropyl alcohol:water medium, delivered by a peristaltic pump, and the eluate from the column are mixed. All three are simultaneously delivered and an additional chan-

Figure 9 Schematic depiction of the zero-dead-volume CL detection system for liquid chromatography. (From Ref. 48.)

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nel is used to keep the volume of the reaction mixture in the cell constant. The resulting CL response is monitored in real time by the PMT. Isopropyl alcohol is used as cosolvent to improve the solubility of the aryl oxalate solution (ethyl acetate) in water. This device allows more flexible manipulation of the reactant concentrations and flow rates relative to the mobile phase.

The analytical potential of this zero-dead-volume detection system was evaluated in the HPLC determination of polycyclic aromatic hydrocarbons (PAHs) [48]. The cell volume, or, specifically, the volume of the reaction mixture in the cell, was found to be the most influential variable on its performance. Thus, the minimum volume used to ensure reproducible results was 100 L, and a reaction volume of 350 L was recommended as a compromise between sensitivity and band broadening (peak width only 1.5 min) in the determination. Various PAHs were thus determined over a wide concentration range (0.2–6000 ng), with good precision (RSD 3.5–5.7%). The ensuing method is more sensitive than the gas chromatography/mass spectrometry tandem and supercritical fluid chromatography with high-resolution laser-induced fluorescence detection in PAH determinations. This integrated derivatization-CL detection system has also been used for the HPLC determination of hallucinogenic alkaloids at the picomole to nanomole level using DNPO as aryl oxalate reagent in this case [49]. The method allows the determination of these alkaloids (the identification and quantification of some were reported for the first time) in Heliconiini butterfly specimens with good results.

In summary, this zero-dead-volume integrated CL detection approach provides two major advantages, namely:

1.Increased sensitivity by virtue of the high hydrogen peroxide-to-aryl oxalate ratios used, which facilitate suppression of background emission and thus raise the signal-to-noise ratios.

2.a zero-dead time between mixing of the reactants and reaction with the fluorophore/analyte, which is unfeasible in a typical flow system. This results in increased efficiency of the PO-CL reaction and avoids the usual instability of some aryl oxalate solutions (e.g., those of DNPO) in the presence of hydrogen peroxide.

3.4Analyte Pulse Perturbation (with oscillating reactions)

The last unconventional approach considered in this chapter is low-pressure ‘‘analyte pulse perturbation-CL spectroscopy’’ (APP-CLS). This approach is highly dynamic as it relies on the combination of an oscillating reaction, which is a particular case of far-from-equilibrium dynamic systems, and a CL reaction.

The APP technique, recently introduced [50, 51], uses a continuous stirring tank reactor (CSTR) and relies on the sequential perturbation of an oscillating

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reaction by successive addition of analytes (or standards) after regular oscillations are restored. The perturbation results in a change in the oscillation period or amplitude the magnitude of which is proportional to the analyte concentration. Provided the optimum experimental conditions are maintained, the system remains in an oscillating state, acting as a continuous indicator system, for at least 8 h. This provides a rapid, simple method for performing many determinations on the same oscillation system. This operating mode offers obvious advantages over classical discrete systems and endows the APP technique with a high practical potential.

The potential of the APP technique with CL detection has been assessed by coupling the following reactions [52]: the Epstein-Orban chemical oscillator reaction, the ingredients of which are hydrogen peroxide, potassium thiocyanate, and copper sulfate in the presence of sodium hydroxide, and the CL reaction involving the oxidation of luminol by hydrogen peroxide, catalyzed by copper(II) in alkaline medium. In this case, the CL reaction acts as detector or indicator system of the far-from-equilibrium dynamic system. In fact, the CL oscillating response may be due to interaction between the yellow hydroxyl radical–cuprous ion complex involved in the positive-negative feedback loop of the oscillating reaction and luminol to give the light-emitting form of luminol according to the following reaction:

HO2 Cu(I)yellow LumH → Cu2 2OH Lum*

(6)

When this oscillating system is perturbed by a pulse of an analyte such as vitamin B6, it undergoes a change in its amplitude or period (amplitude for this vitamin) that is proportional to the concentration and can be used to construct a calibration plot.

Figure 10 shows the instrumental setup used to implement the APP-CLS approach. It consists of: (a) a CSTR that is a thermostated 10-mL glass reaction vessel accommodated in a commercially available spectrofluorimeter (a Hitachi F2000 model in this case); (b) a four-channel peristaltic pump with three channels used to dispense the reagent solutions and the fourth to keep the volume of the reaction mixture in the CSTR constant; the three reagent solutions are as follows:

(1) 0.15 M hydrogen peroxide; (2) 0.15 M sodium thiocyanate, 0.15 M sodium hydroxide, and 1.95 10 3 M luminol; and (3) 6.0 10 4 M copper(II) sulfate;

(c)an autoburette from which pulses (µL) of the analyte solution are added; and

(d)a compatible computer for recording and processing the CL oscillating signal. Figure 11 shows typical CL oscillating responses of this system as per-

turbed by vitamin B6 pulses, which decrease the oscillation amplitude. Arrowheads indicate the times at which analyte pulses were introduced. Zone A corresponds to the oscillating steady state; zone B to the response of the oscillating system to vitamin B6 perturbations; and zone C to the recovery following each perturbation (second response cycle), which was the measured parameter. This

Figure 10 Experimental setup for implementation of CL oscillating reaction-based determinations. CSTR, continuous stirring tank reactor. (From Ref. 52.)

Figure 11 Typical profiles for the H2 O2-KSCN-CuSO4-NaOH-luminol CL oscillating reaction in the presence and absence of a vitamin B6 perturbation. Arrows indicate the times at which oscillations were perturbed. (From Ref. 52.)

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system allows determination of this vitamin at the mol level (0.5–20) with good precision (RSD, 3%) and acceptable throughput (9 samples/h). After evaluating potential interfering effects from structurally related species on the performance of the method, this was satisfactory validated by determining the vitamin in real samples such as pharmaceutical preparations.

4.RECENT APPROACHES TO MULTICOMPONENT CHEMILUMINESCENCE-BASED DETERMINATIONS

As stated in previous sections, the use of kinetic CL-based determinations has grown considerably in recent years as a result of their high sensitivity and expeditiousness, and also of their instrumental simplicity. In this context, SF-CLS and CAR-CLS have been two important supports for development as they provide kinetic information of a higher quality than does the low-pressure continuousflow alternative. Despite these developments, the low selectivity achieved in some CL-based determinations restricts their scope. Although this problem can be solved by using coupled CL reactions as detection systems, with chromatographic techniques, such as liquid chromatography (and, recently, capillary electrophoresis), a more inexpensive alternative exists that solves the analytical problem with major practical advantages. This section deals with the potential of kinetic methods of analysis for multicomponent determinations based on CL-time primary data.

Multicomponent CL-based determinations are based on different principles depending on the particular system used to mix the sample and reagents to generate the CL signal, and also on the half-life of the CL reaction involved. Thus, when the CL reaction is carried out in a low-pressure flow system (i.e., by using the so-called CL-FIA), the manifold configuration is the actual key to accomplishing discrimination among the analytes in the sample. In this case, an instrumental approach, rather than differences in reaction rate among the analytes, enables the multicomponent determination, even though some kinetic aspects are involved owing to the transient nature of the CL signal. In other cases, a kinetic discrimination is the actual basis for the multicomponent CL-based determination, which can be approached in two ways depending on the relative half-lives of the CL reactions for each analyte in the mixture. Thus, when the measured CL intensity for each analyte peaks at a very different time, the sequential determination is feasible and the approach is designated time-resolved CL. When the kinetic behavior of the mixture components is very similar, then the resulting complex dynamic system is generally modeled with computational aids based on mathematical algorithms. This field of kinetic methods of analysis, which combines chemometric and kinetic aspects, has recently been termed kinetometrics [53].

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4.1 Instrumental Discrimination

Although the subject of CL in FIA is treated elsewhere in this book, it might be of interest to briefly comment on the most salient features of the resolution of mixtures using these flow systems. Thus, the simultaneous flow injection determination of acetylcholine and choline [54], and that of lysine and glucose [55], are based on measurements of the hydrogen peroxide produced from the analytes in enzyme reactions, which is detected by CL using the luminol-cobalt(II) system. The enzyme reactors (used to generate the active species in the CL reaction) and the delay coils (employed to resolve CL signals in time for their sequential detection) before the detector allow the mixture to be successfully resolved (Fig. 12a). A similar principle has also been used for the speciation of metal ions such as ferric-ferrous ions [56], and chromium(III) and chromium(VI) [57] (see Fig. 12b). Because only iron(II) and chromium(III) are catalytically active on the luminoldissolved oxygen and luminol-H2 O2 reaction, respectively, the resolution of the

Figure 12 General flow injection manifold used for the simultaneous determination of

(a) organic species involved in enzymatic reactions; and (b) inorganic ions, using a reduction column both with CL detection. IV, injection valve.

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mixtures involves two steps: first, the iron(II) and chromium(III) are directly determined; and second, the sample is passed through a reduction column (copper-plated zinc granules and copper-coated zinc granules, respectively) to convert iron(III) to iron(II) and chromium(VI) to chromium(III). CL is again measured and the concentrations of oxidized species are determined by difference. The methods provide good analytical performance; e.g., iron can be determined at the nM level with a high sample throughput (60 runs/h); they were validated with real samples such as cobalt in waste water from a chromiumplating factory.

Sequential injection analysis (SIA) is a further development of flow injection methods [58] with a high potential for simultaneous determinations. An SIA system with CL detection typically consists of a double-piston pump, a multiposition valve, a holding coil, enzyme reactors (for enzymatic determinations), and a PMT detector [59]. Basically, two operations are performed: (1) the sample and reagents are aspirated in a given sequence into the holding coil; and (2) the plug of partially layered reagents and sample is dispensed to the detector. Between the first aspiration of sample and reagents into the holding coil and the final dispensing of the well-mixed plug to the detector, there may be several intermediate stages to develop additional suitable reactions—enzymatic reactions, stopped-flow, etc.—and back into the holding coil. This approach has mainly been used for the simultaneous determination of species based on enzymatic reactions such as those of glucose-lactic acid-penicillin [59], glucosepenicillin [60], and H2 O2-glucose [61], using the CL luminol system with good analytical results and minimal reagent consumption.

4.2 Time-Resolved Chemiluminescence

This approach uses a kinetic sequential principle to carry out multicomponent CL-based determinations. In fact, when the half-lives of the CL reactions involved in the determination of the analytes in mixture are appreciably different, the CL intensity-versus-time curve exhibits two peaks that are separate in time (in the case of a binary mixture); this allows both analytes to be directly determined from their corresponding calibration plots. In general, commercially available ‘‘chemiluminometers’’ have been used in these determinations, so the CL reaction was initially started by addition of one or two reaction ingredients. Thus, in the analysis of binary mixtures of cysteine and gluthatione, appropriate timeresolved response curves were obtained provided that equal volumes of peroxidase and luminol were mixed and saturated with oxygen and that copper(II) and aminothiol solutions were simultaneously injected [62, 63].

Table 5 shows the most salient features of reported methods for multicomponent CL-based determinations using time-resolved CL spectroscopy. As can be seen, the CL luminol system has been widely used for this purpose because

Table 5	Multicomponent CL-Based Determinations Using Time-Resolved CL Spectroscopy
								Dynamic	Precision
Mixtures		CL chemical system						range ( M)	(RSD, %)	Comments	Ref.
Cysteine/glutathione		Luminol-peroxidase-Cu(II)						1–50/3–50	2.5/2.8	Solutions were saturated with	62, 63
										oxygen
Cobalt(II)/copper(II)		Luminol-H	2	O		-cysteine		0.5–10/2–20	2.5/3.0	Metal ion solutions were satu-	64
					2
										rated with oxygen
Gold(III)/silver(I)		Luminol-K	2	S	2	O	-ethylenediamine	0.05–5/0.1–10	—/—	A YHF-1 liquid CL analyzer	65
						8
										was used
Ascorbic acid/tartaric		Tris(bipyridyl)ruthenium(II)-Ce(IV)						0.05–1.7/1.4–140	4.8/1.7	The method was applied to	66
acid										wine samples
Oxalic acid/tartaric		Tris(bipyridyl)ruthenium(II)-Ce(IV)						0.14–14/2.9–150	2.4/4.8	The method was applied to syn-	67
acid										thetic urine

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it provides appropriate time-resolved response curves (i.e., CL curves in which the two peaks are well apart in many cases). Thus, in the simultaneous determination of cobalt(II) and copper(II) with the luminol-hydrogen peroxide-cysteine system, light emission appearing immediately in the presence of cobalt(II) and after 7 min in that of copper(II) [64]. Although this situation is less favorable with other CL systems such as the tris(bipyridyl)ruthenium(II)-Ce(IV) one, where the CL intensity for ascorbic and tartaric acids peaks at 2 and 40 s, respectively, the multicomponent determination is also feasible [66]. On the other hand, the linear dynamic ranges for the analytes in Table 5 are in the M region and precision is quite good. In some cases, a short half-life in the first peak results in poor reproducibility; such is the case with the above-mentioned determination of ascorbic acid in mixtures with tartaric acid [66], where the RSD was 4.8% versus 1.7% for tartaric acid.

4.3 Kinetometric Approaches

One other way of improving selectivity in kinetic CL-based determinations is by mathematical discrimination using kinetometric approaches. In general, chemical systems involved in multicomponent CL-based determinations have drawbacks that make the use of statistical methods inadvisable. Thus, the differential equations that describe these chemical systems are unknown; also, synergistic effects are often present. Because of this marked nonlinearity, only powerful chemometric tools provide adequate accuracy to resolve these mixtures. Among them, least-squares matrix methodology [68] and computational neural networks (CNNs) [69] have been used for this purpose. On the other hand, when the CL system behaves linearly, various approaches allow one or more components in mixtures to be determined from kinetic differences. Such is the case with the resolution of mixtures of oxalate and proline with electrogenerated tris(bipyri- dine)-ruthenium(III) [70], in which a differential reaction rate method based on least-squares regression of the first decay data obtained by the stopped-flow technique was used to resolve the analytes. The method should be useful for the simultaneous determination of unresolved analytes of pharmaceutical and environmental interest.

Kinetic analysis usually employs concentration as the independent variable in equations that express the relationships between the parameter being measured and initial concentrations of the components. Such is the case with simultaneous determinations based on the use of the classical least-squares method but not for nonlinear multicomponent analyses. However, the problem is simplified if the measured parameter is used as the independent variable; also, this method resolves for the concentration of the components of interest being measured as a function of a measurable quantity. This model, which can be used to fit data that are far from linear, has been used for the resolution of mixtures of protocatechuic

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and caffeic acids, which act as enhancers in the CL reaction of luminol induced by hexacyanoferrate(III) [68]. The phenolic acids can be quantified both separately and together—in the presence of synergistic effects—using the stoppedflow technique and monitoring the intensity at 0.3 s and the initial rate. A twofactor factorial design was used to obtain the experimental response and a firstorder equation was employed to fit it as closely as possible. The method allows the determination of the analytes in mixtures in concentration ratios ([protocatechuic acid]:[caffeic acid]) from ca. 1:3 to 2:1 with good sensitivity (at the nM level) and recoveries between 95 and 112%.

CNNs are among the most exciting recent developments in computational science [71] and have grown enormously in popularity in different scientific fields including analytical chemistry [72–74]. However, CNNs have scarcely been used in connection with kinetic methods of analysis; our group has employed this kinetometric approach for the estimation of kinetic analytical parameters [75] and also in multicomponent kinetic analyses with photometric detection [76–78]. Based on the good results achieved with these methods, and taking into account that multilayer feed-forward neural networks based on different versions of standard back-propagation learning algorithm have been used by several authors as highly powerful tools to study uniform approximation of an unknown continuous function that can be derived up to a p-th order (CP-function), we developed a new methodology for multicomponent CL-based determinations.

The simultaneous determination of trimeprazine and methotrimeprazine in mixtures using the classical peroxyoxalate system based on the reaction between TCPO and hydrogen peroxide was used to validate the new methodology. The reaction was implemented by using the CAR technique, which increased nonlinearity in the chemical system studied by virtue of its second-order kinetic nature. In addition, both drugs exhibited a similar kinetic behavior and synergistic effects on each other, as can be inferred from the individual and combined (real and theoretical) CL-versus-time response curves.

One important kinetic problem in this simultaneous determination is selecting an appropriate time domain of the CL curve as the source of inputs to CNN. In this case, the optimal interval of the CL signal-versus-time plot for data preprocessing (selection of CNN inputs) is the initial concave portion of the plot and part of the linear portion (see Fig. 13). When the linear portion is wider, the additional information collected does not contribute to increased discrimination. Using this time domain, the filtration step (data preprocessing) was carried out by using principal component analysis (PCA), a widely employed technique for reducing the dimensions of multivariate data while preserving most of the variance. By using a heuristic method, nine significant principal components were selected as input to CNNs, the final architecture being 9:5s:2/ (see Fig. 13).

Under these conditions, and to test the generalizing capacity of the selected network for the simultaneous determination of phenothiazine derivatives subject