Chemiluminescence in Analytical Chemistry

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depending on the way the irradiation penetrated, which in fact had already been observed many years before from inorganic ‘‘phosphors.’’ He demonstrated that there was no dispersion, superficial or internal, rejecting at the same time the term ‘‘dispersion’’ suggesting to replace the latter by ‘‘fluorescence,’’ as derived from a certain variety of fluorspar (fluorite) that showed blue reflections, similar to what was observed from solutions of quinine sulfate [12, 14, 15].

Numerous substances that produced fluorescence were examined by Stokes: plant extracts (e.g., chestnut rind, chlorophyll in water), glass, paper, animal material, uranium compounds, etc., and he pointed out that ‘‘the rays produced by the fluorescence process were much more ‘‘refrangible’’ than the rays initiating them.’’

Yellow light is much more deflected by a prism than blue light, as a result of which more pronounced yellow fluorescence is induced by the blue, but never the other way round. Hence violet and ultraviolet radiation are most active in many cases of fluorescence [12].

In the previously mentioned publication by Stokes, apart from introduction of the term ‘‘fluorescence,’’ the concept of fluorescence being emission of light was proposed, being the first to clearly define fluorescence as a process of emission. He worked out the technique for observing fluorescence using filters of various colors, one to allow the exciting light to impinge on the compound, and one to observe the emitted fluorescence, and he developed the physical statement that is actually known as the ‘‘Stokes law’’; namely the wavelength of the emitted light is higher than that of the exciting light. It is worth mentioning that the Stokes law from 1852 is valid, for example, for the phosphorescence from the Bolonian stone as well as for the fluorescence of solutions of quinine sulfate, and that, for the first time, two types of (photo)luminescent phenomena were comprised, being phosphorescence and fluorescence, until then considered independent [15, 16].

Stokes observed that the fluorescent emissions from certain crystals were polarized, although he did not detect polarized fluorescence emerging from solutions [17].

In a later work, Stokes established the relationship between the intensity of fluorescence and the concentration, pointing out that the emission intensity depended on the concentration of the sample (analyte), but that attenuation of the signal occurred at higher concentrations as well as in the presence of foreign substances. He actually was the first to propose, in 1864, the application of fluorescence as an analytical tool, based on its sensitivity, on the occasion of a conference given previously in the Chemical Society and the Royal Institution, and entitled ‘‘On the Application of the Optical Properties to the Detection and Discrimination of Organic Substances’’ [5].

In 1867 Goppelsro¨der introduced the term ‘‘Fluoreszenzanalyse’’ (analysis by fluorescence or fluorimetry) and proposed the first fluorimetric analysis in

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history: the determination of Al(III) by the fluorescence of its chelate with morin [5]. In 1889 he proposed the capillary analytical technique using ultraviolet light. This technique, which was frequently used in paper and thin-layer chromatography, was again put into practice by Danckwortt and Pfau in 1928 [4].

By the end of the nineteenth century around 600 fluorescent compounds had been identified [3], including fluorescein (A. von Baeyer, 1871), eosine (H. Garo, 1874), and polycyclic aromatic hydrocarbons (C. Liebermann, 1880) [5]. Although it is generally accepted that fluorescence markers are relatively new analytical benefits, it is surprising to note that their chemical synthesis is rather old, such as the fluorescein reported by Baeyer, the 2,5-diphenyloxazole by Fisher in 1896, and the fluorene by Berthelot in 1867 [18].

In 1888, Walter studied the quenching of fluorescence, by the concentration effect, of fluorescein solutions. Nicols and Merrit observed in 1907, in solutions of eosine and resorufine, the symmetry existing between their absorption and fluorescence spectra. In 1910, Ley and Engelhardt determined the fluorescence quantum yield of various benzene derivatives, values that were still referred to until recent years [18]. The works by Lehmann and Wood, around 1910, marked the beginning of analysis based on fluorescence [4].

Edmond Becquerel (1820–1891) was the nineteenth-century scientist who studied the phosphorescence phenomenon most intensely. Continuing Stokes’s research, he determined the excitation and emission spectra of diverse ‘‘phosphors,’’ determined the influence of temperature and other parameters, and measured the time between excitation and emission of phosphorescence and the duration time of this same phenomenon. For this purpose he constructed in 1858 the first ‘‘phosphoroscope,’’ with which he was capable of measuring lifetimes as short as 10 4 s. It was known that lifetimes considerably varied from one compound to the other, and he demonstrated in this sense that the phosphorescence of Iceland spar stayed visible for some seconds after irradiation, while that of the potassium platinum cyanide ended after 3.10 4 s. In 1861 Becquerel established an exponential law for the decay of phosphorescence, and postulated two different types of decay kinetics, i.e., exponential and hyperbolic, attributing them to monomolecular or bimolecular decay mechanisms. Becquerel criticized the use of the term fluorescence, a term introduced by Stokes, instead of employing the term phosphorescence, already assigned for this use [17, 19, 20]. His son, Henri Becquerel (1852–1908), is assigned a special position in history because of his accidental discovery of radioactivity in 1896, when studying the luminescence of some uranium salts [17].

The term ‘‘luminescence’’ (Latin ‘‘lucifer,’’ meaning ‘‘light carrier,’’ cf. emission of cold light) was introduced in 1888 by Eilhardt Wiedemann to distinguish between the light proceeding from the thermal excitation of substances, and the emission of light by molecules excited by other means without increasing their kinetic energy. He stated that the phosphorescence and fluorescence phe-

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nomena indicate that compounds are capable of emitting light without necessarily having been heated up. In other words, Wiedemann characterized luminescence by the fact that this ‘‘cold light’’ does not obey Kirchoff’s laws of thermal absorption and emission by black bodies. He observed phosphorescence from colored aniline derivatives in ‘‘solid’’ solutions and gelatines. He mentioned that double salts of platinum emitted polarized light when excited by cathode rays, and specified that the luminescence could be initiated by various types of excitation, proposing that along with these modes of excitation, six different classes of luminescence are to be considered. Based on the mechanisms of excitation, which are much better understood nowadays, the present-day classification of luminescence phenomena is essentially the same as proposed by Wiedemann [5, 17].

In fact, an important advance in the phosphorescence theory was realized by Wiedemann in 1889, stating that a ‘‘phosphor’’ exists in two forms, a stable one, A, and an unstable one, B. Light absorption brings along conversion of form A to B, which then returns to A emitting light. This hypothesis was in agreement with the exponential decay law as postulated years before by Becquerel, but who did not provide any information about the nature of both forms [5].

In 1935, after studying the luminescence of various colorants, Jablonski suggested the ‘‘electronic energy diagram’’ of the singlet and triplet states to explain the luminescence processes of excitation and emission. The proposed diagram of molecular electronic energy levels formed the basis of the theoretical interpretation of all luminescent phenomena [21].

Spectroscopists interested in elucidation of the molecular energy schemes studied the phosphorescence emission of over 200 compounds, of which 90 were tabulated by Lewis and Kasha in 1944. They classified phosphorescing substances in two classes, based on the mechanism of phosphorescence production. The first group comprises minerals or crystals named ‘‘phosphors,’’ where the individual molecule is not phosphorescent as such, but emits a shining associated with the presence of some impurity localized in the crystal. This type of phosphorescence cannot be attributed to a concrete substance. The second type of phosphorescence emission is attributed to a specific molecular species, being a pure substance in crystalline form, adsorbed on a suitable surface or dissolved in a specific rigid medium [22].

Lewis and Kasha identified phosphorescence as a forbidden transition from the triplet to the lowest singlet state, and suggested the phosphorescence emission spectrum as an analytical tool to assess molecular identification. Each phosphorescence phenomenon is unique with respect to frequency, lifetime, quantum yield, and vibrational pattern (band spacing). Moreover, as phosphorescence characteristics are a unique property of each organic species, they suggested application of this phenomenon for identification of mixtures [22]. Hence, the development of phosphorescence as analytical technique was showing up in the analytical investigations describing metastable phosphorescence or triplet-state

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emission together with a description of the paramagnetic nature of this luminescence phenomenon [22–25].

James Dewar observed in 1894 phosphorescence from frozen solutions utilizing liquid air [5]. Jean Becquerel discovers in 1907 that samples frozen at liquid air temperatures considerably narrow the spectral shape and increased information is obtained from the luminescence spectra [26].

Lewis et al. stated in 1941 that when a liquid is frozen the phosphorescence intensity increases along with an increase of viscosity [27]. Some of the works on phosphorescence were done, before 1940, in frozen aqueous solutions, and during the fifties for studying compounds of biochemical importance [28, 29]. However, it was demonstrated quite rapidly that it was more advantageous to use organic solvents to create transparent, rigid glassy matrices to measure phosphorescence in. In fact, the requirement of freezing conditions for phosphorescence measurements represented a major disadvantage in the use of this luminescence-based analytical technique. Later developments applied the technique of room temperature phosphorescence (RTP). As a matter of fact, Schmidt already observed in 1896 phosphorescence from colorants, adsorbed on solid gels. Without any doubt, historically this is the first observation of RTP [30].

It is worth noting some historical aspects in relation to the instrumentation for observing phosphorescence. Harvey describes in his book that pinhole and the prism setup from Newton were used by Zanotti (1748) and Dessaignes (1811) to study inorganic phosphors, and by Priestley (1767) for the observation of electroluminescence [3]. None of them were capable of obtaining a spectrum utilizing Newton’s apparatus; that is, improved instrumentation was required for further spectroscopic developments. Of practical use for the observation of luminescence were the spectroscopes from Willaston (1802) and Frauenhofer (1814) [13].

Before the nineteenth century and during part of the latter, the observations of fluorescence occurred visually and in the course of this century photographic observation is being proposed. However, for measuring the intensity of fluorescence, until around 1930 methods based on visual comparison were used. Desha realizes in 1920 the first quantitative measurement of fluorescence, using a nephelometer with variable optical pathway (similar to the Duboscq colorimeter), and in a later work he states that at low concentrations, the fluorescence intensity increases linearly with the concentration [31]. Utilizing an instrument of the visual comparison type—a prism spectrometer—Bayle, Fabre, and George measure in 1925, employing a tedious procedure, the fluorescence emission spectra of a great number of drugs [5]. Gaviola mentions in 1927 the construction of the first phase fluorimeter, based on the phenomenon that the intensity of the exciting source radiation modulates sinusoidally. With this apparatus he measured the lifetime of rhodamine B (2 ns) and of fluorescein (4.5 ns), values that are still accepted [32].

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The first photoelectric fluorimeter was described by Jette and West in 1928. The instrument, which used two photoemissive cells, was employed for studying the quantitative effects of electrolytes upon the fluorescence of a series of substances, including quinine sulfate [5]. In 1935, Cohen provides a review of the first photoelectric fluorimeters developed until then and describes his own apparatus using a very simple scheme. With the latter he obtained a typical analytical calibration curve, thus confirming the findings of Desha [33]. The sensitivity of these photoelectric instruments was limited, and as a result utilization of the photomultiplier tube, invented by Zworykin and Rajchman in 1939 [34], was an important step forward in the development of suitable and more sensitive fluorometers. The pulse fluorimeter, which can be used for direct measurements of fluorescence decay times and polarization, was developed around 1950, and was initiated by the commercialization of an adequate photomultiplier [35].

The first complete commercial spectrofluorimeter was manufactured by the American Instrument Company (Aminco), based on a design published by Bowman, Caulfield, and Udenfriend in 1955. The appearance of this commercial model instrument was of utmost importance for spectrofluorimetric investigations by numerous chemists, biologists, and biochemists [5].

In spite of the suggestions made by Lewis and Kasha in 1944, the analytical applications of phosphorescence appeared in the next decade only, when suitable phosphoroscopes were available, employing practical modifications of the Becquerel phosphoroscope, whose scheme can be found in the monograph by Bernard [20]. Keirs et al. created and utilized a resolution phosphoroscope, based on suggestions by their laboratory companion M. Kasha, which allowed them to distinguish the phosphorescence emission from the exciting light, and the simple resolution of mixtures of phosphorescent compounds based on their lifetimes. In 1957 they published the first work on quantitative phosphorescence, stressing two important aspects: obtaining phosphorimetric analytical curves of diverse organic molecules and the quantitative analysis of mixtures of two or three components by means of selective excitation, phosphoroscopic resolution, and simultaneous equations. They could phosphoroscopically and spectroscopically resolve mixtures of benzaldehyde, benzophenone, and 4-nitrobiphenyl; the phosphoroscopic resolution of mixtures of acetophenone and benzophenone; and the determination of mixtures of diphenylamine and triphenylamine by means of selective excitation. As solvent they used EPA (a mixture of ethyl ether, isopentane, and ethyl alcohol in a volume ratio of 5:5:2) at liquid nitrogen temperatures, behaving as a clear and transparent glass [36].

In 1962, Parker and Hatchard described a photoelectric spectrometer for phosphorescence measurements with which they were capable of obtaining phosphorescence spectra, and of determining lifetimes and quantum efficiencies of a large number of organic compounds. This work stimulated intensely the interest in the phosphorimetry of diverse chemical analytes [5], and one year later, Wine-

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fordner and Latz proposed a phosphorimetric method for determination of aspirin in blood [37]. The development of a phosphorimetric method for aspirin in blood was the first application to a ‘‘real’’ sample and it contributed very much to the further acceptance of phosphorimetry [5]. Since then, phosphorimetry has been developing as a full analytical technique, which, when compared to fluorimetry, often is more sensitive for specific organic molecules and sometimes provides complementary information about the structure, reactivity, and surrounding requirements.

However, since the second half of the eighties, practically no more phosphorescence appears, at least in the analytical literature for quantitative estimations, being nearly completely substituted by sophisticated fluorescence and laserinduced fluorescence methods, mostly applied as detection tools in diverse flowing streams.

2.THE DISCOVERY OF BIOLUMINESCENCE AND CHEMILUMINESCENCE PHENOMENA

The significance of oxygen in bioluminescence (BL) was first established by Robert Boyle in 1669, who carried out experiments with shining wood, fish, and flesh and found that the emitted light was largely reduced and in some cases disappeared on removal of air [38, 39]. Although Boyle was not aware of the existence of oxygen, as it was discovered independently by Scheele and Priestley over 100 years later, this was the first experimental demonstration that oxygen, or one of its derivatives, is required in all known bioluminescent reactions and most artificial organic CL. By that period, it was not realized that living organisms were responsible for the shining of wood and flesh. In fact, proof that the glowing or shining of the latter was caused by a luminous fungus and luminous bacteria, respectively, was first reported by Heller in 1843. The requirements for oxygen in the bioluminescent reactions can be explained by the very high affinity for oxygen of some enzymes involved in this kind of luminescent processes.

Explanation of the mechanisms of BL systems begins in 1821, when Macaire suggested that the source of light in the glowworm might be some organic compound, rather than the inorganic phosphor, as previously assumed. In his studies, he observed that all chemical reagents that caused albumin to coagulate also extinguish the glowworm’s light and concluded that the luminous material was composed mainly of albumin and required oxygen [40]. Following the study of living organisms that emit light, Pasteur reported in 1864 the spectrum of light from the tropical luminous beetle Phyrophorus as continuous, without a dark or light band [3]. In 1885, Dubois stimulated the interest in BL by carrying out a series of experiments using these luminous beetles and the luminous rock-boring bivalve Pholas [41–43]. He obtained a cold-water and hot-water extract form

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Phyrophorus, which, when mixed together, reacted to produce light. He showed that luminescence was the result of a chemical reaction that requires a heat-stable factor, named luciferine, and a heat-labile factor, named luciferase. He was able to demonstrate that both compounds comprised an enzyme-substrate system, which required the presence of oxygen. From this period on, in which luminescence bacteria were used analytically for the first time by Beijerinck to detect small amounts of oxygen, BL reactions have been widely studied in this kind of organism, mainly by Harvey. He traveled around the world observing, collecting, and describing bioluminescent organisms and his publications still provide the most comprehensive description to date of the distribution of luminescence in nature [3, 44, 45].

3. STUDY OF THE CHEMILUMINESCENT SYSTEMS

In the mid-nineteenth century the chemiluminogenic capacity of simple organic compounds was discovered. By 1880, Radziszewski elaborated a long list including synthetic chemiluminescing organic compounds and compounds of biological origin, such as terpenes, cholates, and fatty acids and in the same year he was able to obtain the first CL spectrum of a synthetic organic compound.

In his paper dating from 1877, Radziszewski reported for the first time on the CL exhibited by the synthetic organic compound lophine (2,4,5-triphenylimi- dazole). He found that lophine emitted green light when it reacted with oxygen in the presence of strong base [46]. In the same year, Eder accidentally observed the luminescence of alkaline pyrogallol when it was employed as a developer for photographic plates [47] (Fig. 1).

The term ‘‘chemiluminescence’’ was not introduced until 1888, when Wiedemann defined the term ‘‘luminescence.’’ He was able to classify luminescence phenomena of six different kinds, according to the manner of excitation: photoluminescence, caused by the absorption of light, electroluminescence, produced in

Figure 1 Chemical structure of (A) lophine (2,4,5-triphenylimidazol) and (B) pyrogallol.

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gases by an electric discharge; thermoluminescence, produced by slight heating; triboluminescence, as a result of friction; cristalloluminescence, as a result of crystallization; and chemiluminescence, caused by a chemical reaction [48].

Many more CL reactions were discovered during the early twentieth century. In 1905, Trauzt described in an extensive report the known examples of CL and systematically reported the luminescent properties of the reactions from several hundred organic compounds with various oxidants. In this study, some of the earliest investigations of the spectral distribution of the emitted light were performed and the emission was attributed to some form of ‘‘activated’’ oxygen [49]. In the same period, Wedekind reported the first luminescent assay with a Grignard reagent. He described the brilliant green emission observed when an ether solution of phenylmagnesium bromide (Fig. 2) or iodide reacted with chloropicrin [50]. Following these studies, Hezcko reported that Grignard reagents emitted visible light in the presence of oxygen [51]. He carried out his luminescent experiment during a lecture demonstration in front of a large auditorium. Some years later, Dufford, Evans, and co-workers systematically investigated the CL properties of a large number of Grignard compounds to establish a relation between the intensity of light emission and the chemical structure [52–54]. Also, in 1927 light emitted during electrolysis was observed by Dufford et al. [55] for solutions of Grignard compounds in anhydrous ether.

In 1912 Delepine observed light generated in the gaseous phase from the vapors of some phosphorus-sulfur compounds in the presence of oxygen [56]. Two years later, Bancroft published a paper on the luminescence generated at mercury and other electrodes in the electrolysis of halides [57].

The chemistry of siloxenes and their light emission properties were studied by Kautsky et al. [58–60]. These complex silicon compounds are highly polymerized solids that have a permutoid structure, forming an isolated network that has a thickness the size of a molecule and where all reacting groups are quantitatively accessible for external agents (Fig. 3). These substances were first prepared more than a century ago by Wo¨hler, using calcium silicide and concentrated hydrochloric acid [61]. They emitted a bright light when a suspension of the siloxene in dilute acid was treated with strong oxidizing agents such as permanganate, ceric compounds, or nitric acid, the color and intensity of the emission varying strikingly with time.

Figure 2 A Grignard compound: p-chlorophenylmagnesium bromide.

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Figure 3 Chemical structure of (A) siloxene and (B) aesculin.

Mallet reported in 1927 that the intensity of light emitted in the reaction of hydrogen peroxide and the hypochlorite ion was enhanced when eosin, fluorescein, anthracene, quinine sulfate, or aesculin (Fig. 3) was added to the reaction medium [62].

Although the synthetic substance luminol was discovered in the midnineteenth century, it was not until 1928 that it was reported by Albrecht, who described the intense luminescence associated with the alkaline oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and other N,N-diacylhydrazides [63] (Fig. 4). Soon after, Harvey [64] observed the light emitted during its electrolysis in alkaline solution at the anode. In 1934, the name luminol was given to this compound [65] and in 1936, confirmation of previously reported findings was made; it was reported that the reactions with hematin were the most intense [66]. The first proposal for the use of luminol in medicolegal investigations as a presumptive test for blood was reported by Specht in 1937 [67] and studied and confirmed in 1939 [68]. Most notable was the finding that dried, decomposed, and generally older bloodstains produced a much more brilliant and longer-lasting reaction with luminol than did fresh blood. Applying fresh luminol spraying— after allowing the previous applications to dry—can reactivate the luminescence; hematin can be detected in a dilution of 1:108. Luminol is best employed to

Figure 4 Chemical structure of (A) luminol (5-amino-2,3- dihydro-1,4-phthalazinedi- one) and (B) isoluminol.

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Figure 5 Chemical structure of lucigenin (10,10′- dimethyl-9,9′-biscridinium (nitrate)).

detect trace quantities of blood that are not visible to the naked eye, e.g., areas intentionally wiped clean of blood, washed clothes, dark surfaces, cracks and crevices, plumbing segments, and large areas to be screened, being used in criminal investigations [69]. Since then, several luminol derivatives have been synthesized, the largest CL quantum yield being shown from a benzoperylene derivative [70]. Also, isoluminol derivatives such as aminobutylethylisoluminol (ABEI) were synthesized in 1978 by Schroeder et al. [71] and subsequently widely applied in analytical chemistry.

One of the more efficient CL substances, lucigenin (10,10′-dimethyl-9,9′- biscridinium nitrate), was discovered by Gleu and Petsch in 1935 (Fig. 5). They observed an intense green emission when lucigenin was oxidized in an alkaline medium [72]. Other acridinium derivatives were shown to produce CL emission upon hydrogen peroxide oxidation of aqueous alkaline solutions. The main reaction product was N-methylacridone, acting as an active intermediate in the mechanism proposed by Rauhut et al. [73, 74] (Fig. 6).

Figure 6 Chemiluminescent mechanism of acridinium salts.