red-Chemiluminescent

Effect of some aminoanthraquinone derivatives as red fluorescers on chemiluminescence systems originating from bis-(2,4,6-trichlorophenyl) oxalate and lucigenin

Mojtaba Shamsipura, Corresponding Author Contact Information, E-mail The Corresponding Author, Mohammad Javad Chaichib, Ali Reza Karamic and Hashem Sharghid

aDepartment of Chemistry, Razi University, Kermanshah, Iran

bDepartment of Chemistry, Mazandaran University, Babolsar, Iran

cDepartment of Chemistry, Tarbiat Modarres University, Tehran, Iran

dDepartment of Chemistry, Shiraz University, Shiraz, Iran
Received 13 November 2004;
revised 15 December 2004;
accepted 13 January 2005.
Available online 7 April 2005.

Abstract

Peroxyoxalates such as bis-(2,4,6-trichlorophenyl) oxalate (TCPO) can transfer energy to fluorescer molecules via formation of dioxetanedione intermediate. The aminoanthraquinone derivatives used in this study found to produce a red light in the chemiluminescence systems containing TCPO or lucigenin. Both the fluorescence and chemiluminescence spectra of each of aminoanthraquinone fluorescer system were obtained and the influence of anthraquinone derivatives on the λmax of fluorescer was investigated. The blue light of chemiluminescenct system containing lucigenin is of direct chemilumiescence type, while the red chemiluminescence produced in the presence of anthraquinones is indirect in nature.

Keywords: Alkylaminoanthraquinone; Chemiluminescence; TCPO; Lucigenin; Red fluorescer
Article Outline

1. Introduction
2. Experimental

2.1. Apparatus
2.2. Reagents
2.3. Procedures

3. Results and discussion
References

1. Introduction

The essential requirements for chemiluminescence (CL) include (1) a chemical reaction enable to generate a high energy intermediate, (2) decomposition of the intermediate with concurrent transfer of excess chemical energy to a receptor molecule which becomes electronically excited, and (3) light emission as the excited receptor molecule relaxes to the ground state.

Two classical mechanisms have been proposed for explanation of the chemiexcitation phenomenon. The first is one in which a high energy molecule is converted through a thermal reaction to an electronically excited state. This electronically excited state may then emit a photon of light either as fluorescence or phosphorescence (direct chemiluminescence) or undergo a chemical reaction. Alternatively, this electronically excited state may transfer energy to a suitable acceptor (fluorescer) and the thus formed excited acceptor may then go on to emit a photon of light (indirect chemiluminescence) or undergo a chemical reaction [1], [2], [3] and [4]. Indirect CL has been reported in a bioluminescent system of coelentrates [4] and [5]. In several of the luminescent coelentrates, the color of the light emission of the living organism is green due to green fluorescent protein (indirect CL), whereas the emission of the isolated in vitro reaction is blue (direct CL) [4] and [5]. Aromatic fluorescent compounds can accept energy and change the bacterial bioluminescent spectra [6]. This indicates that there is an energy transfer from the excited molecule to a second species followed by emission from its excited state.

In this paper, we studied the influence of four recently synthesized aminoanthraquinone derivatives 1-amino-2-methyl-9,10-anthraquinone (A1), 1-amino-2-ethyl-9,10-anthraquinone (A2), 1-amino-2,3-dimethyl-9,10-anthraquinone (A3) and 1-amino-2,4-dimethyl-9,10-anthraquinone (A4) on the chemiluminescence systems containing bis-(2,4,6-trichlorophenyl) oxalate (TCPO) and lucigenin.

The aminoanthraquinones used found to produce a red light in both chemiluminescence systems employed, while, TCPO and lucigenin produced a blue-green light. In fact, the aminoanthraquinone derivatives acted as fluorescers and accepted energy from TCPO or lucigenin and emitted red light. The proposed mechanisms for the direct and indirect CL are summarized as follows:

Direct chemiluminescence:

A + B → C* + D



C* → C + light (blue-green)

Indirect chemiluminescence:

A + B → C* + D



C* + F → F* + C



F* → F + light (red)

Some derivatives of polycyclic aromatic hydrocarbons have already been introduced as red fluorescers [7] and [8].
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2. Experimental
2.1. Apparatus

All fluorescence and chemiluminescence spectra were recorded on a Model LS-50B Perkin-Elmer instrument. The xenon lamp of spectrofluorometer was off when the CL spectra were obtained in the visible region. The fluorescence spectra for aminoanthraquinones in the presence and absence of TCPO and lucigenin (i.e., before and after CL reaction) were recorded at excitation wavelengths of 254 and 366 nm, respectively.
2.2. Reagents

The aminoanthraquinone derivatives A1–A4 were synthesized [9] and used after recrystallization from reagent grade benzene and vacuum-drying. The purities (>99.99%) of aminoanthraquinone derivatives were confirmed by spectroscopic data and elemental analysis. All other reagents and solvents were of reagent grade and purchased from Fluka (CH-9470 Buchs, Switzerland). Bis-(2,4,6-trichlorophenyl) oxalate (TCPO) was prepared by the method of Mohan and Turro [10] from reaction of 2,4,6-trichlorophenol and oxalyl chloride in the presence of triethylamine and recrystallised from ethyl acetate. Hydrogen peroxide (30%) was concentrated via freeze drying (using a model FD-1 Eyela freeze dryer, Tokyo, Japan) up to 60% and mixed with dimethyl phthalate in a 1:1 v/v portions and shook well on an electrical shaker. After about 10 h, the organic phase was separated, dried on anhydrous Na2SO4, and the H2O2 concentration was determined by a standard potassium permangenate solution. Standard solutions of hydrogen peroxide (1.5 M in 80:20 dimethyl phthalate:tert-butyl alcohol containing 0.005 M sodium salycilate) were prepared from this solution.
2.3. Procedures

To a fluorometric cell containing 1 ml TCPO (0.01 M in ethyl acetate) and 1 ml of aminoanthraquinone (0.01 M in ethyl acetate) was added 0.2 ml hydrogen peroxide (1.5 M in 80:20 dimethylphthalate:tert-butyl alcohol containing 0.005 M sodium salicylate) and after vigorous stirring both the fluorescence and chemiluminescence spectra were recorded.

To a fluorometric cell containing 1 ml lucigenin (0.002 M in methanol) and 1 ml of aminoanthraquinone (0.01 M in ethyl acetate) and 0.2 ml hydrogen peroxide (10–20% in water) was added 0.2 ml of a NaOH solution (0.1 M in water) and after vigorous stirring both the fluorescence or chemiluminescence spectra were recorded.
3. Results and discussion

The initial example of peroxyoxalate CL was reported in 1963 by Chandross [11] who found that oxalyl chloride, when treated with hydrogen peroxide in the presence of a fluorescent compound such as 9,10-diphenylantracene, produces a bright, short-lived blue emission corresponding to the fluorescence of the hydrocarbon. The energy produced in the chemical reaction was transferred to the fluorescer, producing the excited singlet state of the fluorescer, which emits in a typical fluorescence process. Rauhut and his co-workers at Cyanamid subsequently developed the chemiluminescent reactions of aryl oxalate esters and hydrogen peroxide with a variety of fluorescers in quest of a long-lived light source for commercial and military used [12], [13], [14], [15], [16] and [17].

Peroxyoxalate–chemiluminescence (PO–CL) is well-known as one of the most efficient non-biological light producing systems. Like many chemiluminescence reactions, the PO–CL reaction can be schematized in three basic steps [4], [15], [16], [18], [19] and [20] (Scheme 1). In the first step, an aryl oxalate ester like TCPO reacts with H2O2 to produce a key chemical intermediate, C2O4, containing the necessary excitation energy. The second step involves the chemiexcitation of a fluorophore, like aminoanthraquinone, to electronically excited states by the reactive intermediate via conversion of the chemical energy into electronic excitation energy. The final step is the emission of light energy by returning the excited fluorophore molecule to the ground state.



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Scheme 1. The mechanism of PO–CL reaction.

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In preliminary experiments, it was found that the addition of a few drops of the stock solution of H2O2 to an ethyl acetate solution containing TCPO and aminoanthraquinone results in a relatively intense red light. The sensitized PO–CL spectrum of aminoanthraquinone A1 is shown in Fig. 1. Similar PO–CL spectra were obtained for compounds A2–A4. The wavelengths of maximum intensity for the resulting fluorescence and CL spectra are tabulated in Table 1. As it is seen, since the light emission steps for both chemiluminescence and fluorescence are essentially analogous, the emission wavelength maxima in both processes are similar [13] and [21]. Hence, the color of the CL emission can be manipulated readily by introducing fluorescent additives with known emission characteristics. For example, a purple light can be achieved with 9,10-diphenylanthracene (λmax = 430 nm), blue with perylene (λmax = 471 nm), green with 9,10-diphenylanthracene (λmax = 513 nm) and orange with rubrene (λmax = 556 nm) [4], [7], [8], [16] and [22].



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Fig. 1. PO–CL in the presence of A1.

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Table 1.

Wavelengths of maximum intensity (λmax) for the fluorescence and chemiluminescence spectra of alkylaminoanthraquinones in the peroxyoxalate chemiluminescence system
Compound λmax (nm)
FL FL in reaction mixture FL after reaction CL
A1 558.3 552.2 579.4 576.1
A2 569.4 567.3 582.5 582.1
A3 583.5 574.4 590.5 586.1
A4 572.3 570.3 577.4 575.1
Full-size table

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As expected, the intensity of the PO–CL emission peak was found to be proportional to the initial concentration of the reactants. Sample of the corresponding plots for H2O2–TCPO–L1 system are shown in Fig. 2. In the presence of sodium salicylate (0.0023 M), the light intensity is much higher than that in the absence of the base. The observed behavior is clearly indicative of the catalytic effect of sodium salicylate on the PO–CL system studied [15], [16] and [23]. However, further addition of sodium salicylate revealed a gradual decrease of the CL intensity. This is most probably due to the quenching effect of the base at higher concentrations, which begins to decompose the reactive intermediate, dioxetanedione, and hence reduces the CL light. As is obvious from Fig. 2, there was also a nice linear correlation between the CL intensity and the hydrogen peroxide and TCPO concentrations. The basis for such linear correlation has already been discussed in detail in the literature [24]. Meanwhile, as it has been clearly shown before [25], there is an exponential increase in chemiluminescence of the H2O2-TCPO–L1 system with increasing concentration of the fluorescer.



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Fig. 2. Effect of concentrations of A1 (A), H2O2 (B) and TCPO on the PO–CL of TCPO–H2O2–A1 system.

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Lucigenin (N,N′-dimethyl-9,9′-diacridinium nitrate) undergoes a CL reaction with H2O2 in alkaline solution to yield N-methylacridone (NMA) and light. Several side products can also be formed. Due to the formation of the excited state N-methylacridone (NMA), which emits from about 420–550 nm with a maximum at about 440 nm, the emission is green. In the presence of fluorescers, energy can be transfered and emitted in other spectral region [4] and [16]. Lucigenin CL reaction is shown in Scheme 2.



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Scheme 2. The mechanism of lucigenin CL reaction.

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When hydrogen peroxide was added to a solution containing TCPO and an aminoanthraquinone derivative, there appeared an intense reddish indirect CL. In the lucigenin CL system, a blue-violet light was observed. The intensity of the emission peaks was found to be proportional to the initial concentration of the reactants. The fluorescence spectra of aminoanthraquinones before and after CL reaction in the TCPO and lucigenin systems revealed that these compounds act as fluorescers, which are stable in the solution and can be commercially used as suitable red emitter fluorescers.

As the light emission steps for both fluorescence and CL are essentially analogous, the emission wavelength maxima in both processes are similar for the same emitter and the same concentration range (Table 1). The fluorescers’ stability in reaction medium is dependent on their resistance to hydrogen peroxide, which might result in some small shifts in the resulting FL spectra taken in the reaction mixture, as they are compared with the corresponding FL spectra recorded after the CL reaction. The low CL quantum yield of lucigenin and a broad peak of direct CL for this compound was affected the energy transfer process. As we can observe from the CL of lucigenin in the presence of 1-amino-2,3-dimethylantraquinone, the shoulder observed at about 582.1 nm belongs to the resulting indirect CL (Fig. 3). This low intensity peak was also observed in the flurescence spectrum of the reaction mixture and has about the same wavelength (585.1 nm) with respect to the CL spectrum.



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Fig. 3. Lucigenin–CL in the presence of A3.

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As it is obvious from Table 1, the wavelengths of maximum intensity for both the FL and PO–CL spectra varied with the position and number of substituted alkyl groups on the frame of 1-amino-9,10-anthraquinone. The λmax of both the CL and FL spectra increases in the order A1 < A2 < A4 < A3. Because the ethyl group is a more electron donor than the methyl group, the CL and FL of A2 shows some bathochromic shift with respect to A1. The influence of the substitution of two donating methyl groups on A3 and A4, as it compared to A1 containing only one methyl substitution, is quite obvious from Table 1. As expected, they show maximum intensity at longer wavelengths. The most bathochromic shift belongs to A3, which has two methyl substituents on 2- and 3-positions. The longer emission wavelengths for A3 is due to the facilitated hyperconjugation effect of the two substituted methyl groups on 2- and 3-positions. While, in the case of compound A4 with two methyl groups in 2- and 4-postions, there is only one methyl group located in the para position in relation to one of the carbonyl groups suitable for hyperconjucation. It is also interesting to note that the λmax values of the CL and FL emissions of A3 in the lucigenin are very close to those of the TCPO system.
References

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[2] G.B. Schuster, B. Dixon, J. Koo, S.P. Schmidt and J.P. Smith, Photochem. Photobiol. 30 (1979), p. 17. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (0)

[3] E.H. White, E. Rapaport, H.H. Seliger and T.A. Hopkins, Bioorg. Chem. 1 (1971), p. 92. Article | PDF (2230 K) | View Record in Scopus | Cited By in Scopus (74)

[4] A.K. Campbell, Chemiluminescence, VCH, New York (1988).

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[6] N.S. Kudryasheva, E.V. Nemtseva, A.G. Sizykh, A.G. Shishkin and A.S. Provorov In: A. Roda, M. Pazzagly, L.J. Kricka and P.E. Stanley, Editors, Bioluminescence and Chemiluminescence: Perspectives for the 21st Century, Wiley, Chichester (1988), pp. 585–588.

[7] P.J. Hanhela and D.B. Paul, Aust. J. Chem. 34 (1981), p. 1687.

[8] P.J. Hanhela and D.B. Paul, Aust. J. Chem. 34 (1981), p. 1701.

[9] R. Rezaei, MS Thesis, Shiraz University, Shiraz, Iran, 1992.

[10] A.G. Mohan and N.J. Turro, J. Chem. Edu. 51 (1974), p. 528. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (40)

[11] E.A. Chandross, Tetrahedron Lett. (1963), p. 761. Abstract | PDF (187 K) | View Record in Scopus | Cited By in Scopus (45)

[12] M.M. Rauhut, Acc. Chem. Res. 2 (1969), p. 80. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (47)

[13] M.M. Rauhut, B.G. Roberts and A.M. Semsel, J. Am. Chem. Soc. 88 (1966), p. 3604. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (25)

[14] M.M. Rauhut, L.J. Bollyky, B.G. Roberts, M. Loy, R.H. Whitman, A.V. Iannotta, A.M. Semsel and R.A. Clark, J. Am. Chem. Soc. 89 (1967), p. 6515. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (43)

[15] A.G. Mohan In: J.G. Burr, Editor, Chemi and Bioluminescence, Marcel Dekker, New York (1985), pp. 245–258.

[16] R.S. Givens and R.L. Schowen In: J.W. Birks, Editor, Chemiluminescence and Photochemical Reaction Detection in Chromatography, VCH, New York (1989), pp. 125–147 (Chapter 5).

[17] A.G. Hadd, A. Seeber and J.W. Birks, J. Org. Chem. 65 (2000), p. 2675. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (25)

[18] K. Reszka and J.W. Lown, Photochem. Photobiol. 50 (1989), p. 297. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (14)

[19] K.W. Sigvardson, J.M. Kennish and J.W. Birks, Anal. Chem. 56 (1984), p. 1096. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (39)

[20] F.J. Aiba and J.R. Daban, Photochem. Photobiol. 69 (1999), p. 405.

[21] R.M. Hochstrasser and G.B. Porter, Quat. Rev. (London) 14 (1960), p. 146. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)

[22] S.K. Gill, Aldrichimica. Acta 16 (1983), p. 59.

[23] M. Stigbrand, T. Jonsson, E. Ponten, K. Irgum and R. Bos In: A.M. La Campana and W.J. Baeyens, Editors, Chemiluminescence in Analytical Chemistry, Marcel Dekker, New York (2000).

[24] C.L.R. Catherall, T.F. Palmer and R.B. Cundall, J. Chem. Soc., Faraday Trans. 2 80 (1984), p. 823. Full Text via CrossRef

[25] C.L.R. Catherall, T.F. Palmer and R.B. Cundall, J. Chem. Soc., Faraday Trans. 2 80 (1984), p. 837. Full Text via CrossRef

TCPO

http://en.wikipedia.org/wiki/TCPO


TCPO is one of the ingredients in glow sticks. When combined with 9,10-bis(phenylethynyl)anthracene, a solvent (such as diethyl phthalate), and a weak base (usually sodium acetate or sodium salicylate), and hydrogen peroxide, the mixture will start to glow an aqua-green color.

http://www.shsu.edu/~chm_tgc/JPPdir/JPP1999/

One of the suggested reaction sequences in the reaction of peroxyoxalates, of which bis(2,4,6-trichlorophenyl)oxlate (TCPO) is the most prominent example, follows. It involves the fuel (TCPO) plus the oxidant (H₂O₂) reacting to produce a proposed intermediate, in this example shown as a dioxetane; although, this reaction probably produces many intermediates, and others, such as hydroperoxyoxalate, have been proposed (Milofsky and Birks, 1991; Choksi et al., 1990).

Dioxetanedione ---> Light" vspace="10" width="465" align="RIGHT" border="1" height="162" hspace="10">

The intermediate, shown here as 1,2-dioxetanedione, excites a fluorophore. In the included movie demonstrating TCPO chemiluminescence, 9,10-diphenylanthracene acts as the fluorophore; its lambda max is 425 nm in the solvent used, tetrahydrofuran. Its reaction with the intermediate produces the excited state product which quickly emits light.

The process of transferring the energy of the initial reaction, the chemical reaction of hydrogen peroxide with TCPO, to light emission from the excited state fluorophore (fluorophore*) can be sidetracked along the way by loses in each step of the process: the initial oxidation to produce the intermediate, the reaction of the intermediate with a fluorophore, and the reaction of the excited fluorophore to produce light (Orosz et al., 1996).

Light + CO2" vspace="10" width="467" align="RIGHT" border="1" height="66" hspace="10">

The initial oxidation can yield the high energy intermediate or

• TCPO can be hydrolyzed instead or
• oxidation can occur that doesn't yield chemiluminescent products.

The high energy intermediate can react to excite the fluorophore or

• the intermediate can react with a quencher more easily oxidized than the fluorophore
• the intermediate and fluorophore can react without yielding excited fluorophore
• the intermediate can decompose or be further oxidized by residual H₂O₂.

Finally the excited fluorophore can loose energy by emission of light or

• the excited fluorophore can de-excited by production of heat instead of light.

In normal chromatographic (HPLC) procedures, these alternate mechanistic routes can be effected by solvent and buffers (Orosz, 1989; Jennings and Capomacchia, 1988); pH (de Jong et al., 1986); catalyst (Orlovic et al., 1989; Alverez et al., 1986); and type of fuel (Orlovic et al., 1989; Orosz, 1989), oxidant (Orlovic et al., 1989), and fluorophore concentration and identify. Possibly most important for chromatographers, eluent and reagent flows (Givens and Schowen, 1989; Kwakman and Brinkman, 1992), detector volume and geometry (de Jong et al., 1990; Grayeski and Weber, 1984), and mixing parameters (Kobayashi and Imai, 1980; Sugiura et al., 1993) can all effect this method's light production.

This, therefore, sets the stage for analytical methods whereby manipulating the appropriate parameter allows for the sensitive determination of hydrogen peroxide (Pontén et al., 1996; Stigbrand et al., 1994) or fluorophore content.

Recently, for example, Hamachi et al. (1999) determined the concentration of propentofylline in hypocampus extracts from rats by derivitizing the analyte to create a fluorophore which would chemiluminesce with another peroxyoxalate, TDPO [bis(2-(3,6,9-trioadecanyloxycarbonyl)-4-nitrophenyl)oxalate, and hydrogen peroxide following HPLC. Propentofylline is a reported inhibitor of dopamine released during low oxygenation events in the cerebellum. The derivatization of propentofylline was carried out in trifluoracetic acid/acetonitrile solution using DBD-H (a benzoaxadiazole). The detection limit for the analyte, 31 fg/injection, was about 200 times better than comparable HPLC-UV methods.

Emission Spectrum of Diphenylanthracene as Chemiluminescent Fluorophore
A solution of TCPO and 9,10-diphenylanthracene (DPA; Aldrich Chemicals Co., Milwaukee, WI USA) both in the 1 x 10^-3 M concentration range dissolved in tetrahydrofuran (THF) were mixed with a dilute solution of H₂O₂ in THF (~0.3%) at ~25^oC. The resulting emission spectrum was recorded on a fluorescence spectrometer (Hitachi F-4500; 1 cm quartz cell) in chemiluminescence mode (with no excitation source). The slit and PMT voltage were adjusted to allow for the detection of a strong signal without overloading the detector. The components were mixed and the emission spectrum scanned immediately (1200 nm/min). As the Figure below shows, the emission was centered around 425 nm. This is, of course, similar to DPA's "normal" fluorescent emission.

Movie of TCPO + H₂O₂ + Diphenylanthracene Chemiluminescence Reaction
The movie included here involves that same solution, TCPO and 9,10-diphenylanthracene dissolved in THF. If you look closely you may be able to see the milky consistency of the slightly yellow, initial mixture--shown under fluorescent lights, before hydrogen peroxide was added. Without a mixed solvent system, the solubility of each of these components is relatively low and so the solution is basically saturated with each of these reagents (but still in the low millimolar concentration range).

In the dark, a solution of ~0.3% H₂O₂ in THF was added dropwise to approximately 8 mL of the fuel + fluorophore in THF (~25^oC) in an open-topped vial. The reaction(s) immediately produces light from the excited fluorophore. The emission is relatively short lived but since H₂O₂ is apparently limiting, a second and third dropwise addition of the oxidant yields additional bursts of light. If you will look carefully at the end of the movie you will see a clear--yet still yellow--solution in which all precipitates have dissolved. Also apparent to the experimenter, but undetectable in the movie, was the formation of a gas produced by the reaction; this appeared as a bubbling that could be seen while the reaction was still producing light yet which stopped as the reaction reached completion, about 30 seconds after the last (excess) H₂O₂ addition. This kind of gas production has been used as evidence for the production of CO₂ as a product from the 1,2-dioxetandione intermediate as detailed in the figure above. Further peroxide addition does not yield more bubbling so this is not simply H₂O₂ decomposition. The process of filming this reaction is described below.

Luminol Chemiluminescence

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Luminol is also widely used as a chemiluminescent reagent, but unlike the peroxyoxalate systems does not require an organic/mixed solvent system. The chemiluminescent emitter is a "direct descendent" of the oxidation of luminol (or an isomer like isoluminol) by an oxidant in basic aqueous solution. Probably the most useful oxidant is also hydrogen peroxide similar to peroxyoxalate chemiluminescence; however, other oxidants have been used such as perborate, permanganate (Lu and Lu, 1992), hypochlorite (Cunningham et al., 1998), and iodine (Seitz, 1981). If the fuel is luminol, the emitting species is 3-aminophthalate (see below); however, luminol-derivatized analytes allow for determination of compounds that would not normally chemiluminescence in this system and presumably have slightly different emitters (Edwards et al., 1995; Kawasaki et al., 1985; Lippman, 1980; Nakazone et al., 1992; Pontén et al., 1996).

3-APA + Light" vspace="10" width="455" align="RIGHT" border="1" height="115" hspace="10">

The presence of a catalyst is paramount to this chemiluminescent method as an analytical tool. Many metal cations catalyze the reaction of luminol, H₂O₂, and OH^- in aqueous solution to increase light emission or at least to increase the speed of the oxidation to produce the emitter and therefore the onset/intensity of light production. [Some metals, however, repress chemiluminescence at different concentrations (Yuan and Shiller, 1999; see below.] This therefore can be the foundation of significantly different analytical determinations. For instance, this system can be used:

• to determine luminol itself by holding other variables constant
• to determine luminol-like derivatives similarly (Edwards et al., 1995; Kawasaki et al., 1985; Lippman, 1980; Nakazone et al., 1992; Pontén et al., 1996)
• to determine hydrogen peroxide or the progress of reactions that produce H₂O₂ (Yuan and Shiller, 1999; Tsukagoshi et al., 1998)
• to determine the concentrations of metal cations (Kyaw et al., 1998; O'Sullivan et al., 1995; Robards and Worsfold, 1992; TheingiKyaw et al. 1999)
• or to determine analytes that effect the concentration of metal catalysts.

This last is particularly powerful feature of this system because many compounds complex metallic cations and thereby make themselves "known." Amino acids (Koerner and Nieman, 1987), fructose and tagatose (Valeri et al., 1997), glycerol (Robards and Worsfold, 1992), thiols (Sano and Nakamura, 1998), and serum albumin (Tie et. al., 1995) among many others have been determined using luminol chemiluminescence.

Most recently, Yuan and Shiller (1999) report a subnanomolar detection limit for H₂O₂ using luminol chemiluminescence. Their method, which was used to determine hydrogen peroxide content in sea water, was based on the cobalt(II) catalytic oxidation of luminol. While Co is the most sensitive luminol metal catalyst, it is also present in sea water at very low concentrations. The pH of the luminol solution used in this work was 10.15, and interferences from seven different metals were investigated. Interestingly some metals interfered positively and some negatively, and Fe(III) interfered positively at one concentration and negatively at another. Finally, very low concentrations of iron(II) showed a significant positive interference in determination of H₂O₂, but the authors used the relatively short half life of Fe(II) in marine water as a means of eliminating Fe(II) interference in the determination of hydrogen peroxide in their analysis by storing samples for over 1 hr before analysis.

Light emission from 3-APA
Approximately 15 ml of a solution containing luminol, copper catalyst, and pH controllers were placed in a glass vial at ~25^oC (1 x 10^-3 M luminol; 0.05 M sodium carbonate; 0.3 M sodium bicarbonate; 5 x 10^-3 M ammonium carbonate; 1.5 x 10^-3 M Cu(II) added as sulfate salt). An aqueous solution of approximately 0.25% H₂O₂ was added dropwise.

The emission spectrum was taken as before using a fluorescence spectrometer with the excitation source off. The light intensity-time decay data were taken immediately after mixing the reagents and for 60 seconds. The lambda max is at approximately 445 nm, slightly longer wavelength than the TCPO/DPA system described above. Online presentations of the light intensity-time decay aspects of the luminol reaction with hydrogen peroxide and differing concentrations of Cu(II) as catalyst are also available elsewhere (Iwata and Locker, 1998); however, with this reagent mixture the onset of emission was almost instantaneous and reached a maximum within a few seconds.

As the figure shows the light intensity decayed to approximately 50% of maximum at about 8 seconds. Iwata and Locker found that both the initial intensity and rate of decay in this kind of system was dependent on Cu(II) content. In the TCPO system described above, Orosz et al. (1996) reported that decay rate, rise constant, maximal light intensity, and quantum efficiency depended on hydrogen peroxide concentration. These authors present a comprehensive review of efforts to model the optimization of reagent flow rates and concentrations on HPLC detector responses with the TCPO reaction(s).

Movie of Luminol Chemiluminescence
The luminol reaction described above was carried out by placing approximately 15 mL of a solution containing the fuel (luminol), Cu²⁺ (1.5 x 10^-3 M as the sulfate), and buffers detailed above in a open-topped glass vial (~25^oC). The initial solution is visible at the movie's beginning as light blue in color under the laboratory's fluorescent light due to aqueous copper cations. In the dark, aqueous hydrogen peroxide (~0.3%) was added dropwise four times (small 1 or 2 mL squirts is probably a better description). The light emission is also, as before with TCPO, almost simultaneous upon mixing. The light produced appears white/blue and, as in the TCPO/DPA movie, since fuel is initially in excess, multiple injections of the limiting H₂O₂ reagent are necessary to take the reaction nearer to completion. Finally, after the fourth addition, the mixture was allowed to decay undisturbed and the light intensity drops off rather quickly (see the time decay data above). Approximately 80 seconds after the initial mixing began, the overhead fluorescent light were turned on and the final frame shows that solution. The light blue solution then appear green with a finely dispersed, black precipitate.

Process for producing the movies

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The chemiluminescence reactions were filmed with a Panasonic PV-5540 video camera onto VHS tape. The camera's iris setting was manually opened to approximately 75% of full. The reaction sequences were digitized from tape using the onboard video of a 300 MHz G3 Macintosh (frame rate ~ 24/sec) directly onto an Ultra Wide SCSI hard drive using Apple Video Player (v1.7.1). The movie files, initially saved in QuickTime format (v4.0) were saved in AVI format with no compression using QuickTime Player (v4.0). The files for the 2^nd Internet Conference of Photochemistry and Photobiology were compressed using the Emblaze system from the uncompressed AVI files.

Bibliography

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What is Chemiluminescence?

http://www.chm.bris.ac.uk/webprojects2002/fleming/intro.htm

Chemiluminescence is the production of light from a chemical reaction in excess of the black body radiation expected from that body. As such, it is often referred to as “cold light”.
Normally, chemiluminescence involves the production of an electronically excited species from a number of reactants which goes on to release visible light in order to revert to its ground state energy.

Applications of Chemiluminescence

• Pharmaceutical Industry (analysis and quality control)
• Clinical Science
• Alumina Industry
• Forensics
• Detecting the photoactivity of water through its H₂O₂ Concentration.
• Detecting Nitric Oxide in the breath of Asthma Patients.
• HPLC (High Performance Liquid Chromatography)
• This has nothing to do with luminol but has to be seen!

The Difference Between Fluorescence, Phosphorescence and Chemiluminescence

There are three main types of “cold light” reactions in chemistry which are often confused but are in fact very different. It is important to make sure that you know the difference.

Fluorescence
You have probably covered this in your course. Here higher energy radiation is absorbed by a species and stored for long enough that the molecules have collided and lost vibrational energy in collisions. When the excited molecule re-emits, the photon is of less energy depending on the species and is therefore of a lower frequency.
A good example of this phenomena is your white clothes. Detergent manufacturers put fluorescers in with their mixture to make your clothes really “whiter than white” as they absorb UV radiation in sunlight and emit it in the visible region causing the net light reflected from your clothes to be higher than expected. The same fluorescers are to blame for your white clothes glowing blue under UV lights as the re-emitted wavelength falls in the blue portion of the spectrum. Note that Fluorescence is normally emitting from a singlet state whereas phosphorescence (below) occurs from triplet states.

Phosphorescence
Here light is absorbed by a molecule which becomes excited to a higher singlet state. The light can either be re-emitted straight away or (very-rarely), the electron can switch to a triplet state (this is technically forbidden but can happen often enough through a process known as intersystem crossing). Transitions between different multiplicities are forbidden by quantum selection rules and as the ground state of the molecule will often be a singlet state (recall all closed shells are singlets), the molecule is not allowed to emit a photon that will get it directly to the ground state. The molecule must wait a long time (in quantum chemistry) until it can make the forbidden transition.

Chemiluminescence
The mechanism for this reaction is outlined extensively throughout this site but the key difference to note is that no radiation is absorbed – the energy required to emit light comes from the energetics of the chemical reaction. The emission could proceed either from a singlet (fluorescence) or triplet (phosphorescence) state. "Light Sticks" are a popular example of Chemiluminescence.

Electrochemiluminescence

Electrochemiluminescence

http://www.chm.bris.ac.uk/webprojects2002/fleming/popelectro.html

in 1929, Newton Harvey reported "I have recently observed a quite bright luminescence that can be used as a test for active oxygen, formed on anodes during the passage of an electric current. This involves the use of a chemiluminescent compound, aminophthalichydrazid [today; Luminol]".

The high sensitivity (picomlar) of this reaction today (with peroxide) is used in HPLC and immunoassays.

The left hand peak shows peak emission occurs at around 424nm as with normal chemiluminescence (this would be expected). The right hand graph shows the intensity of luminescence with voltage. The first appearance of luminescence follows closely the oxidation potential of luminol to its 3-APA supporting further the mechanisms proposed.

Reaction Mechanisms

These are based on the Liverpool Electrochemiluminescent Group's work.

There are two main mechanisms to luminol electrochemiluminescence

1. The reaction with oxygen proceeds exactly as in the normal reaction.
2. A second reaction occurs with hydrogen peroxide where the hydrogen peroxide and luminol become oxidised to radicals before following the first reaction.

Experimental Suggestions

A simple demonstration of this phenomena can be performed by taking a small amount of my luminol solution and passing a DC current through it. A blue luminescence will appear at one of the lectrodes (think about which one and why) and diffuse out, lingering after removal of current.

This experiment works with copper, iron and nickel electrodes but not with zinc.

chemiluminescent -1 Luminol

Cited from web site: http://www.chm.bris.ac.uk/webprojects2002/fleming/mechanism.htm

A sensible mechanism of the reaction is as follows. Base removes the nitrogen protons leaving a negative charge which moves onto the carbonyl oxygen to form what is known as an enolate (a charged version of the keto-enol tautomerisation you should be familiar with). The oxygen next performs a cyclic addition to the two (previously) carbonyl carbons. Nitrogen is an excellent leaving group because its own bonds are so strong (and as a gas, it is entropically favoured too) so the charge on the oxygens come back down to form carboxylate anions by expelling nitrogen gas. This leaves 3-APA*.

An important point to take note of is the formation of the cyclic peroxide which converts directly to 3-APA*. Chemiluminescent reactions often involve the cleavage of an organic peroxide because this bond is particularly weak and there is much energy to be gained by cleavage and subsequent reorganisation of bonds. This works with the leaving group effect of the nitrogen (above) to form a particularly efficient reaction.

This sequence of events has been confirmed by using radiolabelled ¹⁸O in the oxygen source which was incorporated into the product as predicted.

e.g. in sodium hydroxide,

Luminol + 2NaOH + O₂ N₂ + Na₂APA + hv

White et al have shown that the chemiluminescence of luminol has an emission spectra with two peaks indicating two similar species that emit light. This has been attributed to a 3-APA* hydrogen bonded to water or protonated fully which emits at 424 nm. The other peak arises from a 3-APA* not bonded in this way (485 nm).

The same team have also shown, through comparison with other known fluorescence and phosphorescence reactions that the chemiluminescence of luminol proceeds from a singlet state like fluorescence. The strength of emission has been shown to directly correlate with the addition of electron donating substituents (e.g. CH₃O-) on the benzene ring.

Molecular oxygen is in a triplet state (to find out why follow this Link). It has been suggested that adducts of oxygen exist as triplet states. Loss of nitrogen leads to a vibrationally excited triplet state of 3-APA which undergoes intersystem crossing to form a singlet state from which it can emit a photon.

Note that if you are using fluorescers then this changes the reaction kinetics drastically as two main types of reaction occur and the introduction of third bodies complicate matters somewhat (in this case there are more than ten rate constants to deal with! This paper discusses the mechanism of “photosenitised” chemiluminescence of luminol but the conclusions reached are well beyond the scope of any A-level project.

An example of the colourful reactions possible with fluorescers is shown here.

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