Chemiluminescence in Analytical Chemistry

Recently ECL reactions of Ru(bpy)32 with other reducing agents have been documented, such as various β-diketone and some methylene compounds that have cyano and carbonyl groups [40]. Hence with further research, analytical applications should arise for many classes of compounds other than amines that can act as reductants, or electrochemical precursors of reductants, capable of reacting with Ru(bpy)33 to produce ECL.

2.2.5Methods of Generation of Ru(bpy)33

The various approaches to the generation of the active ECL reagent Ru(bpy)33 have been reviewed by both Lee [14] and Gerardi et al. [16]. Methods of generation include purely chemical, photochemical, external electrochemical, and in situ electrochemical approaches.

Chemical oxidants such as lead dioxide and cerium(IV) are commonly used to produce Ru(bpy)33 from Ru(bpy)32 , and such methods are rapid and relatively simple. Ru(bpy)33 can be prepared in bulk, offline, using a batch procedure, or online, using flow methods, the latter either by combining a stream of Ru(bpy)32 with that of an oxidant at a mixing T, or by passing Ru(bpy)32 over an immobilized oxidant, such as lead dioxide on silica gel. However, the majority of applications use electrochemical means to produce Ru(bpy)33 , owing to problems of maintaining a stable and reproducible supply of reagent.

External electrochemical generation of Ru(bpy)33 involves the bulk electrolysis of a reservoir of acidified Ru(bpy)32 , at about 1.1–1.3 V (vs. Ag/AgCl), over a period of typically 30–45 min, during which time the solution will change color from orange to green. Working electrodes are fabricated from materials with a large surface area, such as platinum gauze or glassy carbon sponge, to maximize conversion. The counterelectrode is usually kept separate from the bulk solution to prevent reduction and electrodeposition of ruthenium at this electrode. Owing to the instability of the reagent, solutions of Ru(bpy)33 are often maintained at around 0°C. This method of reagent production is time consuming, and the reagent, once formed, has a limited lifetime. Hence Uchikura developed a flow-through, in-line, electrochemical reactor for continuous Ru(bpy)33 generation [16]. This device employs a working electrode consisting in part of a porous plug of tightly packed glassy carbon particles though which acidified Ru(bpy)32 is pumped. More commonly in situ generation is used, producing both Ru(bpy)33 and the CL reaction with the analyte, together at the electrode surface. Ru(bpy)32 may be in the bulk solution or immobilized at the electrode surface.

These different approaches to reagent generation have recently been compared by Lee and Nieman, for analytical determinations of a range of analytes using flowing streams [41]. The external generation mode gave the most intense emissions and highest sensitivity; however, working curves had poor linearity and ECL intensities were heavily dependent on experimental variables such as

flow rate. Using in situ immobilized reagents produced the widest linear dynamic ranges and consumed the least amount of reagent, but gave the lowest sensitivity. In situ generation from solution-phase reagents proved the most satisfactory, in terms of convenience, rapidity, and reproducibility, and it is from this method that many of the advantages of ECL, discussed in Sec. 1.2, arise.

2.3 Electrochemical Generation of Conventional CL

Many conventional CL reactions can be initiated electrochemically. However, the most studied and exploited reaction has been that of luminol due to its versatility in analytical determinations. The mechanism of luminol ECL is thought to be similar to that of its chemiluminescence [42] and has been investigated in detail by Haapakka and Kankare [43]. The generally accepted mechanism is shown in Figure 5. In alkaline solution the luminol anion undergoes a single electron electro-oxidation to form a diazaquinone, which is further oxidized by peroxide or superoxide to give 3-aminophthalate in an excited state, which emits

Figure 5 Proposed mechanism for the ECL reaction of luminol. (Reprinted from Ref. 15, with permission from, and copyright, Elsevier Science.)

light at 425 nm. Luminol ECL has been used for a diverse range of analytical applications [12, 15]. These have included; the determination of luminol or species such as phenylalanine, ibuprofen, and hisidine labeled with luminol derivatives; hydrogen and other peroxides; and biochemical analytes that are substrates for enzymic reactions that produce hydrogen peroxide, such as glucose with glucose oxidase. Luminol ECL can also be used to indirectly determine species that either catalyze the reaction, such as the transition metal ions cobalt(II), copper(II), and nickel(II), or species that inhibit the reaction, such as sulfite. Analytical ECL applications of other conventional CL reactions have been briefly investigated. These include; lucigenin for trace metals, bis(2,4,6-trichlorophenyl) peroxyoxalate (TCPO)/fluorescer for trace O2, and acridinium ester–labeled compounds [12].

Methods of analysis based on electrochemical generation of conventional CL reported in the literature have declined in recent years since it is now generally recognized that where a conventional CL method exists, the added complications in methodology and instrumentation needed to produce an ECL system outweigh the potential advantages. Also in most cases, as with luminol, the CL reagents cannot be electrochemically regenerated.

2.4 Miscellaneous ECL Reactions

A variety of other ECL reactions are known that do not fall into the categories mentioned thus far, and some of these have found analytical applications. Such CL reactions can generally only be readily produced by electrochemical means, and often exhibit only very low ECL intensities, resulting in relatively high limits of detection. Examples include the determination by direct electrolysis of indole and tryptophan in the presence of hydrogen peroxide, and saccharides and alcohols that have neighboring hydroxyl groups; the determination of 2,4- and 3,4- diaminotoluene, biodegradation products of nitrotoluene explosives, which form weakly electrochemiluminescent complexes with gold(I) and copper(II) ions; the determination of antioxidants that inhibit the ultraweak anthracene sensitized ECL from the anodic oxidation of sodium citrate, methanol, and O2; and the determination of yttrium(III) and silver(I) ions that catalyze the ECL reaction of certain phthalazine derivatives [12, 15].

2.5 Cathodic Luminescence

The mechanism of cathodic luminescence is distinctly different from other ECL systems. Light is emitted from oxide-covered, so-called valve metal, electrodes, namely aluminium and tantalum, during the reduction of peroxodisulfate, hydrogen peroxide, or oxygen, in aqueous solution, at relatively low potentials ( 10 V). The mechanism involving persulfate, for example, is as follows. A conduc-

tance band electron is transferred to the peroxodisulfate ion (S2 O82 ) resulting in the formation of a sulfate radical (SO4 •). If its standard reduction potential matches the valence band edge of the semiconductor, it captures an electron from the valence band thus injecting a hole in this band. The recombination of an electron from the conduction band with the valence band hole produces light emission termed band-gap electroluminescence. However, cathodic luminescence having energy less than the semiconductor band-gap energy is often observed caused by recombination via surface states, i.e., energy levels localized at the electrode surface. Many species from inorganic ions to organic compounds can form attachments with the hydroxylated oxide surface of the electrode, and can hence act as surface states to enhance this sub-band-gap ECL, which occurs at 500–700 nm [44].

Haapakka and Kankare have studied this phenomenon and used it to determine various analytes that are active at the electrode surface [44–46]. Some metal ions have been shown to catalyze ECL at oxide-covered aluminum electrodes during the reduction of hydrogen peroxide in particular. These include mercury(I), mercury(II), copper(II), silver(I), and thallium(I), the latter determined to a detection limit of 10 10 M. The emission is enhanced by organic compounds that are themselves fluorescent or that form fluorescent chelates with the aluminum ion. Both salicylic acid and micelle solubilized polyaromatic hydrocarbons have been determined in this way to a limit of detection in the order of 10 8M.

3. ECL INSTRUMENTATION

Unlike conventional CL, no readily available commercial instrumentation exists for developing and utilizing methods of ECL analysis, and this is still a major drawback of the technique. One relatively simple option is to convert existing laboratory instrumentation. For example, by enclosing electrochemical apparatus in a light-tight box and incorporating a light detector, or by placing an electrochemical cell within a spectrophotometer in which the excitation light source has been disabled. However, in most cases researchers have opted to build their own instrumentation. ECL detectors need to be light-tight, yet still allow for easy sample introduction and removal; incorporate working, counter, and reference electrodes and a suitable phototransducer with a geometry such that light is not generated on an electrode surface obscured to the light detector; and be robust yet relatively easy to dismantle for electrode cleaning.

3.1 Configuration and Nature of the Electrodes

Three main electrode configurations have been used in ECL work: rotating ringdisk (RRD), dual and single electrodes. With the RRD electrode, the disk may,

for example, be set at a potential to produce the oxidized species and the ring set to produce a reduced species. The rotation of the disk sweeps the oxidized species out to the ring to react with the reduced species on the face of the ring [47]. This system is useful in the study of ECL reaction kinetics by varying the rotation rate; however, it does not readily lend itself to flow-though systems, which are used for most analytical applications. Dual-electrode configurations use direct-current potentials such that each species needed for the ECL reaction is produced continually at separate electrodes. For stable species, one electrode can be placed downstream of the other. Thus ECL is produced when species formed at the first electrode are transported downstream to react with species formed at the second electrode. In the case of relatively unstable intermediates, the electrodes are placed in close proximity to each other, and species formed at each electrode diffuse and react together in the small interelectrode gap. Two configurations that have been used are interdigitated electrodes and two plate electrodes in thin-layer geometry placed approximately 100 m apart. In the case of the latter, at least one electrode is transparent. Tin oxide on glass has been used, although lack of robustness is a problem [48]. The simplest, most widely adopted solution is to use a single electrode, and where more than one species needs to be electrochemically produced for the ECL reaction, an alternating potential is applied switching between the redox potentials of the particular species desired.

Various materials have been used for the working electrode where the ECL reaction takes place. These include platinum, gold, glassy carbon, and carbon paste. No one particular electrode material is suitable for a specific ECL reaction and similar electrodes have shown marked differences in sensitivity in the hands of different workers. In each case the condition of the electrode surface has a marked effect on the ECL signal, and the intensity and reproducibility of ECL measurements often fall as the surface becomes fouled during continuous use. Relatively little is known about these fouling processes and various approaches are used to minimize the effect, including repolishing, chemical treatment, and redox cycling [34, 49]. After regeneration of the electrode surface, electrodes are generally stable for approximately 40 h of continuous use.

3.2 ECL Flow Cells

Although the earliest ECL work was carried out in simple batch cells, the need for a rapid throughput of samples, and reproducible sampling and mixing with reagents, has led to the almost universal adoption of ECL flow cells. Most are developments of CL laminar flow cells, where the test solution flows in a thin layer, sandwiched between a glass observation window and the working electrode surface, the volume and shape of the cell being defined by an inert spacer. An example devised by Jackson and Bobbitt [34] is shown in Figure 6. Generally

Figure 6 Schematic diagram of an ECL flow cell developed by Jackson and Bobbitt. (A) Front view; (B) Side view. (Reprinted from Ref. 34, with permission from, and copyright, Elsevier Science.)

the counterelectrode is placed downstream of the working electrode, such that any species that may form at the counterelectrode cannot interfere with the ECL reaction. The counterelectrode can be within the main body of the cell or, more commonly, made of a short section of stainless steel tubing forming part of the outflow line from the cell. Standard reference electrodes are used, either incorporated within the body of the cell or positioned close to the output line of the cell. However, pseudoreference electrodes, such as plain silver or platinum metal, are often used. Such electrodes have been shown to provide a stable reference, have the advantages of being simple in construction and easy to refresh, produce minimum sample contamination, and are compatible with both organic and aqueous electrolyte systems.

Standard commercial potentiostats are usually used, although since some ECL methods require rapid switching of potentials and the application of complex waveforms, a flexible computer-controlled potentiostat is preferable. To achieve useful limits of detection with ECL a photomultiplier tube (PMT) is most commonly used as the light detector, although photodiodes have also been successfully employed [50]. Photodiode structures have the advantage that they can become an intrinsic part of the flow cell itself, have peak sensitivity at the red end of the visible spectrum where the ruthenium metal complexes emit, and are small, robust, cheap, and run from a low-voltage source. Wavelength discrimination is rarely used and all light emitted is recorded in the analytical signal. Hence monochromators are not usually a component of ECL devices.

A novel flow cell for the analysis of gaseous analytes has recently been developed by Collins and Rose-Pehrsson [51]. The device incorporates an electrochemical cell containing a Ru(bpy)32 solution contained beneath a Teflon diffusion membrane, over which air is continually sampled. Ru(bpy)33 is generated in situ at a platinum gauze working electrode. The gaseous analyte flowing through the cell passes through the membrane to react with the Ru(bpy)33 , generating an ECL emission in view of a PMT. The cell was used for determination of gas-phase hydrazine and derivatives to low ppb levels. Later designs have used a gold-coated cellulose membrane for the working electrode separating the Ru(bpy)32 solution on one side from the gas flow on the other [52].

3.3 ECL Probes

Recently various ECL probes have been developed. Early designs had a small reservoir of reagents, incorporating the electrodes, facing a fiberoptic bundle to carry the light to a PMT. In a novel design by Kuhn et al. [53] a gold coated optical fiber was polished flat such that the gold formed a micro-ring electrode surrounding the optical fiber. In each case the probes had to be used within a light-tight enclosure. Preston and Nieman [54], however, developed a probe that does not require a dark box, and is similar in construction and operation to a pH probe. The probe contains working, counter, and reference electrodes, an optical fiber, and baffled channels to admit the test solution, while at the same time shielding the optical transducer from ambient light. This device has been successfully used with both Ru(bpy)32 ECL and luminol ECL coupled with an immobilized oxidase enzyme.

4. REAGENT IMMOBILIZATION AND ECL SENSORS

The ability to immobilize ECL reagents on the electrode surface and thus produce sensors is seen as being very advantageous, especially if the activity of the reagent can be electrochemically regenerated following the ECL reaction. This is because the need to continually deliver reagent to the reaction cell is removed, simplifying both the instrumentation and methodology, and dramatically reducing reagent consumption. In ECL this has been achieved with limited success for Ru(bpy)32 . The most favored approach has been to use the perfluorinated, sulfonated cation exchange polymer Nafion, to which Ru(bpy)32 electrostatically binds. Nafion has been widely used for modifying electrodes owing to its high chemical, mechanical, and thermal stability, and selectivity for large cations such as Ru(bpy)32 , while excluding potential anionic interferences such as OH . Electrodes can be simply prepared by brief immersion into a solution of a few percent Nafion dissolved in alcohol and evaporating to dryness, followed by immersion

for approximately 30 min in an acidic solution of 5 mM Ru(bpy)32 . Electrodes prepared in this manner take on a deep-orange color since the polymer film takes up significant amounts of the cation. Rubinstein and Bard first investigated Ru(bpy)32 immobilized in this way [55], and noted that charge transfer through the polymer film partly results from diffusion of the electroactive species [14]. This approach has then been subsequently exploited by Downey and Nieman for the determination of oxalate, alkylamines, and NADH [56].

However, there are significant limitations. Sensors made in this may have been universally observed to have limited long-term stability, in the worst cases losing up to 85% sensitivity when stored overnight in buffer, and total loss of activity if allowed to dry out between measurements [56]. The decrease is speculated to be due to diffusion of Ru(bpy)32 into hydrophobic regions of the Nafion film restricting charge transport, rather than desorption of Ru(bpy)32 .

Another approach, developed by Egashira et al. [57], used a carbon paste electrode. Graphite powder was blended with 2% w/w bis(2,2′-bipyridine)-(4,4′- dinonadecyl-2,2′-bipyridine) ruthenium(II), mineral oil, and a solvent. After thorough mixing the solvent was evaporated and the resulting paste packed into a glass tube. The electrode, as part of a fiberoptic ECL sensor, was used for the determination of oxalate ions, toward which selectivity was enhanced by the hydrophobic environment produced by the long alkyl chains of the ruthenium complex. However, the ECL response rapidly decayed under continuous operation for only 10 min, attributed to the degradation of the complex, and the electrode material had to be removed from the tube, blended, and repacked before being reused. Electropolymerization of monomers based on Ru(bpy)32 has also been carried out, and the resulting polymers show ECL activity. For example, Abrun˜a and Bard demonstrated a chemiluminescent polymer based on tris(4-vinyl-4′- methyl-2,2′-bipyridyl) ruthenium(II) [58]. However, once again the analytical potential of such systems is limited by instability, the ECL emission persisting for only 20 min of continuous operation.

5. ECL IN FLOW INJECTION ANALYSIS

Flow injection analysis (FIA) has been widely adopted in ECL, although primarily for analytes in simple matrices such as synthetic mixtures or pharmaceutical preparations that have few potential interfering species [15, 16]. Flow cells incorporating a working electrode, as described in Sec. 3.2, are employed for methods using in situ generation of ECL reagents, and more conventional CL flow cells are used for methods that employ external electrochemical generation of ECL reagents such as Ru(bpy)33 . Figure 7 shows an example of the FIA calibration and analysis of the diuretic pharmaceutical, hydrochlorothiazide, in tablet form, using external electrochemical generation of Ru(bpy)33 [59]. For in situ genera-

Figure 7 Flow injection peaks of hydrochorothiazide standards and samples, showing reproducibility. (Reprinted from Ref. 59, with permission from, and copyright, Elsevier Science.)

tion of ECL, the electrode is usually permanently charged and the injected sample produces light as it passes over the electrode, which is recorded as a familiar FIA peak response.

Consideration should be given to the flow rate of the sample through the detection cell. Shultz and co-workers have demonstrated the wide variability in reaction kinetics between ECL reactions, and hence the influence of flow rate on ECL intensity [60]. For example, the rate constants (k) of the Ru(bpy)32 ECL reactions of oxalate, tripropylamine, and proline were calculated to be 1.482, 0.071, and 0.011/s, respectively. Maximum ECL emission was obtained at low linear velocities for slow reactions ranging up to high linear velocities for fast reactions. That is, the flow rate and flow cell volume should be optimized such that the light-emitting species produced is still resident within the flow cell, in view of the light detector, when emission occurs.

6. ECL IN LIQUID CHROMATOGRAPHY

Increasingly ECL detection is being coupled with chromatography, to allow the determination of analytes in more complex matrices, such as blood plasma or foodstuffs, and samples containing more than one ECL active compound of interest [15, 16]. However, such methods are complex to optimize, as ideally a me-