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of the CL system. The presence of manganese(III) in the reaction medium is commonplace in manganese-catalyzed reactions.

Based on the analytical figures of merit of the methods in Table 1, the best precision and selectivity are accomplished by using the decay rate rather than the formation rate or conventional CL-measured parameters such as the peak height or area under the CL curve. Table 2 gives the selectivity factor, expressed as decay-rate and peak-height tolerated concentration ratio, for the CL determination of hydrogen peroxide using SF-CLS. As can be seen, the selectivity factor was quite favorable in most instances.

The luminol reaction has also been used for the CL determination of organic substances such as penicillins [32] and tartrate ion [30] in pharmaceutical preparations by their inhibitory effect on the luminol-iodine and luminol-periodate- manganese(II)-TEA system, respectively. As can be seen from Table 1, the results were quite satisfactory. In the indirect determination of penicillins by their inhibitory effect on the luminol-iodine system, the stopped-flow technique improves the accuracy and precision of the analytical information obtained, and also the sample throughput [32]. Thus, in only 2–3 s one can obtain the whole CL signal- versus-time profile and calculate the three measured parameters: formation and

Table 2 Selectivity of Various Measurement Methods Used in the Determination of 20 ng/mL Hydrogen Peroxide

Tolerated concentration, ng/mL

	Decay	Peak	Selectivity
Species tested	rate, VD	height, h	factor, VD/h
Fe(III)	60	20	3.0
Cu(II)	80	20	4.0
Cr(III)	30	10	3.0
Mn(II)	10	10	1.0
Ni(II)	75	30	2.5
Hg(II)	2000	2000	1.0
Ce(IV)	100	50	2.0
MnO4	100	30	3.3
[Fe(CN)6]3	3000	1000	3.0
ClO	120	60	2.0
S2	1000	500	2.0
SO32	1000	500	2.0
NO2	2000	2000	1.0

Source: Adapted from Ref. 27.

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decay rates, and peak height. From these, penicillins such as penicillin G, ampicillin, amoxycillin, and carbenicillin can be determined.

Many of these CL reactions are very fast, so they require the use of the stopped-flow technique. Analytical results can be obtained within seconds, so the sampling frequency is quite high. Figure 5 illustrates the short time needed for the maximum peak in the CL curve to be reached in the previous examples. Such a time is 500 and 700 ms in the hydrogen peroxide and hypochlorite determinations, respectively, and 200 ms in the periodate one. The fastest CL reactions are those of the luminol-dissolved oxygen-Mn(II) and luminol-iodine systems for the determination of manganese and penicillins, respectively. The entire transient CL signal can be acquired within about 300 ms and the maximum peak height is reached at ca. 50 ms.

Other high-pressure stopped-flow/CL systems are based on special mixing modes, the main technical difference of which is that the CL observation cell

Figure 5 CL-versus-time responses for various systems provided by the stopped-flow technique.

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and mixing point are not integrated; in fact, detection takes place at a chamber located before the observation flow cell [33, 34], as shown in Figure 6. Figure 6a depicts a system similar to that of Figure 3 but based on conventional propel- ling—three instead of two—and stopping syringes; however, the mixer and flow cell are separate [33]. In the second scheme (Fig. 6b), the mixer and flow cell are also separate, but three liquid chromatographic pumps—instead of the typical propelling syringes—are used and the stopping syringe is replaced with a valve for stopping the flow before the sample and reagent are mixed [34]. These assemblies also provide the whole CL profile and are used mainly as fitted to a liquid chromatograph in the determination of organic substances using peroxyoxalate systems.

Figure 6 Schematic diagrams of atypical high-pressure SF-CL systems based on special mixing modes: (a) system based on conventional propelling and stopping syringe; and (b) system based on liquid chromatographic (LC) pumps and stopping the flow before the mixer (M). V, six-ports valve. (Adapted from Refs. 33 and 34.)

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3.3 Continuous-Addition-of-Reagent Technique

The continuous-addition-of-reagent (CAR) technique is one other unconventional mixing mode used with CL reactions. This approach, developed in the authors’ laboratory [35], is based on the continuous addition, at low pressure and constant rate, of one reaction ingredient (usually the reagent) over a vessel containing the analyte and the other reaction ingredients. This technique is specially suited to fast reactions and offers a major alternative to high-pressure SF-CLS. In fact, it is more flexible for the kinetic study of these reactions, possesses a higher analytical potential, and uses a more affordable instrumentation [35–37]. As shown below, these features endow the CAR technique with a high analytical usefulness in CL analysis, known as ‘‘continuous-addition-of-reagent CL spectroscopy’’ (CARCLS).

In general, if an irreversible reaction, A R → P (where A is the analyte, R the reagent, and P the products), is developed by using the CAR technique (i.e.,if a solution of the reagent is added at a low pressure and constant rate u to a volume V0 of a solution containing the analyte in a reaction vessel) the overall reaction rate of the process will depend on two factors: one chemical (the reaction rate proper) and the other physical (the rate of dilution of the species present in the reaction vessel). If the reagent concentration in the addition unit, [R]0, is assumed to be much higher than that of the analyte in the reaction vessel, [A]0, and that the reagent uptake always will be much smaller than the amount of reagent added, then the integrated rate equation for this process is given by [35]:

ln	S∞ St	k[R]0 t	k[R]0 V0	ln	V0 ut	ln	V0	ut	(2)
	S∞		u					V0
					V0

where k is the second-order rate constant, and St and S∞ are the signals at time t and ∞ (total reaction development), respectively, provided the reaction is monitored via the product, using spectrophotometric detection (absorbance measurements), which is the usual choice with this technique.

The kinetic profile (signal-vs.-time plot) obtained for a chemical reaction developed by using the CAR technique is peculiar; and as can be seen in Figure 7a, it consists of three distinct portions, namely: (1) an initial, concave segment where the analytical signal is directly proportional to t2, this dependence being the basis for the so-called ‘‘initial-reaction method’’ for the determination of the analyte; (2) a wide, linear, intermediate portion in which changes in the analyte signal with time (maximum reaction rate) depend on various factors according to

dS				√	k[R]0 u
		max	K[A]0				(3)
	dt				V0

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Figure 7 Typical signal-versus-time profiles obtained from: (a) a chemical reaction carried out by the CAR technique with photometric detection; (b) a CL reaction developed by using the SF technique; and (c) a CL reaction performed by using the CAR technique to mix the sample and reagents.

where K is a constant that depends on the detection system used; this linear dependence between the maximum reaction rate and the analyte concentration is the key to kinetic analytical determinations with the CAR technique; (3) a final, convex portion that responds to the dilution of the products formed and is of little analytical interest.

In the case of a CL reaction, such as A R → P hν, the response curve corresponds to two first-order consecutive reaction steps; taking into account the possible rate equations that can be formulated for each reaction step, the integrated equation can be formulated as [27]:

SCL Ck2	[R]0		k1	(e k2 t e k1 t)	(4)
		k1
			k2

where SCL is the CL signal at any time t, k1 and k2 are the rate constants corresponding to two opposite simultaneous first-order processes, and C is a constant related to instrumental features. Figure 7b shows the typical CL profile (signal vs. time) obtained in this case, the peak height, and the area under the CL curve, which are used as measured parameters for quantitative determinations.

When a CL reaction is developed by using the CAR technique, the shape of the resulting CL signal-versus-time plots follows a differential equation that is a combination of the integrated Eq. (2) and (4), and is very difficult to obtain. However, the kinetic curve exhibits the characteristic initial concave and wide linear portions that correspond to a reaction (see Fig. 7c). Therefore, the maxi-

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mum reaction rate can also be used as measured parameter for kinetic determinations in CL analyses with CAR-CLS technique.

The instrumental setup used to implement the CAR technique in CL kinetic-based determinations is very simple and inexpensive (see Fig. 8). It consists of three basic units, such as: the addition unit, the detector housing, and the data acquisition system. The addition unit is composed of an autoburette, a cylindrical glass reaction vessel with a suitable capacity to achieve V0 of ca. 1.0 mL, and a small magnetic stirrer to homogenize the reaction mixture. The most important feature of this addition unit is that the addition rate of the autoburette can be programmed over a wide range according to the requirements (half-life) of the CL reaction under study—it can be changed from sub 0.1 to above 10 mL/min. Two alternatives can be used to detect the generated CL signal: (1) a commercial spectrofluorimeter with its light source switched off the sample holder of which is replaced with the above-mentioned small magnetic stirrer and the reaction vessel; and (2) a homemade photomultiplier (PMT) detector housing (black box) with the corresponding high voltage supply and built-in voltage divider and current-to-voltage conversion circuits, among other components. In both configurations, the distance of the reaction vessel from the PMT is critical to acquire as much emitted CL as possible, which can also be increased by using

Figure 8 Instrumental setup used to implement the CAR technique in CL kinetic-based determinations according to the detection system used: a spectrofluorimeter or a black box including a PMT.

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a mirror in front of the photomultiplier tube. Finally, the processing unit consists of a PC-compatible computer equipped with an analog-to-digital converter. A trigger allows data acquisition to be synchronized with reagent addition from the autoburette. This simple, inexpensive instrumentation—available in many analytical laboratories— is an effective tool for monitoring CL reactions with relatively short emission lifetimes because the CL signal is simultaneously monitored by the PMT during development of the reaction.

The first attempt at assessing the potential of the CAR technique for CL analysis involved determining copper by its catalytic effect on the reaction between luminol and hydrogen peroxide [38]. One important step in implementing the CAR-CLS technique is selecting the appropriate reagent for addition from the autoburette (luminol or hydrogen peroxide in this case). There is no general rule to make the decision, which is based on experimental results (signal-to-noise ratio, precision). One other important consideration in optimizing a CAR system is selecting the reagent concentration and its rate of addition from the autoburette. In fact, both variables are closely related via Eq. (3); the actual reagent concentration [R] in the reaction vessel depends on both according to

	[R]	ut[R]0	(5)
		V0

In this case, it is generally preferable to use a higher concentration of reagent (if it is soluble enough) and add it at a lower rate than the opposite to ensure more reproducible mixing in the reaction vessel.

Under these conditions, a CL intensity-versus-time curve can be obtained to determine cupric ion at the ng/mL level using the luminol/hydrogen peroxide system. Luminol, at a 3.5 10 5 M concentration, was placed in the reaction vessel and the reaction was started by adding a 1.5 M solution of hydrogen peroxide at a rate of 1 mL/min from the autoburette, under stirring. The CL signal was detected at an emission wavelength of 425 nm, using a spectrofluorimeter. The CL response curve spanned a relatively long time interval (between 1 and 30 s), and the peak height was obtained within about 6 s, which is long relative to, for example, commercially available chemiluminometers and SF-CLS (in this latter, the time needed to obtain the peak height in the determination of manganese(II) with the luminol system is only about 50 ms [31]). This can be ascribed to the special way in which sample and reagent are mixed in the CAR technique, which provides a way for CAR-CLS to modify the rate of the CL reaction. In fact, from Eq. (3) it can be inferred that the half-lives of the reactions will be directly proportional to the concentration of reagent, [R]0, and its addition rate. This facilitates data acquisition and improves the analytical performance of the ensuing analytical method. Thus, as can be seen in Table 3, the maximum reaction-rate method provides a wider dynamic linear range and better preci-

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Table 3 Comparison of the Performance of the Reaction-Rate and Peak Height Methods in the CL Determination of Copper(II) Using Luminol and the CAR Technique

	Maximum
	reaction-rate	Peak-height
Figure of merit	method	method
Linear dynamic range (ng/mL)	1–700	1–250
Detection limit (ng/mL)	0.30	0.25
Precision (RSD) (%)	2.48	3.66
Sampling frequency (h 1)	48	30

Source: Adapted from Ref. 38.

sion, throughput, and selectivity (e.g., selectivity factor of 2.0 for copper and manganese) than does the peak-height method (the classical choice in CL analysis) in the CL determination of copper.

The scope of CAR-CLS in analytical determinations has been expanded with one other type of CL reaction (luminol-based CL reactions are restricted to direct determinations of metal ions and some indirect ones). The so-called ‘‘energy transfer CL’’ is one interesting alternative, with a high analytical potential. As stated above, PO-CL systems based on the reaction between an oxalate ester and hydrogen peroxide in the presence of a suitable fluorophore (whether native or derivatized) and an alkaline catalyst are prominent examples of energy transfer CL. This technique has proved a powerful tool for the sensitive (and occasionally selective) determination of fluorophores; its implementation via the CAR technique is discussed in detail later.

One other form of energy transfer CL involves the production of very weak luminescence by an excited state formed in a reaction of the analyte; the resulting emission intensity can be greatly enhanced by adding a luminescent energy acceptor (sensitizer) to the reaction medium. This alternative—of a lower analytical potential than PO-CL reactions—is useful in some cases as it enables some CL analytical determinations that are inaccessible via PO-CL reactions. Such is the case with the CL determination of low-molecular-mass aliphatic tertiary amines using the CAR technique. In fact, these amines are not fluorescent, and also impossible to derivatize to fluorophore with fluorescent probes owing to the lack of an appropriate structure. However, if tertiary aliphatic amines are reacted with sodium hypochlorite in an alkaline medium containing fluorescein as sensitizer, the CL signal is significantly enhanced. Using this reaction, a simple CAR method was developed for the CL determination of these species [39]. A 0.9-M sodium hypochlorite solution was added from the autoburette at 10 mL/min to the reaction vessel containing the analyte and the other reaction ingredients (borate buffer

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and fluorescein as sensitizer). The CL signal was measured by using a laboratoryassembled detector housing (black box). The method thus developed allows the determination of trimethylamine over a wide linear range (10–2500 nmol) with good precision (relative standard deviation, RSD, 2.6%), and compares favorably with recent alternatives to the determination of this amine in fish tissue. Primary and secondary amines such as methyland ethylamine can be indirectly determined at the µmol level by their inhibitory effect on the CL determination of trimethylamine.

PO-CL detection has two major advantages over classical spectrofluorimetry, specifically: (1) improved selectivity resulting from the fact that not all fluorophores are equally efficient as energy acceptors and (2) enhanced detection arising from suppression of source instability and source scatter in fluorescence detection. However, the background CL observed in these reactions in the absence of fluorophore is a serious analytical drawback [40–42]. This background emission has been ascribed to the presence of reaction intermediates, mainly phenoxy radicals generated in the oxidation by hydrogen peroxide of phenolate anion produced during the formation of the excited intermediate [42]. Specifically, the TCPO/hydrogen peroxide system gives a broad emission band centered at 450 nm, together with a weak emission band at 540 nm, both which are formed relatively slowly [41]. In addition, in excess hydrogen peroxide, only the intermediate peaks at 450 nm seem to prevail. Accordingly, background emission can be minimized by (a) keeping the hydrogen peroxide in excess and (b) recording the CL intensity immediately after mixing to avoid the presence of these intermediates, which emit light at 450 nm and are formed relatively slowly. The continuous-addition-of-reagent technique meets these requirements since the aryl oxalate is added from an autoburette over a reaction vessel containing excess hydrogen peroxide and the analyte, and the CL signal is recorded as soon as it is produced, using an integrated mixing/detection system. With kinetic methodology, this takes only about 1 s. Background emission is thus avoided and the signal-to-noise ratio is increased, which ultimately leads to a lower detection limit for the analyte.

Table 4 illustrates the use of the CAR technique to develop CL kineticbased determinations for various analytes in different fields. As can be seen, the dynamic range, limit of detection, precision, and throughput ( 80–100 samples/ h) are all quite good. All determinations are based on the use of the TCPO/ hydrogen peroxide system; by exception, that for β-carboline alkaloids uses TCPO and DNPO. A comparison of the analytical figures of merit for these alkaloids reveals that DNPO results in better sensitivity and lower detection limits. However, it also leads to poorer precision as a result of its extremely fast reactions with the analytes. Finally, psychotropic indole derivatives with a chemical structure derived from tryptamines have also been determined, at very low concentrations, by CAR-CLS albeit following derivatization with dansyl chloride.

Table 4	Analytical Figures of Merit of the CL Kinetic-Based Determination of Drugs and Hallucinogenic Alkaloids Using the CAR
Technique
		Dynamic range	Detection	Precision
Analyte		(ng/mL)	limit (ng/mL)	(RSD, %)	Comments	Ref.
Phenothiazines (acepromazine, propio-		7.5–64,000	2.2–66.5	1.0–3.7	Acepromazine was determined in	43
promazine, promazine, trimepra-					horse plasma with good results; the
zine, methotrimeprazine, thiorida-					method is suitable for pharmacoki-
zine, chlorpromazine)					netic studies
Psychotropic indol derivatives (psilo-		0.6–140 pmol	138–230 pmol	1.5–2.2	Psilocin was quantified in specimens	44
cin, bufetonine, N-methyl-, 5-meth-					of the fungal species Psylocibe
yland α-methyltryptamine)					semilanceata
β-Carboline alkaloids (harmaline, har-		0.3–150	0.25–90	2.9–3.7	Harmaline was determined in plasma	45
malol, harmane, harmol, harmine)					samples; DNPO was used as aryl
					oxalate
DNPO, bis(2,4-dinitrophenyl)oxalate.

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