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206	Pe´rez-Bendito and Silva

Figure 13 Schematic depiction of the foundation and architecture of the CNN used for multicomponent CL kinetic-based determinations.

to synergistic effects, various synthetic mixtures were assayed. The magnitude of the relative errors in the concentration of each component in the mixture is apparent from Table 6, which shows the results for various synthetic binary mixtures containing variable amounts of trimeprazine and methotrimeprazine. Mixtures in ratios from 10:1 to 1:10 were satisfactorily resolved. The relative errors

Table 6 Analysis of Various Trimeprazine/Methotrimeprazine Mixtures Using the Optimal Network Design

Concentration taken ( g/mL)			Trimeprazine			Methotrimeprazine
			Found	Relative		Found	Relative
Trimeprazine	Methotrimeprazine		( g/mL)	error (%)		( g/mL)	error (%)
3.00	3.00		2.99	0.33	2.99		0.33
2.00	4.00		1.99	0.50	3.99		0.25
2.00	5.00		2.01	0.50	5.01		0.20
1.00	3.00		0.98	2.00	2.99		0.33
2.00	8.00		1.92	4.00	8.10		1.25
1.00	10.0		1.12	12.0	10.0		0.00
9.00	1.00		8.76	2.66	1.10		10.0
10.0	2.00		10.01	0.10	2.03		1.50
8.00	2.00		8.00	0.00	2.00		0.00
6.00	2.00		5.97	0.50	1.99		0.50
10.0	4.00		9.99	0.10	4.01		0.25
10.0	5.00		10.05	0.50	5.04		0.80

Source: Ref. 69.

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207

made were less than 5% (except at both ends of the range, where they rose to ca. 10%) and hence quite acceptable for such a wide range of concentration ratios.

ACKNOWLEDGMENT

The authors gratefully acknowledge financial support from Spain’s Direccio´n

´

General Interministerial de Ciencia y Tecnologia (DGICyT) in the framework of Project PB96-0984.

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9

Electrogenerated Chemiluminescence

Andrew W. Knight

University of Manchester Institute of Science and Technology,

Manchester, England

1. INTRODUCTION		212
1.1	Brief Historical Background	212
1.2	The Potential of ECL in Analytical Science	213

2.SURVEY OF ECL MECHANISMS AND ANALYTICAL

	APPLICATIONS		215
	2.1	Organic Ion Annihilation ECL	215
	2.2	Inorganic Electron Transfer ECL	217
	2.3	Electrochemical Generation of Conventional CL	227
	2.4	Miscellaneous ECL Reactions	228
	2.5	Cathodic Luminescence	228
3.	ECL INSTRUMENTATION		229
	3.1	Configuration and Nature of the Electrodes	229
	3.2	ECL Flow Cells	230
	3.3	ECL Probes	232
4.	REAGENT IMMOBILIZATION AND ECL SENSORS		232
5.	ECL IN FLOW INJECTION ANALYSIS		233
6.	ECL IN LIQUID CHROMATOGRAPHY		234
7.	ECL IN CAPILLARY ELECTROPHORESIS		236
8.	ENZYME-COUPLED ECL FOR BIOSENSING		238
9.	ECL IMMUNOASSAY AND DNA PROBES		240

211

212		Knight
10.	ECL IN MINIATURIZED ANALYTICAL SYSTEMS	242
11.	ECL IN ELECTRODE CHARACTERIZATION AND THE
	VISUALIZATION OF ELECTROCHEMICAL PROCESSES	242
12.	CONCLUSIONS	243

1. INTRODUCTION

Electrogenerated chemiluminescence (ECL) is the process whereby a chemiluminescence emission is produced directly, or indirectly, as a result of electrochemical reactions. It is also commonly known as electrochemiluminescence and electroluminescence. In general, electrically generated reactants diffuse from one or more electrodes, and undergo high-energy electron transfer reactions either with one another or with chemicals in the bulk solution. This process yields excitedstate molecules, which produce a chemiluminescent emission in the vicinity of the electrode surface.

Much of the study of ECL reactions has centered on two areas: electron transfer reactions between certain transition metal complexes, and radical ionannihilation reactions between polyaromatic hydrocarbons. ECL also encompasses the electrochemical generation of conventional chemiluminescence (CL) reactions, such as the electrochemical oxidation of luminol. Cathodic luminescence from oxide-covered valve metal electrodes is also termed ECL in the literature, and has found applications in analytical chemistry. Hence this type of ECL will also be covered here.

1.1 Brief Historical Background

Some of the first observations of luminescence accompanying electrolysis were reported by Bancroft in 1914, when halides were electrolyzed at mercury and other anodes [1]. Thirteen years later luminescence was observed by Dufford et al. when Grignard compounds in anhydrous ether were electrolyzed, at the anode or cathode, by applying 500 to 1500 volts [2], and subsequently by Harvey for luminol in alkaline solution at the anode, by applying 2.8 volts [3].

However, ECL was not then studied in detail until 1963 [4, 5]. At this time ECL from solutions of aromatic hydrocarbons was first recorded, and mechanisms involving electron transfer between electrically generated radical anions and cations were proposed. Between the mid-1960s and late 1980s there was considerable interest in the phenomenon of ECL. More than 60 publications in the literature focused almost solely on the mechanism of ECL reactions, identi-

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213

fying the excited states, rate constants, quantum efficiencies, and the effects of temperature and various solvents and electrolytes. These were determined by a variety of spectroscopic and electron spin resonance methods, and by observing the effect of magnetic fields on ECL emissions. Developments during this period have been thoroughly reviewed [6–9]. Over 100 additional papers reported on the use of ECL in the study of novel photochemical and electrochemical properties of a wide range of compounds, complexes, and clusters. These included polyaromatic hydrocarbons and their derivatives; organometallics and other transition metal complexes including those of ruthenium, osmium, platinum, palladium, chromium, iridium, and rare earth elements; and molybdenum and tungsten clusters [10–12].

The potential of ECL in analytical chemistry has only more recently been investigated, but has rapidly gained recognition as both a sensitive and selective method of detection. Most reported applications have utilized the tris(2,2′-bipyri- dyl) ruthenium(II) [Ru(bpy)32 ] ECL reaction, or else the electrochemical initiation of more conventional CL reactions, but many other potentially useful systems have been investigated. The applications of ECL in analytical chemistry have recently been the subject of comprehensive reviews [12–16].

1.2 The Potential of ECL in Analytical Science

While retaining the selectivity and sensitivity inherent to conventional CL analysis, ECL has several potential advantages as an analytical technique that are currently being realized.

1.2.1Advantages from the Electroinitiation of CL Reactions

Since the CL reaction is being produced by an electrochemical stimulus, greater control is gained over the initiation, rate, and course of the CL reaction. Indeed this control can be to the extent that the CL reaction may be ‘‘switched on and off,’’ allowing for synchronous detection, effective background correction, and ready automation with computer control. By careful selection of the electrode material, surface treatment, and applied potential, an additional degree of electrochemical selectivity can also be introduced.

1.2.2Advantages from the Electromanipulation of the CL Reaction

Some CL reagents and intermediates can be electrochemically regenerated allowing them to take part in a CL reaction with analyte molecules once again. This often allows lower concentrations of expensive reagents to be used. In some cases, principally Ru(bpy)32 , such reagents can be immobilized on the electrode surface, removing the need to continually add the reagent to the system and creat-

214

Knight

ing an ECL sensor. Some analytes, such as polyaromatic hydrocarbons, or species labeled with a Ru(bpy)32 derivative, can be electrochemically regenerated to their active form, thus allowing each analyte molecule to produce many photons per measurement cycle. In such cases extremely low limits of detection can be achieved. It may also be possible to electrochemically modify compounds considered inactive for CL to form new species that can take part in a CL reaction, and hence extend the range of analytical applications of a particular CL method.

1.2.3Simplifying Existing CL Methodology

CL analysis methods using flowing streams (e.g., flow injection analysis or HPLC) can be simplified by reducing the number of reagents needed to be pumped separately to the site of mixing and light detection. This is possible because active reagents can be electrochemically produced from passive precursors in the carrier stream. For example, hydrogen peroxide for the luminol CL reaction may be electrochemically generated by reduction of molecular oxygen dissolved in the carrier medium. In many flow-through CL methods the speed and completeness of mixing of the redox reagents in view of a light transducer are crucial to the reproducibility and sensitivity of the technique. However in ECL, it is often the case that all of the reagents can be thoroughly and reproducibly mixed prior to their passage into the detection cell, whereupon the CL reaction is electroinitiated. Similarly in conventional CL methods the optimization of the physical distance between the point of mixing and the light transducer, primarily dependent on the kinetics of the CL reaction, is important. This is in order for the reaction mixture to be passing the light detector at the point of maximum CL emission. In ECL however, the light-producing reaction is largely confined to the immediate vicinity of the electrode surface, which can be shaped and positioned directly in front of the light transducer for maximum sensitivity. In some cases unstable CL reagents or intermediates can be electrochemically generated, and allowed to react as soon as they are formed at the electrode surface, or some point downstream in a flow-through system. In this way reproducibility is improved and the problem of working with analytical reagents that rapidly deteriorate is removed. Examples of this include the electrochemical generation of peroxides, Ru(bpy)33 , and radical species of polyaromatic hydrocarbons.

1.2.4Advantages from the Combination of a Spectroscopic and Electrochemical Technique

ECL gives the opportunity of gaining additional analytical information by monitoring the electrochemical activity of the analyte, by a range of electroanalytical techniques, alongside recording the light output. Similarly measurements of chemiluminescent light output can be used to elucidate and visualize electrochemical processes, and measure reaction kinetics. For example, one particular reaction

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215

that produces a CL emission can be selectively monitored from many other reactions occurring simultaneously at the electrode.

1.2.5Limitations

Electrogenerated chemiluminescence does, however, have some limitations, which have not yet been fully overcome. The combination of electrochemical and CL techniques brings together two sets of species that can potentially interfere with ECL determinations, either constructively or destructively. These include species that preferentially react with the electrogenerated CL reagents and intermediates, in nonchemiluminescent reactions; or that quench excited species; or that are electrochemically active and interfere with, or swamp, electrochemical reactions. As a direct consequence of this, the majority of analytical ECL methods now being developed include a chromatographic separation step prior to ECL detection. Similarly optimization is complicated by the need to find conditions, such as pH or choice of electrolyte, to suit both the electrochemical and CL reactions, and as a result a compromise often has to be reached. Additionally, some factors such as the pH and optimum applied potential also interact and are partially dependent on each other. Optimization is complicated still further if the medium is to support an enzymic reaction or act as a mobile phase for a chromatographic separation. As a result multivariate optimization techniques are often required.

Most ECL reactions involve a complex series of electrochemical and CL reaction steps, such that the exact mechanism of all but the simplest ECL reactions has not been explicitly determined. This may be a problem in the search for new analytical applications, when certain compounds show marked ECL activity, while other closely related compounds are inactive, or when trying to produce a linear calibration for a particular analyte. Many applications have also been hindered by poor reproducibility in the ECL measurements, which can only be overcome by frequent refreshing of the electrode surface, the reasons for which are not well understood.

2.SURVEY OF ECL MECHANISMS AND ANALYTICAL APPLICATIONS

2.1 Organic Ion Annihilation ECL

Energetic electron transfer reactions between electrochemically generated, shortlived, radical cations and anions of polyaromatic hydrocarbons are often accompanied by the emission of light, due to the formation of excited species. Such ECL reactions are carried out in organic solvents such as dimethylformamide or acetonitrile, with typically a tetrabutylammonium salt as a supporting electrolyte. The general mechanism proposed for these reactions is as follows.