Introduction

The brilliant emissions resulting from oxidation of certain oxalic acid derivatives, especially in the presence of a variety of fluorophores, are the bases of the most active area of current interest in chemiluminescence. The example of peroxyoxalate chemiluminescence was reported in 1963 by Chandross [1]. The peroxyoxalate-chemiluminescence (PO-CL) system is one of the most regularly used analytical chemiluminescence schemes [2, 3]. PO-CL is based on the reaction of H2O2 with peroxyoxalate which result an intermediate [4]. The excited intermediate transfers its energy to an efficient fluorophore [5-9] through the chemically initiated electron exchange luminescence (CIEEL) mechanism [10]. This reaction has been successfully used as a highly sensitive detection technique in several procedures developed for low level determinations of various analytes [11, 12], proteins [13], hydrogen peroxide [14, 15], fluorophore labeled amino acids [16] and prostate specific antigen [17] as well as different quencher species [18, 19].

Quinoxalines are well known fluorescent compounds with high quantum yields and have attracted much attention due to their potential functions for high technology applications [20, 21]. These compounds have been utilized as fluorescence probes in some of elaborated chemosensors. Quinoxalines are, in general, comparatively easy to synthesis, and with numerous derivatives which have been designed for potential use as biologically active materials [22]. The classical synthesis of quinoxalines involves the condensation of 1, 2-diamine with a 1,2-dicarbonyl. The reaction is facile and it is the most widely used method for synthesizing both quinoxaline itself and its derivatives [23]. Quinoxalines have shown broad biological activities such as in vitro antitumor activity [24], antiviral [25], herbicidal [26] and anti-inflammatory activity [27], hence they are an important class of nitrogen-containing heterocycles and useful intermediates in organic synthesis [28, 29]. Recently some of these types of molecules have been reported as candidates for treatment of cancer and disorders associated with angiogenesis functions [30-33].

One of the most fascinating questions concerning the peroxyoxlate chemiluminescence reaction is the compound capable that generates light which is related the structure of fluorophore. In this paper, we report chemiluminescence system from reaction of bis-(2, 4, 6-trchlorophenyl)oxalate (TCPO) and hydrogen peroxide using quinoxalines derivatives, as an efficient fluorophores. Two quinoxalines derivatives of 2,3-diphenylquinoxaline (A) and biphenylquinoxaline (B) were used (Fig. 1). Tow quinoxalines derivatives are found intense and useful fluoropher compounds containing aromatic functional group with low-energy п→п* transition level (green light emission). Particularly, compound B is favored in molecules that possess rigid structure. However, lack of rigidity in a molecule A causes an enhanced internal conversion rate (internal rotation about phenyl group) and a consequence increase in the likelihood for radiationless deactivation. Kinetic parameters for the peroxyoxalate-chemiluminescence of two quioxalines were calculated from the computer fitting of the corresponding chemiluminescence intensity-time plots.

Fig. 1
figure 1

Molecular structure of 2,3-diphenylquinoxaline a and biphenylquinoxaline b

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Experimental

Reagents

All chemical compounds were reagent-grade and purchased from Fluka chemical company (CH-9470, Buchs, Switzerland) and used as received without further purification. Quinoxaline derivatives were prepared from the reaction of 4-Nitro-O-phenylen diamine with benzyl and 9,10-phenanthroquinone in glacial acetic acid as a solvent and then were reduced by hydrazine monohydrate as described elsewhere [23, 34].

Apparatus

Chemiluminescence detection was performed with a homemade apparatus equipped with a Model BPY47 photocell (Leybold, Huerth, Germany). The apparatus was connected to a personal computer via a suitable interface (Micropars, Tehran, Iran) as shown in Fig. 2. Experiments were carried out with magnetic stirring (500 rpm) in a light-tight flat-bottom glass cell of 15 mm diameter at 25 °C. Chemiluminescence intensity was recorded as a function of time, the time resolution of the apparatus was 0.6 s.

Fig. 2
figure 2

Shematic representation of batch system for measuring visible chemiluminescence

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Steady-state fluorescence spectra were recorded on a Perkin Elmer, Ls-3B spectrofluorimeter instrument. The excitation monochromators were set at 390 and 440 nm for compounds 2,3-diphenylquinoxaline (A) and biphenylquinoxaline (B), respectively. Spectral bandwidth of 3 nm was used.

Procedures

Solution I was made with mixing 1.0 mL of TCPO (0.01 M) and 0.5 mL of quinoxaline derivatives (various concentrations in ethyl acetate). Solution II contained 2 mL of hydrogen peroxide (3.2 M) and 1.0 mL of catalyst (0.1 M) in methanol. Solution I was transferred into glass cell using polypropene syringes. Then 100 µL of solution II was injected in the glass cell, chemiluminescence spectrum was recorded soon after mixing of solutions. The fluorescence spectra were carried out using freshly prepared solutions containing 3.3 × 10-5 M of fluorophores in ethyl acetate, using a 3-cm quartz cuvette.

Results and discussion

Peroxyoxalate-chemiluminescence (PO-CL) is well-known as one of the most efficient non-biological light producing systems (Scheme 1). Like many chemiluminescence reactions, the PO-CL reaction can be presented in following steps [35-40].

(1)
(2)
$$ {\text{F*}} \to {\text{F}} + {\text{hv}} $$
(3)

where, F is fluorophore.

In the first step, an aryl oxalate ester like TCPO reacts with H2O2 to produce a key chemical intermediate of 1,2-dioxetandione (C2O4) as an excitation source. The second step, excited cyclic C2O4 intermediate transfers its energy to fluorophore (here quinoxaline derivatives were used as fluorophore). The final step is the emission of light energy by returning the excited fluorophore molecule to the ground state. The florescence spectra of compounds A and B are shown in Fig. 3. By comparison fluorescence spectra A and B, it can be deduced that spectrum B is more intense than that A (hyperchromic effect) and also the maximum wavelength (λmax) of spectrum B is shifted to longer wavelength (bathochrom shift). Typically, in chemiluminescence system, soon after mixing of ingredients (e.g., 2,3-Diphenylquinoxaline, bis-(2, 4, 6-trchlorophenyl) oxalate, H2O2 and sodium salysilate) the intensity of emitted light is risen rapidly and then exponential decay of light intensity follows. Figures 4 and 5 show chemiluminescence intensity as a function of time (intensity/time emission profile) for the PO-CL system in the presence of varying concentrations of quinoxaline derivatives A and B, respectively. Plot of chemiluminescence intensity as a function of fluorophore concentration (compounds A and B) is inserted on top of right hand side of Figs. 4 and 5 respectively. Correlation between the CL intensity and the TCPO concentration for both fluorophore is listed in Tables 1 and 2. The influence of H2O2 concentration on the PO-CL for both compounds A and B was studied at constant concentrations of other reagents and presented in Tables 1 and 2. Results revealed that there is a direct linear relationship between concentration of H2O2 (i.e., 0.017–0.085) and PO-CL intensity (Fig 6a and b). It is pertinent to mention that further increase in H2O2 concentration has no more effect on the PO-CL intensity. The intensity/time emission profile of both quinoxaline derivatives under the optimal constant concentrations of TCPO, H2O2 and fluorophore indicated that the emmison intensity is significantly enhanced by addition of sodium salicylate. This confirms a key role of catalyst in the PO-CL system [41]. In order to investigate the optimal concentration of sodium salicylate, the CL response of the H2O2-TCPO-quioxalines systems was measured against the varying concentrations of the base, results are shown in Tables 1 and 2. The PO-CL intensity dramatically increased with increasing concentration of sodium salicylate until a concentration of 6.0 × 10-4 M is reached. However, further addition of sodium salicylate causes to decrease the CL intensity (Fig 7a and b). This, probably due to the quenching effect of the base at higher concentrations, which possibly it leads to decompose the intermediate, dioxetane dione, and hence reduces the PO-CL light [42].

Fig. 3
figure 3

The fluorescence emission spectra of (a): 3.3×10-5 M of 2,3-diphenylquinoxaline with λex = 390 nm, and (b) 3.3×10-5 M of biphenylquinoxaline with λex = 430 nm in ethyl acetate

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Fig. 4
figure 4

Chemiluminescence emission intensity as a function of time for the bis-(2, 4, 6-trchlorophenyl) oxalate (TCPO)-H2O2-quinoxaline derivatives and sodium salysilate system with constant concentration of TCPO (3.2 × 10-3 M), H2O2 (6.8 × 10-2 M), sodium salicylate (1.0 × 10-3 M), and varying concentrations of 2,3-diphenylquinoxaline: (1) 3.2 × 10-5, (2) 6.5 × 10-5, (3) 1.3 × 10-4, (4) 6.5 × 10-4 and (5) 1.6 × 10-3 M. Corner of right hand side: The correlation diagram for the chemiluminescence emission with 2,3-diphenylquinoxaline concentrations

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Fig. 5
figure 5

Chemiluminescence emission intensity as a function of time for the bis-(2, 4, 6-trchlorophenyl) oxalate (TCPO)- H2O2-quinoxaline derivatives and sodium salysilate system with constant concentration of TCPO (3.2 × 10-3 M), H2O2 (6.8 × 10-2 M), sodium salicylate (1.0 × 10-3 M), and varying concentrations of biphenylquinoxaline: (1) 1.6 × 10-5, (2) 3.2 × 10-5, (3) 6.4 × 10-5, (4) 9.6 × 10-5, (5) 1.3 × 10-4, and 1.6 × 10-4 M. Corner of right hand side: correlation diagram for the chemiluminescence emission with biphenylquinoxaline concentrations

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Table 1 CL parameters calculated from computer fitting of the CL intensity–time plots for TCPO–H2O2–2,3-diphenylquinoxaline–sodium salicylate system
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Table 2 CL parameters evaluated from computer fitting of the CL intensity–time plots for TCPO–H2O2–2,3-biphenylquinoxaline 6-amine –sodium salicylate system system
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Fig. 6
figure 6

Dependence of the H2O2 concentration a on the CL intensity of TCPO–H2O2–2,3-diphenylquinoxaline—sodium salicylate system, b on the CL intensity of TCPO-H2O2–biphenylquinoxaline–sodium salicylate system

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Fig. 7
figure 7

Influence of sodium salicylate concentration a on the CL intensity of TCPO–H2O2–2,3-diphenylquinoxaline—sodium salicylate system, b on the CL intensity of TCPO–H2O2–biphenylquinoxaline–sodium salicylate system

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Results revealed that the concentration of TCPO and sodium salicylate have no significant influence on the chemiluminescence system, i.e., the rise (k r ) and fall (k f ) rates constant in different concentrations of TCPO and sodium salicylate have nearly the same values. Nonetheless, the kinetics parameters of the chemiluminescence system are moderately influenced by both concentrations of H2O2 and fluorophore. In the former case, by increasing concentration of H2O2, the values of fall rates constant (k f ) become larger, on the other hand, enhancing concentration of fluorophore caused to decrease of k f .

In order to evaluate the kinetic data for the PO-CL system, a pooled-intermediate model was used [36, 43, 44]. According to this model, the CL reaction is simplified as:

$$ {\text{A}}\xrightarrow{{{K_r}}}{\text{B}}\xrightarrow{{{K_f}}}{\text{C}} $$
(4)

where A, B and C represent pools of reactants, intermediates and products, respectively, and both reaction steps designated by the rate constants k r and k f are irreversible first order reactions. The integrated rate equation for the CL intensity versus time is:

$$ {I_t} = \left[ {\frac{{M{k_r}}}{{{k_f} - {k_r}}}} \right]\left[ {\exp \left( { - {k_r}t} \right) - \exp \left( { - {k_f}t} \right)} \right] $$
(5)

Where, I t is the CL intensity at time t, M is a theoretical maximum level of intensity if the reactants were entirely converted to a CL-generating material, k r and k f are, respectively, the first order rate constants for the rise and fall of the burst of CL. A further advantage of this model is that it not only allows the determination of parameters M, k r and k f , but also it permits an estimate of the intensity at maximum level (J), the time of maximum intensity (Tmax) and the total light yield (Y), as follows:

$$ J = M{\left( {\frac{{{k_f}}}{{{k_r}}}} \right)^{\left[ {{{{k_f}} \mathord{\left/{\vphantom {{{k_f}} {\left( {{k_f} - {k_r}} \right)}}} \right.} {\left( {{k_f} - {k_r}} \right)}}} \right]}} $$
(6)
$$ {\tau_{\max }} = \frac{{\ln \left( {{{{k_f}} \mathord{\left/{\vphantom {{{k_f}} {{k_r}}}} \right.} {{k_r}}}} \right)}}{{{k_f} - {k_r}}} $$
(7)
$$ Y = {\int_0^\infty {I_{t} dt = \frac{M}{{k_{f} }}} } $$
(8)

In this work, a non-linear least-squares curve fitting program KINFIT [45] was used to evaluate the M, k r and k f values from the corresponding CL intensity-time plots. A typical computer fitting of the CL intensity time plots is shown in Figs. 8 and 9. The theoretical values of maximum intensity (J), and corresponding time (Tmax) and Y can be calculated by Eqs. 6, 7 and 8 using parameters of k f , k r and M which were determined from the resulting CL intensity-time plots. All kinetic parameters are listed in Tables 1 and 2. The experimental values of maximum intensity (Imax) and Tmax were extracted from CL intensity-time plots (Figs. 4 and 5) ). The statistical results (R2 and F) obtained by application of SPSS indicate that, there is a satisfactory agreement between the calculated (J) and experimental (Imax) values of the intensity at the maximum CL (Tables 1 and 2).

Fig. 8
figure 8

A typical computer fitting of the CL intensity–time plot for TCPO–H2O2–2,3-diphenylquinoxaline–sodium salicylate system. ([H2O2] = 0.068 M, [sodium salicylate] = 1.0 × 10-3 M, [2,3-diphenylquinoxaline] = 1.6 × 10-3 M) and [TCPO] = 3.2 × 10-3 M.): (×) experimental point; (o) calculated point; (=) experimental and calculated points are the same within the resolution of the plot

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Fig. 9
figure 9

A typical computer fit of the CL intensity–time plot for TCPO–H2O2–2,3-biphenylquinoxaline 6-amine –sodium salicylate system ([H2O2] = 0.068 M, [sodium salicylate] = 1.0 × 10-3, [biphenylquinoxaline] = 1.6 × 10- 4 M) and [TCPO] = 3.2 × 10-3 M.): (×) experimental point; (o) calculated point; (=) experimental and calculated points are the same within the resolution of the plot

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Conclusion

The present study describes a new chemiluminesecence system of H2O2- bis-(2,4,6-trichlorophenyl) oxalate (TCPO) using quioxalines derivatives as fluorophore. In this system a green light is produced in the present of quinoxaline derivatives. The results indicated that how concentrations of the components involved in CL influence on the light emission. Results showed that concentrations of H2O2 TCPO and quioxalines have direction relationship on the light intensity. Kinetic parameters for the peroxyoxalate-chemiluminescence of two quioxalines were calculated from the corresponding chemiluminescence intensity-time plots. A non-linear least-squares curve fitting program KINFIT was used to evaluate the theoretical maximum level of intensity (M), the first order rate constants for the rise k r and fall k f of the burst of CL. Tow quinoxalines derivatives are found intense and useful fluoropher compounds which produce green light emission. Particularly, compound B is favored in molecules that possess rigid structure.