Studies of new peroxyoxalate-H2O2 chemiluminescence system using quinoxaline derivatives as green fluorophores

Article abstract of DOI:10.1007/s10895-010-0601-9

Quinoxaline derivatives are a great interest as fluorescent emitters for peroxyoxalate chemiluminescence. Reaction of peroxyoxalates such as bis-(2,4,6-trichlorophenyl) oxalate with H2O2 can transfer energy to fluorophore via formation of dioxetanedione intermediate. Two quinoxaline derivatives used as a fluorophore in this study which produce a green light in the chemiluminescence systems. The relationship between the chemiluminescence intensity and concentrations of fluorophore, peroxyoxalate, sodium salicylate and hydrogen peroxide was investigated. Kinetic parameters for the peroxyoxalate-chemiluminescence were also calculated from the computer fitting of the corresponding chemiluminescence intensity/time profiles. It was found that the biphenylquinoxaline can be used as an efficient green fluorescent emitter. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s10895-010-0601-9

J Fluoresc (2010) 20:671–679
DOI 10.1007/s10895-010-0601-9
ORIGINAL PAPER
Studies of New Peroxyoxalate-H2O2 Chemiluminescence
System Using Quinoxaline Derivatives
as Green Fluorophores
Abdolraouf Samadi-Maybodi & Reza Akhoondi &
Mohammad Javad Chaichi
Received: 11 October 2009 /Accepted: 29 January 2010 /Published online: 10 March 2010
#
Springer Science+Business Media, LLC 2010
Abstract Quinoxaline derivatives are a great interest as
fluorescent emitters for peroxyoxalate chemiluminescence.
Reaction of peroxyoxalates such as bis-(2,4,6-trichloro-
phenyl) oxalate with H2O2 can transfer energy to fluo-
rophore via formation of dioxetanedione intermediate. Two
quinoxaline derivatives used as a fluorophore in this study
which produce a green light in the chemiluminescence
systems. The relationship between the chemiluminescence
intensity and concentrations of fluorophore, peroxyoxalate,
sodium salicylate and hydrogen peroxide was investigated.
Kinetic parameters for the peroxyoxalate-chemilumines-
cence were also calculated from the computer fitting of the
corresponding chemiluminescence intensity/time profiles. It
was found that the biphenylquinoxaline can be used as an
efficient green fluorescent emitter.
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 success-
fully 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].
.
.
Keywords Quinoxaline derivatives Hydrogen peroxide
.
Chemiluminescence Bis-(2,4,6-trichlorophenyl) oxalate
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, comparative-
ly 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
Introduction
The brilliant emissions resulting from oxidation of certain
oxalic acid derivatives, especially in the presence of a
:
:
A. Samadi-Maybodi (*) R. Akhoondi M. Javad Chaichi
Analytical division, Faculty of Chemistry,
University of Mazandaran,
Babolsar, Iran
e-mail: samadi@umz.ac.ir

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J Fluoresc (2010) 20:671–679
system from reaction of bis-(2, 4, 6-trchlorophenyl)oxalate
(TCPO) and hydrogen peroxide using quinoxalines deriv-
atives, as an efficient fluorophores. Two quinoxalines
derivatives of 2,3-diphenylquinoxaline (A) and biphenyl-
quinoxaline (B) were used (Fig. 1). Tow quinoxalines
derivatives are found intense and useful fluoropher com-
pounds 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-chemilumines-
cence of two quioxalines were calculated from the
computer fitting of the corresponding chemiluminescence
intensity-time plots.
N
N
N
N
H2N
H2N
a
b
Fig. 1 Molecular structure of 2,3-diphenylquinoxaline a and biphe-
nylquinoxaline b
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 angio-
genesis 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
Experimental
Reagents
All chemical compounds were reagent-grade and purchased
from Fluka chemical company (CH-9470, Buchs, Switzer-
land) 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-phenan-
throquinone in glacial acetic acid as a solvent and then were
reduced by hydrazine monohydrate as described elsewhere
[23, 34].
Fig. 2 Shematic representation
of batch system for measuring
visible chemiluminescence

J Fluoresc (2010) 20:671–679
673
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.
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 biphenylqui-
noxaline (B), respectively. Spectral bandwidth of 3 nm was
used.
Fig. 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) 1.6×10-3M. Corner of right hand side: The
correlation diagram for the chemiluminescence emission with 2,3-
diphenylquinoxaline concentrations
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 fluores-
cence 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].
Fig. 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
Fig. 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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J Fluoresc (2010) 20:671–679
Table 1 CL parameters calculated from computer fitting of the CL intensity–time plots for TCPO–H2O2–2,3-diphenylquinoxaline–sodium
salicylate system
Parameter changed
H2O2
Concentration (M) kr (min-1)
kf (min-1)
M
J
Texp (min) Tmax (min)
Y
I
3.4×10-2
5.1×10-2
6.8×10-2
8.5×10-2
3.2×10-4
6.0×10-4
1.2×10-3
1.9×10-3
2.5×10-3
2.0×10-4
4.0×10-4
6.0×10-4
8.0×10-4
1.0×10-3
1.6×10-5
3.2×10-5
9.6×10-5
1.3×10-4
3.2×10-4
6.5×10-4
3.40 0.09 0.55 0.01
3.28 0.09 0.72 0.01
3.54 0.12 0.89 0.02
2.97 0.12 0.90 0.03
3.56 0.11 0.55 0.01
3.32 0.10 0.68 0.01
3.43 0.11 0.72 0.02
3.36 0.10 0.53 0.01
3.53 0.11 0.75 0.02
3.29 0.13 0.69 0.02
3.39 0.14 0.85 0.03
864 11
1368 20
608
893
0.50
0.53
0.46
0.52
0.52
0.47
0.58
0.56
0.51
0.50
0.47
0.45
0.45
0.48
0.34
0.37
0.37
0.38
0.41
0.46
0.63
0.59
0.52
0.57
0.57
0.59
0.57
0.58
0.55
0.60
0.54
0.53
0.50
0.51
0.39
0.40
0.43
0.44
0.46
0.49
1,570
1,894
602
910
1992 37 1,252
2192 56 1,304
2,236 1,358
2,433 1,454
TCPO
374 6
702 11
1040 16
1381 21
253
467
687
911
546
1,029
1,443
1,941
257
469
711
642
1740 27 1,145
1424 30 942
2038 49 1,282
2,308 1,214
2,070 1,138
2,381 1,561
2,456 1,494
2,420 1,462
2,375 1,391
Sodium salycilate
3.58 0.13 0.81 0.02 1 998 40 1,292
3.94 0.13 0.82 0.02 1,976 32 1,309
3.90 0.12 0.80 0.02 1,911 32 1,269
2,3-diphenyl quinoxaline
6-amine
1.32 0.05 4.28 0.22
1.70 0.09 3.49 0.22 1,011 73
4.22 0.18 1.06 0.03 944 24
4.24 0.17 1.01 0.03 1,156 27
778 47
142
248
593
737
181
289
185
321
753
910
882
1,137
4.17 0.14 0.92 0.02 1,736 32 1,129
3.79 0.13 0.89 0.02 1,987 35 1,275
1,870 1,298
2,235 1,397
Statistical parameters R2 =0.981 F=875.069 correlation is significant at the 0.01 level
Cl
Cl
Cl
Cl
OH
OH
O
O
O
O
+
+
OH
H2O2
2
Cl
Cl
O
O
Cl
ð1Þ
O
O
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 chemilumi-
nescence system, soon after mixing of ingredients (e.g.,
2,3-Diphenylquinoxaline, bis-(2, 4, 6-trchlorophenyl) oxa-
late, 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, respec-
O
O
ð2Þ
+
+
F
2
CO2
F
*
O
O
»
F ꢀꢀꢀꢀꢀꢀ! F þ hv
ð3Þ
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

J Fluoresc (2010) 20:671–679
675
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
Parameter changed
H2O2
Concentration (M) kr (min-1)
kf (min-1)
M
J
Texp(min)
(min)
Y
I
Tmax
1.7×10-2
3.4×10-2
5.1×10-2
6.8×10-2
8.5×10-2
3.2×10-4
6.0×10-4
1.2×10-3
1.9×10-3
2.5×10-3
3.2×10-3
2.0×10-4
4.0×10-4
6.0×10-4
8.0×10-4
1.0×10-3
6.4×10-6
9.6×10-6
1.3×10-5
1.6×10-5
3.2×10-5
6.4×10-5
9.6×10-5
1.3×10-4
1.6×10-4
2.86 0.09 0.40 0.01
811 12
578
0.94
0.61
0.48
0.38
0.38
0.41
0.38
0.40
0.39
0.36
0.36
0.64
0.45
0.39
0.39
0.41
0.33
0.32
0.33
0.31
0.32
0.35
0.34
0.35
0.36
0.78
1,850
562
2.99 0.09 0.79 0.02 1,986 33
3.63 0.13 0.91 0.02 2,796 57
4.31 0.18 1.03 0.03 3,527 81
4.51 0.18 1.03 0.03 3,773 85
1,228
1,761
2,251
2,436
287
0.60
0.50
0.43
0.42
0.46
0.42
0.41
0.41
0.40
0.40
0.69
0.50
0.44
0.43
0.44
0.31
0.32
0.36
0.32
0.34
0.40
0.39
0.39
0.40
2,482 1,290
3,077 2,005
3,426 2,720
3,654 2,972
TCPO
3.52 12
1.18 0.04
498 12
953 24
420
747
2,99
606
3.96 0.17 1.27 0.04
556
4.27 0.17 1.24 0.04 1,592 37
4.13 0.19 1.24 0.04 2,320 63
4.26 0.19 1.26 0.05 3,150 88
4.56 0.18 1.14 0.03 3,383 79
2.31 0.09 0.81 0.03 2,634 74
3.59 0.16 0.95 0.04 3,457 90
4.24 0.18 1.00 0.03 3,670 70
4.34 0.18 1.02 0.03 3,659 76
4.34 0.17 0.98 0.03 3,236 67
960
1,281 1,091
1,871 1,652
2,453 2,292
2,966 2,595
3,250 1,692
3,661 2,735
3,656 2,991
3,603 2,924
3,282 2,497
1,385
1,860
2,132
1,496
2,147
2,347
2,349
2,094
123
Sodium salycilate
2,3 biphenyl quinoxaline
6-amine
3.20 0.21 3.20 0.21
3.11 0.22 3.11 0.22
333 22
530 38
104
170
288
278
595
161
250
338
436
836
195
1.47 0.07 4.64 0.27 1,338 94
3.18 0.22 3.18 0.22 885 62
2.86 0.24 2.86 0.24 1,707 144
3.16 .31
249
326
628
1.93 0.17 2,333 190 1,077
1,206 1,410
1,895 2,024
2,286 2,331
2,985 2,884
1.46 0.06 4.17 0.22 7,914 485 1,576
4.22 0.21 1.38 0.05 3,165 101 1,838
4.12 .20
1.30 0.05 3,887 121 2,284
Statistical Parameters R2 =0.992 F=2933.591 Correlation is significant at the 0.01 level
tively. 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 signif-
icantly 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 concentra-
tion 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].
Results revealed that the concentration of TCPO and
sodium salicylate have no significant influence on the
chemiluminescence system, i.e., the rise (kr) and fall (kf)
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

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J Fluoresc (2010) 20:671–679
concentration of H2O2, the values of fall rates constant (kf)
become larger, on the other hand, enhancing concentration
of fluorophore caused to decrease of kf .
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:
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, kr
and kf, 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:
)
ꢆ
ꢇ
k
k ꢀk
½ f =ð rÞꢁ
f
kf
Kr
Kf
J ¼ M
ð6Þ
A ꢀꢀꢀꢀꢀ! B ꢀꢀꢀꢀꢀ! C
ð4Þ
kr
where A, B and C represent pools of reactants, intermedi-
ates and products, respectively, and both reaction steps
designated by the rate constants kr and kf are irreversible
first order reactions. The integrated rate equation for the CL
intensity versus time is:
ꢂ ꢈ ꢃ
ln kf kr
kf ꢀ kr
tmax
¼
ð7Þ
ð8Þ
ꢀ
ꢁ
ꢄ
ꢂ
ꢃꢅ
Z
Mkr
1
M
kf
It ¼
expðꢀkrtÞ ꢀ exp ꢀkf t
ð5Þ
Y ¼
It dt ¼
kf ꢀ kr
0
In this work, a non-linear least-squares curve fitting
program KINFIT [45] was used to evaluate the M, kr and kf
values from the corresponding CL intensity-time plots. A
Where, It 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, kr and kf are,
Fig. 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

J Fluoresc (2010) 20:671–679
677
Fig. 7 Influence of sodium salic-
ylate 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
Fig. 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-diphenyl-
quinoxaline]=1.6×10-3 M) and [TCPO]=3.2×10-3 M.): (×) experi-
mental point; (o) calculated point; (=) experimental and calculated
points are the same within the resolution of the plot

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J Fluoresc (2010) 20:671–679
Fig. 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, [biphenyl-
quinoxaline]=1.6×10- 4 M) and [TCPO]=3.2×10-3 M.): (×) experi-
mental point; (o) calculated point; (=) experimental and calculated
points are the same within the resolution of the plot
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 kf, kr 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).
green light emission. Particularly, compound B is favored in
molecules that possess rigid structure.
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