Chemiluminescence in Analytical Chemistry

116	Rakiciog˘lu et al.

commonly used as a reporter enzyme in various assays. Previously, the only significant chemiluminescent reagent for detecting HRP was the enhanced luminol detection system. However, the level of concentration at which this enzyme can be detected with CL detection is at least three orders of magnitude lower than that of other marker enzymes. A wide variety of N-alkylacridancarboxylic acid derivatives, including esters, thioesters, and sulfonamides, are effectively oxidized by a peroxidase and a peroxide to enzymatically produce the corresponding chemiluminescent acridinium compound. Use of these substrates provides a more sensitive chemiluminescent detection of HRP and also has a longer signal duration compared to the luminol system [58].

A chemiluminescent assay of β-galactosidase in coliform bacteria, using a phenylgalactose-substituted 1,2-dioxetane derivative as a substrate has been developed by Poucke and Nelis. The assay of β-galactosidase is important in clinical, environmental, food, and molecular microbiology. There are chromogenic and fluorogenic tests that are commonly used to assay β-galactosidase; however, they require visual or spectrophotometric detection of colored reaction products, and therefore have a limited sensitivity. This limited sensitivity is of no concern in a confirmatory test of bacterial isolates because the abundance of available cells allows a rapid cleavage of the substrate. However, when a small number of bacteria have to be detected in a sample, such as in drinking water, an incubation period of at least 24 h is needed to allow sufficient bacterial propagation and enzymatic hydrolysis before any color becomes detectable. Using a chemiluminogenic substrate, such as a phenylgalactose-substituted 1,2-dioxetane derivative, as a substrate would create a substantial gain in sensitivity compared to both chromogenic and fluorogenic tests. This is a powerful substrate for the sensitive assay of bacterial β-galactosidase in samples containing small numbers of cells [59].

Marley and Gaffney have constructed a hydrocarbon analyzer on the basis of the CL reaction with ozone. The detector is designed to operate at various temperatures to take advantage of the different rates of reaction of the hydrocarbon classes with ozone to yield a measure of their atmospheric reactivity. This research was initiated by the growing concern with regard to the atmospheric reactivity of hydrocarbon emissions from fossil-fuel-powered machinery. These hydrocarbons have been increasing the levels of tropospheric ozone and other oxidants. Tropospheric ozone is a concern because of its impact on human health and its effects on plants and crops. Other oxidants can play important roles in the formation of acid rain and other global climate effects. Thus, a need to characterize both the reactivities and concentrations of reactive hydrocarbon emissions exists to better evaluate their atmospheric impact. Marley and Gaffney’s chemiluminescent hydrocarbon analyzer represents a new concept in measuring the reactive hydrocarbons in the atmosphere based on the temperature dependences of their reactions with ozone. CL techniques lend themselves readily to real-time

Table 3	Determination of Some Drugs Using Direct Chemiluminescence
Analyte	Method	Limit of detection	Range

Tetracycline	Reaction with bromine in alkaline
	medium, flow injection analysis
Acetaminophen	Reduction of cerium (IV), flow in-
	jection analysis
Cortisone	Sensitization of CL of sulfite by ce-
Hydrocortisone	rium (IV), flow injection anal-
Prednisolone	ysis
Methylprednisolone
Dexamethasone
Betamethasone
Quinine	Sensitization of CL of sulfite by ce-
	rium (IV), flow injection anal-
	ysis
Isoniazid	Oxidation with N-bromosuccini-
	mide
Morphine	Reaction with permanganate, flow
	injection analysis, high-perfor-
	mance liquid chromatography

Loprazolam	Reaction with permanganate, flow
	injection analysis

19 g/mL

5 10

5

–1 10

2

M

(4 10

5

M)

0.070 g/mL

1.00–10.0 g/mL

0.040 g/mL

0.028 g/mL

0.100–1.00 g/mL

0.016 g/mL

0.021 g/mL

0.16 g/mL

0.500–5.00 g/mL

0.30 g/mL

0.64 g/mL

5.00–500 g/mL

0.024 g/mL

0.050–20.0 g/mL

0.7 pg (2 fmol, 1

10

10

M) for

50 ng/mL–500 g/mL

flow injection analysis.

25 ng/mL, 50 ng/mL (aqueous so-

lution, biological fluids, for

high-performance liquid chroma-

tography

mol/50 L)

163 ng (7

10

6

1 10

5

M–1

10

3

M

Buprenorphine HCl Reaction with permanganate, flow	5 ng/mL	1 10	8	–1	10	4	M
injection analysis

Ref.

62

63

64

65

66

67, 68

69

70

Analysis Organic in Chemiluminescence

117

118			Rakiciog˘lu et al.
Table 4 Drugs Analyzed with a Chemiluminescent System
		Limit of
Analyte	Method	detection	Range	Ref.
Penicillin	Enhancement of luminol	100 ng/mol	100 ng/mL–100 g/mL	71, 72
	chemiluminescent re-
	action, batch method
Cephalothin	Enhancement of luminol	—	—	73
sodium	chemiluminescent re-
	action, batch method
Hydrocortisone	Solid-state peroxyoxalate	2 ng/mL	—	74
Digoxin	CL, high-performance	2 ng/mL
Theophyllin	liquid chromatography	4 ng/mL

monitoring of atmospheric species, while being selective due to the relatively small number of compound classes that will produce a chemiluminescent reaction with ozone [60].

Chemiluminescence has become a powerful tool for drug determination. It has a wide range of applications, and low detection limits can be measured with a simple, low-cost instrument. The coupling of flow injection with CL has made this technique more popular with wider applications. Sultan and Almuaibed report the CL of medazepam in an oxidation reaction with permanganate in a sulfuric acid medium. A photomultiplier tube was used to detect emitted light. Compounds commonly used in pharmaceutical preparations, such as starch, glucose, maltose, and lactose, had no effect on CL. The coupling of flow injection with CL has allowed the assay of medazepam. Using the simplex optimization for chemiluminescent measurements they were able to determine optimum conditions for the determination of the drug in aqueous solution [61].

Some drugs will emit light under specific reaction conditions. The discovery of these direct chemiluminescent reactions has come about through trial and error. Table 3 lists some of these drugs and the method used for analysis. Other drugs that are not themselves chemiluminescent can be easily analyzed using a chemiluminescent system (Table 4).

4. ULTRAWEAK CHEMILUMINESCENCE

Some organic reactions produce ultraweak chemiluminescence. Grignard reagents [75, 76] and indole derivatives [13], for example, have very low φCL (10 5– 10 8) when they are oxidized. Many cellular systems also produce this dim or low-level chemiluminescence during phagocytosis [77], chemotaxis, and mitosis

Chemiluminescence in Organic Analysis				119
Table 5 Ultraweak Chemiluminescence of Drugs
Analyte	Method	LOD	Range	Ref.
Imipramine HCl	Auto-oxidation	—	—	80
Clomipramine HCl	Auto-oxidation	—	—	81
Trimipramine maleate
Desipramine HCl
Clocapramine diHCl
Carpipramine diHCl
Pepleomycin	Auto-oxidation	—	—	82
Mytomycin C
Neocarzinostatin	Auto-oxidation	—	—	83

[75, 78, 79]. Several drugs can be analyzed by their ultraweak chemiluminescence. Some examples are given in Table 5.

Other drugs, for example lofepramine HCl, carpipramine, warfarin, and triprolidine HCl, have also shown ultraweak CL intensity [84]. The measurement of ultraweak CL entails integration of the signal over the entire course of the CL reaction to produce analytically useful results. Alternatively, an acceptor fluorophore may be employed for analysis as in the case of dioxetanes. The ultraweak CL is probably generated by the formation of peroxide and hyperperoxide intermediates upon auto-oxidation. Specific functional groups have been tested for ultraweak CL in simple organic compounds [85]. The emissions of alkynes, aliphatic amines, aliphatic aldehydes, epoxides, and peroxides were demonstrated [75, 76]. An ultraweak CL reaction between an amino group and a carbonyl group was also observed [86]. This reaction has been used to quantitate amino acid derivatives.

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6

Application of Chemiluminescence in Inorganic Analysis

Xinrong Zhang

Tsinghua University, Beijing, P.R. China

Ana M. Garcı´a-Campan˜a

University of Granada, Granada, Spain

Willy R. G. Baeyens

Ghent University, Ghent, Belgium

1.	INTRODUCTION		124
2.	POSITION OF INORGANIC CL ANALYSIS AMONG CURRENT
	TECHNIQUES		125
3.	PROGRESS IN ANALYTICAL CL METHODOLOGY		126
	3.1	CL Measurements Based on Direct Oxidation, Catalysis,
		and Inhibition Effects	126
	3.2	CL Measurements Based on Reduction Reactions	128
	3.3	CL Measurements Based on Coupling Reactions	128
	3.4	CL Measurements Based on Time-Resolved Techniques	131
	3.5	CL Measurements on Solid Surfaces	132
	3.6	Hyphenated Techniques: Coupling HPLC/CE to CL
		Detection	133
4.	CONCLUSIONS		136

123

124	Zhang et al.

1. INTRODUCTION

In the past years, chemiluminescence (CL) analysis of inorganic compounds has been extensively developed in both gas and liquid phases. These methods typically rely on the oxidation or reduction of a chemically reactive agent and the subsequent emission of a photon from an electronically excited-state intermediate.

The primary attraction of CL detection of inorganic compounds is the excellent sensitivity that can be obtained over a wide dynamic range using simple instruments. A detection limit of 40 pg/mL gold in ore samples reported by us is a striking example to indicate the advantage of CL detection [1].

The vast majority of work done in the CL field has been devoted to solutionbased flow analysis formats [2, 3]. The classical CL reagents for inorganic analysis include mainly luminol and lucigenin, among others. A number of new CL systems have also been explored in recent years for the determination of inorganic ions. Three types of CL reactions are currently being applied for analytical purposes: (1) based on the CL reaction between a reductant and an oxidant, e.g., luminol and I2; (2) based on the catalytic behavior metal ions, e.g., Cr(III), Co(II), and Cu(II), . . . on the luminol-H2 O2 CL reaction; (3) the determination of inorganic ions based on inhibiting effects (due to the limited number of available CL reactions).

Although CL analysis belongs to the powerful techniques for the determination of inorganic compounds due to high simplicity and rapidity, a limitation to the widespread application of this technique often lies in the poor selectivity of the CL detection as such, which is commonly based on the use of established CL reagents. It is essential to separate the analyte species from other ions or sample matrix components to improve selectivity of CL-based analysis. This can be achieved by online coupling liquid chromatography (LC) [4, 5] or capillary electrophoresis (CE) [6, 7] to a CL detector, although incompatibility of separation conditions and postcolumn reaction conditions may present unexpected problems.

A substantial number of papers have been published between the ’60s and the ’90s on the determination of inorganic analytes by CL-based techniques. The application of established methods to the analysis of inorganic compounds involves the areas of environmental, geographical, and biological sciences. Although many efforts have been undertaken in the past years, there still remains a challenge to apply CL-based techniques to routine analysis of inorganic elements, as the complex matrix of a real sample may cause unexpected effects on CL emission.

The aim of this chapter is to briefly introduce the methodology and application of CL-based techniques to the analysis of inorganic analytes. Since the number of relevant CL papers on inorganic analysis is large, only keynote references

Chemiluminescence in Inorganic Analysis

125

will be given. As many papers published in recent years originate from Chinese scientists, important selected sources from Chinese journals (in Chinese) had to be included.

2.POSITION OF INORGANIC CL ANALYSIS AMONG CURRENT TECHNIQUES

Despite the relative large number of sensitive analytical techniques for element determinations, CL still occupies a position as one of the more sensitive techniques for trace analysis of inorganic compounds. So far, only a few of techniques have been compared with CL detection. Table 1 illustrates the detection limits of inorganic analytes using different techniques including CL, atomic absorption spectrometry (AAS), inductively coupled plasma optical emission spectrometry (ICP-OES), and ICP–mass spectrometry (ICP-MS) for some typical metal ions. Although the techniques listed in Table 1 are considered the most sensitive techniques available today, there still exists no technique that exceeds the detection limits offered by CL for the determination of the elements, e.g., Au, Co, Cr, etc. Worth noticing is that the energy required for CL emission is produced by a chemical reaction; hence an excitation source and a spectral resolving system become unnecessary, leading to relatively simple equipment and low costs of instrumentation. A comparison of the prices of commercially available instruments as indicated in Figure 1 shows this advantage of the CL technique.

However, some intrinsic limitations and drawbacks of the CL technique should be taken into consideration. As listed in Table 1, for most analytes preference is given to only a few CL systems such as the luminol-H2 O2 system. In many cases only poor selectivity is obtained when determining a typical element.

Table 1 Comparison of Detection Limits Offered by Important Analytical Techniques for Selected Inorganic Compounds (ng/mL)

Element	Au	Co	Cr	Cu	Mn
CLa (luminol system)	0.04	0.0074	0.0015	0.009	0.08
AASb	20	2	2	4	0.8
ICP-OESc	0.6	0.21	0.15	0.18	0.05
ICP-MSd	0.06	0.01	0.01	0.02	0.03

aSee Table 2.

bData obtained from W. Robinson, Atomic Spectroscopy, New York: Marcel Dekker, 1990.

cData obtained from the manual of JY-Ultima ICP-OES (John Yvon Emission, Longjumeau Cedex, France).

dData obtained from the manual of Perkin Elmer ICP-MS (Perkin Elmer Corporation, Foster City, CA).