Chemiluminescence in Analytical Chemistry

296	Santana Rodrı´guez

an average diameter of 0.57, 0.78, and 0.95 nm for α, β, and γ cyclodextrins, respectively [34]. The solute molecules that have the correct dimensions can interact with the cyclodextrin cavity, which is nonpolar, and form inclusion complexes.

The cyclodextrins are stable bodies in aqueous solution, unlike the micelles, which are transitory and are in a state of dynamic equilibrium with the monomer surfactants. However, in many aspects the inclusion of analytes in the cyclodextrin cavity is reminiscent of the solubilization of hydrophobic molecules in micelles in aqueous solution.

The reduced polarity and the protection supplied by the cyclodextrin cavity have a strong and often favorable influence on the properties of the included solute. Cyclodextrins have been used extensively in luminescence techniques due to the fact that many analytes, on forming complexes with them, undergo an enhancement of their luminescent efficiency compared to that seen in an aqueous medium [35–37]. The factors responsible for this increase are the protection of the analyte molecule in the complex with the cyclodextrin from quenching of the water molecules or of other molecules present in solution and, moreover, the viscosity increase in the cyclodextrin cavity with a corresponding reduction in quenching caused by oxygen. As a consequence, the cyclodextrin cavity can protect the excited state of the analyte from nonradiative processes and from quenching that normally occur in aqueous solution.

3.INFLUENCE OF THE PRESENCE OF ORGANIZED MEDIA ON CHEMILUMINESCENT REACTIONS: ANALYTICAL APPLICATIONS

3.1 Micellar Media

3.1.1Normal Micelles

In recent years different types of surfactants have been used, in concentrations above their cmc, forming normal micelles, to improve different aspects of chemiluminescent reactions. Though the choice of the best surfactant depends on the characteristics of the chemiluminescent reaction, the surfactants most used have been the cationic (fundamentally quaternary ammonium salts) and to a lesser degree the anionic, the nonionic, and the zwiterionic compounds.

3.1.1.1 Cationic Surfactants

Alkyltrimethylammonium surfactants, such as hexadecyltrimethylammonium chloride (HTAC) [6, 9, 38–41], hexadecyltrimethylammonium hydroxide (HTAH) [38, 42], hexadecyltrimethylammonium bromide (HTAB) [43–46], tetradecyltrimethylammonium bromide (TTAB) [47], pentadecyltriethylammon-

Organized Media in Chemiluminescence

297

ium bromide (PTAB) [48], stearyltrimethylammonium chloride (STAC) [46], cetylethyldimethylammonium bromide (CEDAB) [46], didodecyldimethylammonium bromide (DDDAB) [46], and dioctadecyldimethylammonium bromide (DODAB) [41], have been widely used in various chemiluminescent reactions.

HTAC and HTAH have been used as surfactants in the chemiluminescence reaction of lucigenin (10,10′-dimethyl-9,9′-biacridinium dinitrate) with biological reductants (such as fructose, glucose, ascorbic and uric acid) or hydrogen peroxide [38].

Lucigenin produces a weak light in an alkaline solution, and the intensity of its light is substantially increased by the addition of hydrogen peroxide [49]. As to the reaction mechanism, it has been postulated [50, 51] that lucigenin is oxidized to form a peroxide, which is then decomposed to yield an excited state of N-methylacridine, as shown in Figure 6.

When biological reductants (sugars) are present, the rate-limiting step in the CL reaction between these agents and lucigenin apparently involves a basecatalyzed ketoenol tautomerization in which the sugars (in keto form) are converted to 1,2-enediols (enol form of parent sugar) as shown in Figure 7 for glucose and fructose [52–56]. The 1,2-enediol anion tautomers, which are stronger reducing agents than the parent sugars, subsequently react with lucigenin in a series of rapid steps that lead to CL emission [54].

The results indicate that the enhancement produced by HTAH is greater than that produced by HTAC in CL intensity in the presence of biological reductants. This is due to enhanced micellar catalysis of the rate-limiting step of the lucigenin-reductant CL reaction in HTAH micelles compared to that possible in HTAC. Figure 8 shows the influence of HTAH concentration on CL intensity of lucigenin-glucose and lucigenin-fructose systems. The presence of HTAH as a micellar medium improves the sensitivity and precision for lucigenin CL assay of biological reductants. For example, the detection limits for fructose were 2.3, 7.0, and 9.8 mg/L for the process with HTAH, HTAC, and an aqueous solution,

Figure 6 Chemiluminiscent reaction of lucigenin. (From Ref. 49 with permission.)

298	Santana Rodrı´guez

Figure 7 Ketoenol tautomerization for glucose and fructose.

respectively. With respect to the lucigenin-hydrogen peroxide CL system, both micellar media produced similar effects in the observed CL.

Since most CL assays require basic conditions, the use of a HTAH micellar medium compared to HTAC offers the advantage of providing the micelle-form- ing surfactant (HTA required for the enhancement of CL intensity) and the hydroxide ion (required for efficient CL).

HTAC cationic micelles also markedly enhance the CL intensity of fluorescein (FL) in the oxidation of hydrogen peroxide catalyzed by horseradish peroxidase (HRP) [39]. However, no CL enhancement was observed when anionic micelles of sodium dodecyl sulphate (SDS) or nonionic micelles of polyoxyethylene (23) dodecanol (Brij-35) were used (Fig. 9). CL enhancement is attributed to the electrostatic interaction of the anionic fluorescein with the HTAC micelles. The local concentration of fluorescein on the surface of the micelle increases the efficiency of the energy transferred from the singlet oxygen (which is produced in the peroxidation catalyzed by the HRP) to fluorescein. This chemiluminescent enhancement was applied to the determination of traces of hydrogen peroxide. The detection limit was three times smaller than that obtained in aqueous solution.

Organized Media in Chemiluminescence

299

Figure 8 Dependence of the relative CL intensity upon the final molar concentration of HTAH observed from the reaction of 3.3 10 4M lucigenin with (∆, solid line) 200 mg/L glucose and ( , dashed line) 48 mg/L fructose. (From Ref. 38 with permission.)

Riehl et al. also characterized the CL system lucigenin-hydrogen peroxide- N-methylacridone in the presence of different cationic surfactants such as HTAC, 3-(N-dodecyl-N,N-dimethylammonio) propane-1-sulfonate, and DODAB [41]. Enhancement factors (ratio between CL intensity in the presence of organized medium and CL intensity in the absence of organized medium) of CL intensity were found of 3.4, 2.5, and 1.6, respectively. The alterations in CL intensity are explained in terms of the effect of the different surfactants on the rate of the reaction and on excitation efficiency.

The CL enhancement of the lucigenin reaction with catecholamines in the presence of HTAH micelles was used for determination of dopamine, norepinephrine, and epinephrine [42]. However, the presence of an anionic surfactant, SDS, inhibits the CL of the system. The aforementioned CL enhancement in the presence of HTAH can be explained in the following way: the deprotonated forms of the catecholamines are expected to be the principal species present in aqueous alkaline solution due to the dissociation of the catechol hydroxyl groups, and to react with lucigenin to produce CL. The anionic form of the catecholamines and the hydroxide ion interact electrostatically with and bond to the cationic micelle, to which the lucigenin also bonds. Therefore, the effective concentration of the

300	Santana Rodrı´guez

Figure 9 CL response curves from the oxidation of H2O2 with sodium hypochlorite in the presence of fluorescein and surfactants. (1) Aqueous system without fluorescein; (2) aqueous system; (3) CSDS 2.0 10 2 M; (4) CBrij-35 4.2 10 3 M; (5) CHTAC 3.010 3 M; CH2O2 1.5 10 4 M; CNaOCl 2.0 10 3 M; Cfluorescein 2.7 10 4 M; CNaOH 0.05 M. (From Ref. 39 with permission.)

catecholamines, hydroxide ion, and lucigenin is higher than the stoichiometric concentration in water.

HTAB has been used, on the one hand, to increase the CL intensity of the reaction of 2,6,7-trihydroxy-9-(4′chlorophenyl)-3-fluorene with hydrogen peroxide in alkaline solution, in the presence of traces of Co(II) as a catalyst [43]. As a consequence, a CL method has been established for determination of ultratraces of Co(II). On the other hand, HTAB micelles sensitize the CL oxidation of pyrogallol with N-bromosuccimide in an alkaline medium [44], while anionic and nonionic surfactants inhibit the CL intensity of this reaction (Table 3). This sensitized process allows the determination of pyrogallol by flow injection in an interval of 5 10 7–3 10 5 M.

The CL reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)

(1) is one of the more commonly used nonenzymatic CL reactions and has been extensively studied [49, 57–59]. It is well known that the luminol CL reaction is catalyzed by many kinds of substances, e.g., ozone, halogen-Fe complex, hemin, hemoglobin, persulfate, and oxidized transition metals. The most acceptable scheme is shown in Figure 10. Luminol forms a six-membered ring of peroxide

(3) from a diazaquinone intermediate (2) and then, by the decomposition of 3, N2 gas and the S1-excited state of the phthalate dianion are produced, yielding

Organized Media in Chemiluminescence			301
Table 3 Effect of Surfactant Concentration on CL Intensity
with 1.0 10 5 M Pyrogallol
		Relative CL intensity
Concentration (M)	HTAB	SDS	Triton X-100
0.0	30	30	30
5.0 10 5	44	26	24
1.0 10 4	55	20	18
5.0 10 4	83	13	11
1.0 10 3	120	9	8
5.0 10 3	100	6	4

Source: From Ref. 44 with permission.

emission of light. It is certain that the dianion of 2-aminophtalic acid is an emitter. White et al. [58] confirmed this fact by the following experimental results: (a) luminol yielded 2-aminophtalic acid in 90% yield by the reaction with NaOH and O2 in dimethylsulfoxide; (b) the fluorescence spectrum of aminophthalic dianion agreed well with the resultant CL spectrum; (c) in the reaction system using 18O2, the consumed oxygen was all found in the carbonyl group of the dianion and 1 mol N2 gas was yielded simultaneously.

This CL reaction is most efficient at pH values in the range 10.5–11. This pH limitation is the main handicap of the system when coupled to enzymatic reactions at neutral pH. Abdel-Latif and Guilbault [45] have used HTAB to increase the CL intensity and to save the pH incompatibility of the luminescent

Figure 10 Chemiluminescent reaction of luminol. (From Ref. 49 with permission.)

302	Santana Rodrı´guez

reaction of luminol when it binds to the enzyme reaction glucose/glucose oxidase at neutral pH. The results show the reliability of the simultaneous and efficient operation of both reactions with pH 7.5–8.5, allowing the determination of glucose in an interval of 3 10 7–3 10 4 M with a relative standard deviation of 3.8%.

Other cationic surfactants such as TTAB, DTAB, DODAB, STAC, CEDAB, and DDDAB have been used in CL reactions with less frequency. Thus, tetradecyltrimethylammonium bromide [TTAB] has been used to increase the sensitivity of the method to determine Fe(II) and total Fe based on the catalytic action of Fe(II) in the oxidation of luminol with hydrogen peroxide in an alkaline medium [47]. While other surfactants such as HTAB, hexadecylpiridinium bromide (HPB), Brij-35, and SDS do not enhance the CL intensity, TTAB shows a maximum enhancement at a concentration of 2.7 10 2 M (Fig. 11). At the same time it was found that the catalytic effect of Fe(II) is extremely efficient in the presence of citric acid. With regard to the mechanism of the reaction, it is thought that Fe(II) forms an anionic complex with citric acid, being later concentrated on the surface of the TTAB cationic micelle. The complex reacts with the hydrogen peroxide to form hydroxy radical or superoxide ion on the

Figure 11 Effect of TTAB concentration on the CL intensity in the presence of citric acid. CFe(II) 1.0 10 7 M; Ccitric acid 1.0 10 2 M; Cluminol 1.0 10 5 M; CH202 1.0 10 2 M; pH 9.3 (borate buffer). (From Ref. 47 with permission.)

Organized Media in Chemiluminescence

303

surface of the micelle. In this way the speed of the CL reaction increases, with the CL intensity being enhanced.

Sukhan has used PTAB cationic micelles to enhance the CL reaction of 4- diethylaminophthalohydrazide with oxygen and Co(II) in the presence of fluorescein as sensitizer [48]. This enhancement is mainly due to electron-excited energy transfer from the donor (4-diethylaminophthalohydrazide) to the acceptor (fluorescein). The addition of fluorescein combined with the presence of PTAB reduces the detection limit of Co(II) by a factor of 6. The method was successfully applied in the determination of Co in tap water samples.

Finally, Yamada and Suzuki made a comparative study of the use of DDAB, HTAB, STAC, and CEDAB to improve the sensitivity and selectivity of the determination of ultratraces of Cu(II) by means of the CL reaction of 1,10phenanthroline with hydrogen peroxide and sodium hydroxide, used as detection in a flow injection system [46]. Of the four cited surfactants it was found that CEDAB causes the greatest enhancement of the chemiluminescent signal (Fig. 12) (an enhancement factor of 140 with respect to the absence of surfactant).

Figure 12 CL enhancement versus surfactant concentration. ( ) DDAB; ( ) HTAB, (*)STAC; ( ) CEDAB. CCu(II) 2.0 10 8 M; CH2O2 5%; C1,10-phenantroline 6.0 10 5 M; CNaOH 0.1 M. (From Ref. 46 with permission.)

304	Santana Rodrı´guez

The CL enhancement may be interpreted as follows: 1,10-phenantroline is repelled from the cationic micellar interface owing to its charge; it therefore seems that 1,10-phenantroline exists as a nonionic species in the alkaline solution and migrates to the micellar surfaces, i.e., to the Stern layer, which is significantly less polar than water. On the other hand, superoxide radical anion •O2 , the oxidant in the present system, which is produced from the hydrogen peroxide decomposition catalyzed by the copper–1,10-phenantroline complex in the alkaline solution, migrates easily on the positively charged micellar surface. Consequently, in the hydrophobic environment of the Stern layer the nonionic species may react more effectively with •O2 to form a dioxetane intermediate that decomposes via an exoenergic route producing an emitter. The CL enhancement is therefore attributed to higher excitation efficiency of the dioxetane decomposition in the less polar environment of the Stern layer. In the presence of CEDAB a detection limit of 0.3 pg is obtained, which is much lower than the detection limit obtained by other CL methods and is comparable to that obtained by methods that employ atomic spectrometry. At the same time, it was found that the established method for determination of Cu(II) using CEDAB is highly selective.

3.1.1.2 Anionic Surfactants

From an analytical point of view, the use of anionic surfactants as enhancers of CL reactions is most limited. One of the most recent examples is the use of sodium dodecylbenzene sulfonate (SDBS) as a CL enhancer of the system Ru(bpy)32 SO32 KBrO3 (bpy 2,2′-bipyridyl) [60]. The authors of this work propose the following mechanism for the chemiluminescent system:

SO32 BrO3 → •SO3 Ru(bpy)32 BrO3 → Ru(bpy)33

Ru(bpy)33 •SO3 → Ru(bpy)32 * •SO5

•SO3 O2 → •SO5

•SO5 SO32 → SO52 •SO3

SO52 SO32 → 2SO42 Ru(bpy)32 * → Ru(bpy)32 hν

•SO3 → 3SO2* 3SO2* → SO2 hν,

where Ru(bpy)32 * and 3SO2* are excited state and triplet state of both species, respectively. When this system was studied in the presence of different anionic, cationic, and nonionic surfactants, it was found that SDBS produces the highest enhancement (Table 4). This is due to the fact that Ru(bpy)32 exists as a cationic complex in aqueous solution, where it is surrounded by the anionic surfactant SDBS, which prevents extinction of CL by the water and increases the excited-

Organized Media in Chemiluminescence			305
Table 4 Effect of Different Surfactants on the Chemiluminiscent System
Ru(bpy)32 -SO32 -KBrO3
Surfactant	Intensity/mV	Surfactant	Intensity/mV
Water	3.8	Triton X-100	4.3
SDBS	126	TPB	7.7
Tween-20	7.9	HPB	5.6
Tween-40	7.7	HTAB	3.5
Tween-80	4.2

TPB, tetradecylpyridine bromide.

Source: From Ref. 60 with permission.

state lifetime of the complex Ru(bpy)32 . This chemiluminescent system in the presence of SDBS has been successfully applied to determination of sulfite in sugar.

However, surfactants have been used not only to enhance the signal of CL systems, but also to avoid problems of solubility in these systems. Thus, Klopf and Nieman have used SDS, at a submicellar concentration, to solubilize the product (N-methylacridone) of the CL reaction of lucigenin, due to its insolubility in water [9]. In this way the appearance is avoided of solid deposits in the observation cell and in other components of the flow system.

3.1.1.3 Nonionic Surfactants

One of the nonionic surfactants most used as an enhancer of chemiluminescent reactions is Brij-35. This surfactant increases the reaction of lucigenin with catecholamines by a factor of 2.6 compared with the CL intensity in an aqueous medium [42]. This enhancement can be explained in the following way: it is known that oxygen from the polyoxyethylene chains in Brij-35 can react with sodium ion to form an oxonium ion, by which means the polyoxyethylene chains act as an oxonium cation. In this way the increase in CL intensity due to Brij35 can be attributed to the same effect described for the micelles of a cationic surfactant.

Dan et al. found that Brij-35 also produces an enhancement of the CL intensity of the reaction of bis[N-[2-(N′-methyl-2′-pyridiniumyl)ethyl]-N- [(trifluoromethyl)sulfonyl]]oxamide with hydrogen peroxide in the presence of some fluorophors [40]. To be precise, the CL intensity increased by a factor of 130 for 8-aniline-1-naphthalenesulfonic acid (ANS) and 5.6 for rhodamine B (RH B), compared to the CL intensity in the absence of surfactant. This leads to an increase of 2–3 orders of magnitude in the linear dynamic ranges and a more precise determination of these analytes. However, improvement of the detection