Chemiluminescence in Analytical Chemistry

216

Radical formation:

A → A • e

B e → B •

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Electrochemical oxidation	(1)
Electrochemical reduction	(2)

Two main CL pathways are then possible: If sufficient energy is available, an electron transfer reaction where the singlet excited state of A is accessible (3); otherwise an energy-deficient route whereby the energy from two triplet excitedstate species are ‘‘pooled,’’ to provide sufficient energy to form the singlet excited state, in what is termed a ‘‘triplet-triplet annihilation reaction,’’ (4) and (5).

A • B • → 1A* B	(3)
or
A • B • → 3A* B	(4)
3A* 3A* → 1A* A	(5)
Followed by light emission,
1A* → A hν	(6)

where A is a polyaromatic hydrocarbon or aromatic derivative, B is the same or another polyaromatic hydrocarbon, 1A* and 3A* are the singlet and triplet excited states of A, respectively, and hν is a fluorescence emission [12].

The reactions can be carried out by producing the radical cations and anions at separate electrodes in close proximity to each other, or more elegantly using a single electrode and applying a square-wave potential alternating between the oxidation and reduction potentials of the species concerned. Either A or B can be the analyte species in the reaction, and in most cases after the CL reaction has occurred, barring destructive side reactions, both the original analyte and reagent species are regenerated. Common molecules used for these reactions include rubrene, 9,10-diphenylanthracene, N,N,N′,N′-tetramethylphenylenedia- mine, and benzophenone.

Organic ion annihilation ECL (IAECL) reactions have not, however, found significant numbers of applications in analytical chemistry. This is principally due to the practical problems arising because these reactions take place only in organic solvents, from which water and dissolved oxygen have been rigorously excluded, to prevent quenching of the CL reaction. This invariably involves thoroughly drying solvents, recrystallizing electrolytes, and using enclosed apparatus. Additionally, analysis of the emission intensity from these short-lived radical species involves complex mathematical treatment of the electrode diffusion process, which takes into account species stability. Hence quantitative data are difficult to establish. Nevertheless during the 1980s Hill et al. developed IAECL as a detection technique for reverse-phase HPLC, using acetonitrile as the mobile

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phase, and tetrabutylammonium perchlorate and chloride as the supporting electrolyte [17]. Chromatograms were obtained for a variety of polyaromatic hydrocarbons and aromatic pesticide compounds including, DDT, DDE, and Barban, and calibrations performed in the concentration range 10–1000 ng/mL. The limitation of incompatibility with aqueous solutions has recently been partially overcome by Richards and Bard using water-soluble derivatives of polyaromatic hydrocarbons [18]. They observed that the anodic oxidation of sodium 9,10-di- phenylanthracene-2-sulfonate and 1- and 2-thianthrene-carboxylic acid in the presence of tripropylamine as a coreactant produced ECL in aqueous solution. It was speculated that these compounds may be useful as ECL labels in immunoassay or DNA probe studies.

The use of IAECL for analytical applications has now almost entirely been surpassed by techniques based on certain transition metal complexes, from which ECL reactions can occur in aqueous solution.

2.2 Inorganic Electron Transfer ECL

Analytical applications of inorganic electron transfer ECL have almost exclusively focused on the reactions of tris(2,2′-bipyridine) ruthenium(II), shown in Figure 1, or its derivatives. This is because this compound undergoes fully reversible, one-electron, electrochemical redox reactions, at easily attainable potentials, forming stable reduced or oxidized species. These species can then take part in a host of ECL reactions, producing a phosphorescence emission from the excited state of Ru(bpy)32 , which is regenerated in the reaction. Such ECL reactions can be performed in fully aqueous solutions, a range of organic solvents, or mixtures of the two. The reactions take place over a wide pH range, in the presence of oxygen and many other impurities and at room temperature. Ru(bpy)32 ECL reactions exhibit very high efficiencies, and hence are often used as standards

Figure 1 Tris(2,2′-bipyridine) ruthenium(II).

218

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in determining the ECL efficiency of other compounds [19]. Ru(bpy)32 is also relatively nontoxic and not prohibitively expensive. Analytical applications involving Ru(bpy)32 ECL and CL have been the subject of several recent fundamental reviews [12, 14–16].

Ru(bpy)32 itself can undergo electron transfer reactions to produce ECL in an analogous fashion to polyaromatic hydrocarbons, thus;

Ru(bpy)32 → Ru(bpy)33 e	Electrochemical oxidation	(7)
Ru(bpy)32 e → Ru(bpy)3	Electrochemical reduction	(8)
Ru(bpy)33 Ru(bpy)3 → Ru(bpy)32 * Ru(bpy)32	Electron transfer	(9)
Ru(bpy)32 * → Ru(bpy)32 hν (λmax 620 nm)	Chemiluminescence	(10)

The analytical usefulness of this reaction, stems mainly from that fact that the electrochemically generated Ru(bpy)33 species can be reduced by a large number of potential analyte compounds, or their electrochemical derivatives, via highenergy electron transfer reactions, to produce the Ru(bpy)32 * excited species, without the need for an electrochemical reduction step. The converse is also true. The reduction of peroxodisulfate (S2 O82 ) for example, in the presence of Ru(bpy)32 , produces the Ru(bpy)32 * excited species and an ECL emission, from the reaction of Ru(bpy)3 and SO4 • [20]. Although this latter system has been used for the determination of both Ru(bpy)32 [21] and S2 O82 [22], the vast majority of analytical applications use the co-oxidation route.

2.2.1Determination of Oxalate and Other Organic Acid Salts

One of the first analytically useful ECL reactions of Ru(bpy)32 was documented by Chang et al. [23] for the simultaneous oxidation of Ru(bpy)32 and oxalate. The mechanism later proposed by Rubinstein and Bard [24] is as follows;

Ru(bpy)32 → Ru(bpy)33 e	Electrochemical oxidation	(7)
Ru(bpy)33 C2 O42 → Ru(bpy)32 C2 O4 •		(11)
C2 O4 • → CO2 • CO2		(12)
followed by either;
Ru(bpy)33 CO2 • → Ru(bpy)32 * CO2	Electron transfer	(13)
or
Ru(bpy)32 CO2 • → Ru(bpy)3 CO2	Electron transfer	(14)
Ru(bpy)33 Ru(bpy)3 → Ru(bpy)32 * Ru(bpy)32	Electron transfer	(9)
and finally,
Ru(bpy)32 * → Ru(bpy)32 hν	Chemiluminescence	(10)

Electrogenerated Chemiluminescence

219

Oxalate has been determined using this reaction in a diverse range of biological and industrial samples, including vegetable matter [25], urine, blood plasma [26], and alumina process liquors [27]. Other examples include ascorbic acid, which has been determined in soft drinks and fruit juices post HPLC separation by Chen and Sato [28], ECL in this case arising from the reaction of Ru(bpy)33 and a product of the electrochemical oxidation of ascorbic acid. Only a few organic acid salts are capable of being oxidized directly by Ru(bpy)33 in a single electron reaction to form a reducing radical anion. However, the reaction can be extended to related organic acid salts if a more powerful oxidizing agent than Ru(bpy)33 is used. For example, pyruvate has been determined by simultaneously oxidizing Ru(bpy)32 and cerium(III) nitrate in the presence of pyruvate in dilute sulfuric acid [29]. A detection limit of 3 10 7 M was achieved in synthetic samples. Ce(III) is oxidized to Ce(IV), which, unlike Ru(bpy)33 , is able to oxidize the pyruvate to initiate an ECL reaction analogous to that for oxalate. The reaction proceeds as follows;

Ru(bpy)32 → Ru(bpy)33 e	Electrochemical oxidation	(7)
Ce3 → Ce4 e	Electrochemical oxidation	(15)
Ce4 CH3 COCO2 → Ce3 CH3 COCO2 •		(16)
CH3 COCO2 • → CH3 CO • CO2		(17)
Ru(bpy)33 CH3CO • H2O
→ Ru(bpy)32 * CH3 CO2H H	Electron transfer	(18)
Ru(bpy)32 * → Ru(bpy)32 hν	Chemiluminescence	(10)

2.2.2Determination of Amines

The greatest exploitation of Ru(bpy)32 ECL has been in the determination of analytes with an amine functional group, with the highest sensitivities achieved for tertiary and secondary aliphatic amines. A broad range of potential analytes contain amine functionality including many pharmaceuticals, alkaloids, amino acids, and other biologically important molecules. Trialkylamines and closely related compounds are difficult to detect by other methods since they do not absorb well in the ultraviolet/visible region of the spectrum, and are extremely difficult to derivatize. Many amines can be determined by Ru(bpy)32 ECL since, like oxalate, they can be readily oxidized to form species that subsequently react to produce radical products, which in turn are capable of reducing Ru(bpy)33 back to Ru(bpy)32 in an excited state. The mechanism of this reaction has been investigated by many workers [16, 30] and is believed to be as indicated in Figure 2.

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Figure 2 General reaction mechanism for the ECL reaction of Ru(bpy)32 with a tertiary amine.

Since the critical step in the reaction is the oxidation and subsequent deprotonation of the amine, the efficiency of ECL reactions of amines is generally very pH dependent. For example, the variation of ECL intensity with pH for tripropylamine in phosphate buffer is shown in Figure 3. The pH must be sufficiently high to promote the deprotonation reaction of the amine; however, since the maximum ECL signal is generally obtained below the pKa of the amine, it is likely that the acidity of the amine radical cation, and not the basicity of the amine, is most important in determining the pH dependence of ECL [30]. The optimum pH varies from compound to compound but is generally in the range pH 4–9. Higher pHs are generally to be avoided since the CL reaction of Ru(bpy)33 with hydroxide ions produces a significant background signal [16].

The greatest area of applications of this type of ECL has been in the analysis of pharmaceutical compounds with amine functionality. The reader is directed toward the previously mentioned review articles and Table 1 for further details [12, 14–16]. Many methods have also been successfully applied to real samples in the form of body fluids or pharmaceutical preparations, although sample pretreatment such as deproteinization, centrifugation, and neutralization followed by a chromatographic step to remove interfering species is often required. Limits of detection are typically in the range 10 9–10 12 M. Figure 4 shows examples of some classes of pharmaceutical compounds that have been determined by Ru(bpy)32 ECL.

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221

Figure 3 Variation of ECL intensity with pH for the ECL reaction of tripropylamine with Ru(bpy)32 . (From Ref. 31.)

It is not the case, however, that all amine compounds take part in ECL reactions with Ru(bpy)32 . Many amines produce intense ECL, while other closely related compounds produce virtually no ECL emission. While there are no strict rules governing ECL activity in amines, several workers have observed general trends that appear to explain some of these variations [31]. In general, ECL efficiencies increase in the order primary secondary tertiary amines, with tertiary amines having the lowest detection limits. Primary aliphatic amines have been determined by ECL, but most sensitively after prior derivatization with divinyl sulfone, (CH2 CH)2 SO2, to form a cyclic tertiary amine [32]. It is also known that the nature of α-carbon substituents on the amine affects ECL activity. In general, electron-withdrawing substituents, such as carbonyl, halogen, or hydroxyl groups, tend to cause a reduction of ECL activity, probably by making the lone pair of electrons on the nitrogen less available for reaction, or by destabilizing the radical intermediate. Electron-donating groups such as alkyl chains have the opposite effect. Aromatic amines and those with a double bond that can conjugate the radical intermediate consistently give a very low ECL response. This may be due to resonance stabilization of the radical intermediate, rendering it less active toward Ru(bpy)33 . Molecules that are hindered in attaining planar geometry after oxidation, such as quinine, show ECL intensity orders of magni-

Table 1

Survey Demonstrating the Range of Analytes Detectable by ECL, with Examples of Sample Matrices Used and Typical

Limits of Detection

Class of compound

Selected examples

Samples analyzed

Typical LOD

a

Ru(bpy)

3

2

ECL reactions

13

1

Ru(bpy)

2

itself

Ru(bpy)

2

Synthetic

10

mol L

3

3

Organic acids

Oxalate

Synthetic

10

6

mol

L

1

Urine

10

6

mol

L

1

6

1

Blood plasma

10

mol

L

Pyruvate

Synthetic

27 ppb

Amines

Ascorbic acid

Fruit juice

2 pmol

Trialkylamines

Tripropylamine

Synthetic

0.28 pmol

Alkaloids

Nicotine

Synthetic

0.4 pmol

Atropine

Synthetic

1.5 pmol

Codeine

Synthetic

10

11

mol L

1

Industrial process streams

5

10

9

mol

L

1

Pharmaceuticals

Thiazides

Pharmaceutical capsules

0.06 pmol (hydroflumethiazide),

Antihistamines

Pharmaceutical tablets,

2.1 pmol (cyclothiazide)

9 pmol (brompheniramine)

Tricyclic antidepressants

syrup/urine

10

Synthetic

3

7

mol

L

1

(amitriptyline)

Local anesthetics

Pharmaceutical preparation

7

10

8

mol

L

1

(lignocaine)

β blockers

Synthetic

35 nmol L

1

(oxprenolol)

Antibiotics

Urine/blood plasma

0.05 µmol

L

1

(erythromycin)

Amino acids

D-, L-Amino acids

Pharmaceutical preparations

10 ppb (clindamycin)

Synthetic

100 fmol–22 pmol (arginine, va-

Tryptophan

line, leucine, proline)

Blood plasma

0.2 pmol

Dansylated amino acids

Synthetic

0.1 µmol

L

1

(glutamine)

Primary amino acids

Synthetic

0.06 pmol (alanine)–1 pmol (ser-

ine) (derivatized with divinyl

Other biochemicals

NADH

Synthetic

10

sulfone)

6

mol

L

1

Glucose (enzymatically via NADH)

Synthetic

10

6

mol

L

1

Thyrotropin (TSH)

Serum

mIU

L

1

DNA PCR products

Synthetic

10–200 amol

HIV-1 viral RNA

Plasma

6

10

2

RNA copies per 0.1 mL

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Miscellaneous

Ethanol (enzymatically via NADH)

Synthetic

10 µmol

L

1

Benzene (by enhancement of

Synthetic

µg L

1

level

Ru(bpy)

3

2

/oxalate ECL)

0.035 µg

1

Hydrazine (gas phase)

Synthetic

L

1

Virulent bacteria

Food and environmental

1000 cells mL

(Escherichia coli

samples

O157, Salmonella, Bacillus an-

Radical ion–annihilation ECL

thrax spores)

Polyaromatic hydrocarbons

Synthetic

10

5

–10

7

mol L

1

(anthracene,

Barban/DDT/DDE

Synthetic

rubrene, pyrene)

10 ng mL

1

Luminol ECL

mol L

Luminol itself

Luminol

Synthetic

1

10

10

1

Luminol-labeled species

Ibuprofen

Synthetic

0.15 pmol

Peroxides

Histamine

Synthetic

1.5 pmol

Hydrogen peroxide

Synthetic

66 pmol

Metal ion catalysts

Methyl linoleate hydroperoxide

Synthetic

0.3 nmol

Cobalt (II)

Synthetic

10

8

mol

L

1

Copper (II)

Synthetic

10

7

mol

L

1

Biochemicals

Glucose (enzymatically via hydro-

Fruit juice/human serum

8 µg mL

1

Miscellaneous

gen peroxide)

1 µmol L

Sulfite (via inhibition of luminol

Synthetic

1

ECL)

Cathodic luminescence

Atrazine (enzymatic immunoassay)

Synthetic

0.1 ppb

Metal ions

Thallium (I)

Synthetic

10

10

mol

L

1

Copper (II)

Synthetic

5

10

9

mol

L

1

Terbium (III)

Synthetic

10

13

mol

L

1

Organic compounds

Salicylic acid

Synthetic

3

10

8

mol

L

1

Diphenylanthracene

Synthetic

10

8

mol

L

1

Miscellaneous ECL reactions

Antioxidants

Synthetic

1 µmol

1

(α-tocopherol,

Indole/tryptophan

β-carotene)

Synthetic

1

10

7

mol

L

1

Biodegradation products of

Synthetic

1 ppm

	explosives
a	As quoted in the original publication. The list has been compiled from Ref. 12, 15, and 16, but is not exhaustive. The reader is directed to these review
	articles for other examples and further experimental details of those analytes shown here.

Chemiluminescence Electrogenerated

223

Figure 4

224

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Examples of classes of pharmaceutical compounds with amine functionality, determined by Ru(bpy)	3	2	ECL.

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225

tude less than other amines that are able to tend toward trigonal planar geometry, resulting in effective delocalization of the charge. Finally amines that have other functional groups that are more readily oxidized than the nitrogen atom may take part in alternative, ultimately nonchemiluminescent reactions.

2.2.3Determination of Amino Acids

The determination of amino acids by ECL is seen as having many potential applications, including protein sequencing, and has been investigated extensively by Bobbitt et al. [33, 34] among others [35]. Most amino acids have no inherent chromophore or fluorophore, and hence usually have to be derivatized prior to conventional spectroscopic determination. However, amino acids encompass a diverse group of compounds, with a correspondingly diverse range of ECL activities. Proline, a secondary aliphatic amine, has been determined with the highest sensitivity by Ru(bpy)32 ECL, with a detection limit of 100 fmol M [34]. Many other amino acids have been determined in the range 10 6–10 9 M. Again, in general, amino acids with electron-withdrawing α-carbon substituents and aromatic amines give lower ECL responses. Amino acids containing indole or imadazole functional groups, however, generally give higher ECL responses. For example, Uchikura and Kirisawa [36] determined D- and L-tryptophan enantiomers in plasma by HPLC-ECL, with a limit of detection of 0.2 10 12 M. Interestingly, their method is selective for tryptophan under acidic conditions, approximately pH 3, where interference from other amino acids and amines is diminished.

Enhancements in the sensitivity with which amino acids containing a primary amine group can be determined have been achieved by derivatization. Chen and Sato [37] reported derivatization with divinyl-sulfone-reduced limits of detection by several orders of magnitude, while Lee and Nieman [38] reported derivatization with dansyl-chloride-reduced limits of detection by a factor of three.

2.2.4Other Ru(bpy)32 Analytical Applications

NADH, containing a tertiary amine functional group, has been readily determined by Ru(bpy)32 ECL. However the oxidized form, NAD , containing an aromatic secondary amine group produces virtually no ECL signal. This had led to a variety of indirect enzymic methods of analysis, where the activity of the enzyme results in the conversions between NAD and NADH. These are discussed in Sec. 8.

Ru(bpy)32 itself can be determined with great sensitivity in an excess of an amine to subpicomolar levels [39]. This has led to the development of electrochemiluminescent labels based on Ru(bpy)32 derivatives that have found successful applications in ECL immunoassay and DNA probe analysis. These are discussed in Sec. 9.