Mechanistic Studies on the Salicylate-Catalyzed Peroxyoxalate Chemiluminescence in Aqueous Medium

Article abstract of DOI:10.1111/php.13180

The peroxyoxalate reaction is one of the most efficient chemiluminescence transformations known and the only system occurring by an intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism with confirmed high quantum yields. The peroxyoxalate chemiluminescence (PO-CL) is mainly studied in anhydrous organic medium; however, for bioanalytical application, it should be performed in aqueous media. In the present work, we study the peroxyoxalate system in a binary 1,2-dimethoxyethane/water mixture with bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and bis[2-(methoxycarbonyl)phenyl] oxalate (DMO), catalyzed by sodium salicylate, in the presence of rhodamine 6G as activator. Reproducible kinetic results are obtained for all systems; emission decay rate constants depend on the salicylate as well as hydrogen peroxide concentration, and the occurrence of a specific base catalysis is verified. Although singlet quantum yields determined are lower than in anhydrous media in comparable conditions, they are still considerably high and adequate for analytical applications. The highest singlet quantum yields are obtained for the “ecologically friendly” derivative DMO indicating that this derivative might be the most adequate substrate for the use of the peroxyoxalate system in bioanalytical applications.

Full text of DOI:10.1111/php.13180

PROF. WILHELM JOSEF BAADER (Orcid ID : 0000-0001-7678-8576)
Article type
: Research Article
Mechanistic Studies on the Salicylate-Catalyzed
Peroxyoxalate Chemiluminescence in Aqueous
Medium
Maidileyvis C. Cabello, Glalci A. Souza, Liena V. Bello and Wilhelm J. Baader*
Departamento de Química Fundamental, Instituto de Química, Universidade
de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000, São Paulo, Brazil.
*Corresponding author e-mail: wjbaader@iq.usp.br (Wilhelm J. Baader)
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/PHP.13180
This article is protected by copyright. All rights reserved

ABSTRACT
The peroxyoxalate reaction is one of the most efficient chemiluminescence
transformations known and the on system occurring by an intermolecular
Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism
with confirmed high quantum yields. The peroxyoxalate chemiluminescence
(PO-CL) is mainly studied in anhydrous organic medium, however for
bioanalytical application it should be performed in aqueous media. In the
present work, we study the peroxyoxalate system in a binary 1,2-
dimethoxyethane/water mixture with bis(2,4,6-trichlorophenhyl) oxalate
(TCPO), bis(4-methylphenyl) oxalate (BMePO) and bis[2-(methoxy-
carbonyl)phenyl] oxalate (DMO), catalyzed by sodium salicylate, in the
presence of rhodamine 6G as activator. Reproducible kinetic results are
obtained for all systems; emission decay rate constants depend on the
salicylate as well as hydrogen peroxide concentration and the occurrence of a
specific base catalysis is verified. Although singlet quantum yields determined
are lower than in anhydrous media in comparable conditions, they are still
considerably high and adequate for analytical applications. The highest singlet
quantum yields are obtained for the ‘ecologically friendly’ derivative DMO
indicating that this derivative might be the most adequate substrate for the
use of the peroxyoxalate system in bioanalytical applications.
This article is protected by copyright. All rights reserved

INTRODUCTION
Peroxyoxalate chemiluminescence (PO-CL) is being continuously studied even
more than 50 years after its discovery (1) and initial characterization and
utilization (2–4), approaching mechanistic aspect of the transformation as
well as widespread analytical and bioanalytical application (5–8). PO-CL has
been widely used as a detection system in several analysis systems, such as
liquid chromatography, capillary electrophoresis and flow injection analysis
(5,9–11), which are highly sensitive for the detection of hydrogen peroxide
produced in enzymatic reactions, fluorescent compounds and other molecules
of biological and medicinal interest (12). Using the peroxyoxalate system
coupled to liquid chromatography or capillary electrophoresis, amino acids,
carboxylic acids, carbonyl compounds and catecholamines have been detected
directly or after derivatization (13,14). Detection of hydrogen peroxide can be
achieved under various reaction conditions, and PO-CL can be used in several
systems that produce or consume hydrogen peroxide (9,15–17). Additionally,
Albrecht et al. have developed an analytical method to detect oxalate in
human fluids and cells in the 100 nmol L-1 concentration using PO-CL in
specific reaction conditions (18,19). Nanoparticles and quantum dots were
used in PO-CL for the determination of commercially and environmentally
important analytes as well as a large number of pharmaceutical compounds
(15,20).
Kinetic studies on the PO-CL system in anhydrous organic solvents have
largely clarified the chemical transformations occurring in the reaction of
oxalic esters with hydrogen peroxide catalyzed by various nucleophilic or non-
This article is protected by copyright. All rights reserved

nucleophilic base catalysts, which lead to the formation of a high-energy
intermediate (HEI), whose interaction with an added CL activator (ACT) leads
to electronically excited state formation and consequent CL emission (21–30).
Imidazole is a widely utilized base and nucleophilic catalyst, which leads to
reproducible reaction kinetics, however, it is also known that it can interact
with the HEI formed, leading to a decrease in the CL quantum yields in high
concentrations (28,31). Therefore, other base catalysts have been utilized for
the reaction however without significant improvement with respect to the
quantum yields (5,29,32). Recently, a mechanistic study on the PO-CL in
organic medium using sodium salicylate as catalyst has been performed
indicating its role as general or specific base, but not nucleophilic, catalyst
(33). Several different structures have been proposed for the HEI, which
leads to chemiexcitation of the ACT (23,24,34–38), however, it appears that
1,2-dioxetanedione is the most probable structure of this intermediate (39–
42). The interaction of this intermediate with the ACT, generally
polycondensed aromatic hydrocarbons with low oxidation potentials and high
fluorescence quantum yields, has been shown to occur by the CIEEL
mechanism (25,43,44). This mechanism had been proposed for the catalyzed
decomposition of cyclic peroxides occurring with efficient singlet excited state
formation (45,46)
and then widely utilized to rationalize excited state
formation in a wide variety of CL and bioluminescence (BL) transformations
(29,47–50). However, it has more recently been verified that the
chemiexcitation efficiency of the catalyzed decomposition of model peroxides
for the CIEEL mechanism is much lower than initially determined, possibly
This article is protected by copyright. All rights reserved

due to steric hindrance of the interaction between the HEI and the ACT
(51,52). Therefore, the peroxyoxalate system appears to be the only
chemiexcitation system involving the intermolecular CIEEL mechanism with
high chemiexcitation quantum yields (2,53,54), underlining its importance
from the mechanistic point of view, apart from its analytical importance.
Even so important for analytical and bioanalytical applications, very view
mechanistic studies of the PO-CL have been reported in aqueous media (5). A
recent study established experimental conditions in aqueous phosphate
buffered medium with reproducible reaction kinetics, where, depending on
medium pH, general acid as well as base catalysis by phosphate ions could be
verified (55). Additionally, the mechanism of the PO-CL has been studied
using 2,6-lutidine (2,6-dimethylpyridine) as non-nucleophilic base catalyst in
organic as well as aqueous media (32).
In the present work, we report our results on a kinetic study of the PO-CL
using three oxalic esters with different reactivity such as bis(2,4,6-
trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and
bis[2-(methoxycarbonyl)phenyl] oxalate (DMO) (Figure 1) and sodium
salicylate as base catalyst in partially aqueous medium in order to elucidate
the general reaction mechanism and establish adequate experimental
conditions for potential analytical applications.
<Figure 1>
MATERIALS AND METHODS
This article is protected by copyright. All rights reserved

Materials. Bis(2,4,6-trichrolophenyl) oxalate (TCPO) was purchased from
Sigma (99% purity) and recrystallized from a chloroform/n-hexane (14:5
(v/v)) mixture (m.p. 191-192 °C) (56). Bis(4-methylphenyl) oxalate (BMePO)
was synthesized by the reaction of p-methylphenol (Aldrich, 99%) with oxalyl
chloride (previously distilled, b.p. = 63°C) according to the literature
1
13
procedure (4). BMePO was identified by H- and C-NMR spectra as well as
elementary analysis, as shown in a previous work (55). Bis[2-
(methoxycarbonyl)phenyl] oxalate (DMO) was synthesized from the reaction
between oxalyl chloride (Sigma Aldrich ≥ 99%) and methyl salicylate (Sigma
1
Aldrich ≥ 99%) catalyzed by pyridine and characterized by H NMR spectra
and melting point following the procedures reported in the literature (57).
Benzophenone (Vesicol), sodium salicylate (Sigma Aldrich, ≥ 99.5%) and
rhodamine 6G (Sigma Aldrich) were used without further purification.
Hydrogen peroxide was donated by Solvay Peroxidos do Brasil, São Paulo,
Brazil as a 60% (w/w) unstabilized aqueous solution; H2O2 stock solutions
were prepared in anhydrous 1,2-dimethoxyethane (DME) by diluting 1.0 mL
of 60% (w/w) hydrogen peroxide solution in 20.0 mL of organic solvent and
dried with anhydrous MgSO4 for 12 hours at ca. 4°C; final peroxide
concentrations
were
determined
by
a
iodometric
assay
using
-
spectrophotometrical quantification of I3 formed at 353 nm (353nm = 2.55 104
L mol-1 cm-1) (58). The solvent DME for the kinetic assays was purchased
from Sigma-Aldrich (99.5% purity) and made anhydrous by initial distillation,
followed by addition of sodium wire, refluxing in the presence of
This article is protected by copyright. All rights reserved

benzophenone and final distillation of the dark blue mixture, under a nitrogen
atmosphere.
Kinetic measurements. Chemiluminescent assays for the reaction of TCPO,
BMePO and DMO with hydrogen peroxide and sodium salicylate, using
rhodamine 6G (Rhod 6G) as activator, were performed in a binary 1:1 (v/v)
DME/H2O mixture. Kinetic assays involving TCPO and DMO were performed on
a Varian Cary Eclipse spectrofluorimeter with a Hamamatsu photomultiplier
R928, operating at 1000 V, using an emission slit from 10 to 20 nm, at (25.0
 0.2) °C. Kinetic assays with BMePO were performed in a Berthold Sirius
Luminometer due to the lower emission intensity of this system, at (25.0 
0.2) °C.
Oxalic esters and hydrogen peroxide stock solutions were prepared in
anhydrous DME, the stock solutions of sodium salicylate and rhodamine 6G
were prepared in Milli-Q water. In a typical essay the solvent mixture (1:1
(v/v) DME/H2O), H2O2, and salicylate stock solutions were added to a quartz
fluorescence cell. The reaction was started with the addition of the oxalic
esters stock solution (final concentration 0.100 mmol L-1). The borate buffer
(100 mmol L-1), used in experiments where the pH was maintained constant,
was prepared from a 0.1 mol L−1 H3BO3 solution and a 0.1 mol L−1
Na2B4O7·10H2O, the pH of the final solution was adjusted to 8.2 by addition of
the appropriate amount of Na2B4O7·10H2O solution (0.1 mol L−1).
Data analysis. All mathematical adjustments were made with MicroCal
software Origin 8.0 (2008). The emission intensity decay curves obtained in
the kinetic assays (Figure S1) were fitted with a monoexponential function in
This article is protected by copyright. All rights reserved

order to determine the decay time (eq. 1) and observed rate constants (kobs)
values, which correspond to the inverse of decay time (kobs = 1/t).
Chemiluminescence quantum yields (ΦCL) were obtained using the luminol
standard for photomultiplier calibration (54).
y = y0 + A1 e-xt (eq. 1)
RESULTS
The kinetics of the sodium salicylate catalyzed reaction of bis(2,4,6-
trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and
bis[2-(methoxycarbonyl)phenyl] oxalate (DMO) with H2O2, using rhodamine
6G (Rhod 6G) as activator (ACT), were studied in a binary DME/H2O (1:1,
v/v) mixture in conditions leading to reproducible experimental results. The
salicylate catalyzed reaction of the oxalic esters with hydrogen peroxide in the
presence of the ACT leads to light emission, whereas concurrent ester
hydrolysis, also catalyzed by salicylate, does not result in light emission
(Scheme 1).
This article is protected by copyright. All rights reserved

O
ONa
OH
kper
ArO
O
O
2 ArOH
+ 2 CO2 + hv
+ H2O2
OAr
Rhod 6G
khyd
H2O
HO
O
O
+ 2 ArOH
OH
Products without light emission
Scheme 1: General reaction scheme for the sodium salicylate catalyzed
chemiluminescent reaction of TCPO, BMePO and DMO with hydrogen peroxide
and the concurrent ester hydrolysis, leading to dark products.
The observed rate constants (kobs), obtained from the emission intensity
versus time curves by monoexponential curve fitting (Figure S1), show linear
correlation with the salicylate concentration ([Sal]) for all esters in the low
concentration range, from which the bimolecular rate constant for the rate-
limiting step of the interaction of TCPO, BMePO and DMO with salicylate
TCPO
BMePO
(kSal
= 0.083 L mol-1 s-1, kSal
= 0.251 L mol-1 s-1, kSalDMO = 0.13 L mol-
1
s-1) can be obtained (Figure 2). For BMePO, the rate constants show to be
independent of the base concentration for [Sal] > 100 mmol L-1, with a
BMePO
maximum rate constant of kSal/max
~ 0.025 s-1. The kobs values obtained
for DMO do not fit to the linear correlation for [Sal] > 100 mmol L-1, but no
constant maximum value is obtained. From the intercept of each correlation,
This article is protected by copyright. All rights reserved

TCPO
BMePO
unimolecular rate constants of k0
= 0.0131 s-1, k0
= 0.00292 s-1 and
DMO
k0
= 0.00079 s-1 for the reaction of TCPO, BMePO and DMO not catalyzed
by salicylate can be obtained, respectively (Figure 2, Table1, Table S1).
<Figure 2>
<Table 1>
The kobs show linear dependence on the [H2O2] in the whole concentration
range studied in the reaction of TCPO (Figure 3A, Table S2), while in the
reaction of BMePO and DMO, the kobs values show a linear dependence on the
[H2O2] up to 160 mmol L-1 (Figure 3B, 3C). A bimolecular perhydrolysis and
a unimolecular hydrolysis rate constant are obtained from the slope and the
intercept of these linear regressions respectively (Table 2). No significant
hydrolysis of DMO is observed in this medium and the bimolecular
perhydrolysis constant shows to be similar to that of the other esters. Despite
the higher reactivity of TCPO compared to BMePO in anhydrous medium, the
hydrolysis and perhydrolysis constants are similar for both esters in the
aqueous medium used here; in fact the perhydrolysis rate constant is three
times higher for BMePO than TCPO (Table 2).
<Figure 3>
This article is protected by copyright. All rights reserved

<Table 2>
The highest quantum yields were obtained for the reaction with DMO, with
values of up to 3 10-3 E mol-1, for low salycilate concentrations. The singlet
quantum yields (S) for the peroxyoxalate reaction with TCPO showed to be
only one order of magnitude lower (S  10-4 E mol-1) in the aqueous medium
utilized here, as compared to the salicylate catalyzed reaction performed in
anhydrous organic medium (S  10-3 E mol-1) (33). In the case of the
reaction with BMePO, the yields were several orders of magnitude smaller
(10-7 E mol-1) than with the other esters. The quantum yields have similar
behavior for the three reactions, showing a decrease with increasing salicylate
concentrations. (Figure 4, Table S3).
<Figure 4>
This article is protected by copyright. All rights reserved

The s values increase with increasing peroxide concentration for the reaction
of TCPO until 100 mmol L-1, at which they remained constant, with a
maximum value of around 6 10-3 E mol-1 (Figure 5A). For the reactions with
BMePO and DMO the s decrease continuously with increasing [H2O2] (Figure
5, Table S4).
<Figure 5>
In order to verify the occurrence of a general or specific base catalysis by
salicylate, experiments were performed in aqueous media where the pH value
was kept constant (pH = 8.2), while the [Sal] was varied. In these conditions,
the kobs values showed to be independent of the salicylate concentration for
the whole range studied, with average values of 0.019 s-1 and 0.0035 s-1 for
the reactions with TCPO and BMePO, respectively (Table S5). Interestingly,
in these conditions the reaction of TCPO is faster than that of BMePO, as
would be expected from the leaving-group abilities of their phenolic
substituents. The quantum yields are significantly lower in these experiments
for both esters than that reported above, however, it should be mentioned
that here a totally aqueous system has been used to allow a better pH
control, which might be responsible for the quantum yield reduction (Table
S5). For TCPO the S values decrease with increasing [Sal] by almost a factor
of ten, however, for BMePO the already very low S values do not further
decrease with increasing [Sal] (Table S5).
This article is protected by copyright. All rights reserved

DISCUSSION
The experimental results obtained in our kinetic study on the peroxyoxalate
reaction in a binary water / organic solvent mixture with three different
oxalate esters revealed the first-order dependence of the observed rate
constants on both salicylate and hydrogen peroxide concentrations, indicating
that both species are involved in the rate-limiting step of these
transformations. This observation excludes preliminarily the occurrence of a
nucleophilic catalysis by salicylate, as observed before in the peroxyoxalate
reaction of TCPO catalyzed by imidazole in ethyl acetate (27,28).
The reaction mechanism occurring in this medium could be tentatively
explained by the occurrence of a general (GBC) or specific (SBG) base
catalysis, which includes both reagents as rate-limiting. However, the results
obtained in aqueous borate buffer in the presence of different salicylate
concentrations show that the observed rate constants are independent of the
base concentration with a constant medium pH value of 8.2 (Figure 6, Table
S5).
<Figure 6>
Therefore, on the basis of the kinetic results obtained, the salicylate-catalyzed
reaction of oxalic esters with hydrogen peroxide should proceed by specific
base catalysis, where the hydroperoxy anion is formed in a fast pre-
equilibrium (Scheme 2(a)), followed by hydroperoxy anion attack to the
This article is protected by copyright. All rights reserved

oxalic ester leading to the formation of a monoperoxalic ester derivative (1)
in the slow, rate-limiting reaction step (Scheme 2(b)). This mechanistic
proposal is also in agreement with the fact that the observed rate constants
(kobs) show linear dependence with the salicylate as well as hydrogen
peroxide concentration (Figures 2 and 3).
The reaction proceeds with an intramolecular attack of the peracid to the
adjacent oxalic ester carboxyl function, in a fast reaction step, leading to the
formation of the high-energy intermediate (HEI), which is most probably 1,2-
dioxetanedione (2) (Scheme 2(c)) (38–42). This step should also be
catalyzed by salicylate, as indicate by a previous work involving PO-CL with
TCPO and sodium salicylate in anhydrous medium, which shows the
occurrence of base catalysis in the cyclization step (33). This reaction step,
also generally much faster than the preceding one, might become limiting at
very high hydrogen peroxide and base concentrations, as indicated by the
saturation behavior of the peroxide and base concentration dependence of the
kobs values (Figures 2 and 3).
The last step corresponds to the interaction of the HEI (2) with the ACT
through the intermolecular CIEEL mechanism, leading to the formations of the
ACT’s singlet excited state, which decays to its ground state emitting light.
This step is known to involve electron transfer, bond-cleavage and electron
back-transfer steps and the cyclic peroxide is transformed into two carbon
dioxide molecules (Scheme 2(d)).
In
concurrence
to
the
perhydrolysis
reaction,
which
leads
to
chemiluminescence emission, the oxalic ester derivatives also suffer
This article is protected by copyright. All rights reserved

hydrolysis in the aqueous medium used, also catalyzed by salicylate, which
lead to the formation of two molecules of the corresponding substituted
phenol and oxalic acid, without light emission (Scheme 2(e)). The
contribution of hydrolysis is more pronounce for TCPO and BMePO, whereas
DMO, does not show considerable hydrolysis in the reaction medium used
(Table 2).
Perhydrolysis
O
ONa
OH
O
OH
OH
K
+ H2O2
HO2
OH
+
Na
+
(a)
(b)
Sal
O
OAr
+ HO2
O
O
+ ArO
slow
ArO
O
ArO
O
O
1
oxalic ester
O
ONa
OH
O
O
O
O
OH
+ ArOH
(c)
(d)
fast
O
O
ArO
2
1
O
O
2 CO2 + ACT + hv
+
ACT
CIEEL
O
O
2
Hydrolysis
O
ONa
OH
O
OH
O
O
OAr
+ 2 H2O
2 ArOH
+
(e)
HO
ArO
O
oxalic acid
oxalic ester
Scheme 2: Mechanism for the peroxyoxalate reaction between an oxalic ester and
This article is protected by copyright. All rights reserved

H2O2, occurring with specific base catalysis (SBC) by salicylate, in the presence of an
ACT, performed in a binary DME/H2O mixture, as well as concurrent ester hydrolysis.
The values of the singlet quantum yields (S) decrease with increasing
salicylate concentrations for all esters studied, although in the absence of Sal
the quantum yields are lower for all three esters and for TCPO the yields
increase up to [Sal] = 50 mmol L-1, followed by a steady decrease (Figure
4). The lower S in the absence of base may be rationalized by a lower
efficiency of the uncatalyzed perhydrolysis reaction as compared to
uncatalyzed hydrolysis. The steady decrease in S with increasing base
concentration at higher [Sal] has also observed before in the peroxyoxalate
reaction with TCPO using imidazole and 2,6-lutidine as bases (28,32). These
results should be related to the destruction of the IAE by the action of the
base, as verified before (27,28,31–33). On the other hand, for the reaction
with TCPO, the s increase with increasing [H2O2] and present a saturation
region for high concentrations (Figure 5A), a behavior different from that for
the same reaction catalyzed by salicylate in ethyl acetate, where the quantum
yields decreased with increasing base concentrations (33). The behavior
observed here can be understood considering the occurrence of the
concurrent hydrolysis reaction in the partially aqueous medium, which does
not lead to light emission. Therefore, increasing the peroxide concentration
will lead to a shift from the hydrolysis to the perhydrolysis reaction, which
occurs with light emission, and thereby to an increase in the singlet quantum
yields. However, for the reaction with BMePO and DMO, the ΦS values
This article is protected by copyright. All rights reserved

decreased significantly with increasing [H2O2] (Figure 5). This behavior might
indicate a possible formation of diperoxyoxalic acid for higher peroxide
concentrations, which does not lead to light emission, as evidenced also
before (27,28). Additionally, as these esters are less reactive than TCPO, the
concurrent hydrolysis reaction should be of less importance, therefore no
initial increase in the ΦS values with the peroxide concentration will be
expected.
Although the range of salicylate concentrations in the assays presented
previously involving TCPO in ethyl acetate differs significantly from that used
in these studies, the S values in aqueous medium are only one order of
magnitude lower than in anhydrous conditions. It means that the salicylate
catalyzed peroxyoxalate reaction is of similar efficiency in the DME/H2O binary
system as in purely organic systems.
For the less reactive esters BMePO the quantum yields are significantly lower
than for TCPO, however for this substrate no ΦS values in anhydrous media
have been reported yet. The highest quantum yields were obtained for the
reaction with DMO, with values of up to 3 10-3 E mol-1 for low salicylate
concentrations, indicating that this ester might be the most adequate one to
be utilized in analytical applications in aqueous media. Furthermore, this
derivative does not show significant hydrolysis in the conditions used, as
indicated by the peroxide dependence of the rate constants (Figure 3C).
CONCLUSIONS
This article is protected by copyright. All rights reserved

(i) The experimental results obtained in the present work show that PO-CL
can be reproducible performed in a binary 1,2-dimethoxyethane / water (1:1)
mixture with sodium salicylate as catalyst for bis(2,4,6-trichlorophenyl)
oxalate
(TCPO),
bis(4-methyphenyl)
oxalate
(BMePO)
and
bis[2-
(methoxycarbonyl)phenyl] oxalate (DMO) as oxalic ester derivatives with
different reactivity.
(ii) In all cases, both hydrogen peroxide and salicylate determine the rate
constants of the transformation in the partially aqueous medium and
salicylate acts as a specific base catalyst.
(iii) The role of salicylate proposed for these systems is different from that
verified when using imidazole as catalyst, which acts as a basic and
nucleophilic catalyst. However, the mechanism proposed here is in agreement
with that previously outlined for the sodium salicylate catalyzed reaction of
TCPO in pure organic media.
(iv) Singlet quantum yields decrease with increasing salicylate concentrations
(for TCPO after reaching a maximum for low [Sal]), indicating an interaction
of salicylate with the high-energy intermediate. The variation of ΦS with the
peroxide concentration is distinct for the different derivatives, what can be
explained by the different reactivity of the oxalic esters.
(v) DMO shows the highest quantum yields in the aqueous medium used,
which is advantageous for the use of PO-CL assays for analytical and
bioanalytical purposes.
ACKNOWLEDGEMENTS
This article is protected by copyright. All rights reserved

The authors would like to thank Coordenadoria de Aperfeiçoamento de
Pessoal de Ensino Superior (CAPES, programa 33002010191P0) and Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq, GAS, MCC
(147138/2016-7) and LVB (141816/2017-1)) for financial support in the form
of PhD fellowships. Furthermore, the generous financial support for a research
project for WJB (grant 2014/22136-4) by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) is gratefully acknowledged. Finally, we thank
Peroxidos do Brasil for a generous gift of concentrated hydrogen peroxide.
REFERENCES
1.
2.
3.
Chandross, E. A. A New Chemiluminescent System. Tetrahedron Lett.
12, 761–765 (1963).
Rauhut, M. M. Chemiluminescence from Concerted Peroxide
Decomposition Reactions. Acc. Chem. Res. 2, 80–87 (1969).
Rauhut, M. M., Roberts, B. G. & Semsel, A. M. A Study of
Chemiluminescence from Reactions of Oxalyl Chloride, Hydrogen
Peroxide, and Fluorescent Compounds. J. Am. Chem. Soc. 88, 3604–
3617 (1966).
4.
Rauhut, M. M. Bollyky, L. J. Roberts, B. G. Loy, M. Whitman, R. H.
Iannotta, A. V. Semsel, A. M. Clarke, R. A. Chemiluminescence from
Reactions of Electronegatively Substituted Aryl Oxalates with Hydrogen
Peroxide and Fluorescent Compounds. J. Am. Chem. Soc. 89, 6515–
6522 (1967).
5.
Ciscato, L. F. M. L. et al. The chemiluminescent peroxyoxalate system:
This article is protected by copyright. All rights reserved

State of the art almost 50 years from its discovery. Arkivoc 2012, 391–
430 (2012).
6.
Romanyuk, A. V, Grozdova, I. D., Ezhov, A. A. & Melik-nubarov, N. S.
Peroxyoxalate Chemiluminescent Reaction as a Tool for Elimination of
Tumour Cells Under Oxidative Stress. Sci. Reports 7 Article Nu, 1–13
(2017).
7.
8.
9.
Lee, Y. et al. Dye / Peroxalate Aggregated Nanoparticles with Enhanced
and Tunable Chemiluminescence for Biomedical Imaging of Hydrogen
Peroxide. 6759–6766 (2012).
Romanyuk, A. V. & Melik-Nubarov, N. S. Micelles of amphiphilic
copolymers as a medium for peroxyoxalate chemiluminescent reaction in
water environment. Polym. Sci. Ser. B 57, 360–369 (2015).
Huertas-Pérez, J. F., Moreno-González, D., Airado-Rodríguez, D., Lara,
F. J. & García-Campaña, A. M. Advances in the application of
chemiluminescence detection in liquid chromatography. TrAC - Trends
Anal. Chem. 75, 35–48 (2016).
10. Lara, F. J., Airado-Rodríguez, D., Moreno-González, D., Huertas-Pérez, J.
F. & García-Campaña, A. M. Applications of capillary electrophoresis with
chemiluminescence detection in clinical, environmental and food
analysis. A review. Anal. Chim. Acta 913, 22–40 (2016).
11. Rocha, D. L., Kamogawa, M. Y. & Rocha, F. R. P. A critical review on
photochemical conversions in flow analysis. Anal. Chim. Acta 896, 11–
33 (2015).
12. Waseem, A., Yaqoob, M. & Nabi, A. Analytical Applications of Flow
This article is protected by copyright. All rights reserved

Injection
Chemiluminescence
for
the
Determination
of
Pharmaceuticals&#8211;A Review. Curr. Pharm. Anal. 9, 363–395
(2013).
13. Hu, H. M., Yin, X. F., Wang, X. Z. & Shen, H. A study on the system of
nonaqueous microchip electrophoresis with on-line peroxyoxalate
chemiluminescence detection. J. Sep. Sci. 36, 713–720 (2013).
14. García-Campaña, A. M., Gámiz-Gracia, L., Baeyens, W. R. G. & Alés
Barrero, F. Derivatization of biomolecules for chemiluminescent
detection in capillary electrophoresis. J. Chromatogr. B Anal. Technol.
Biomed. Life Sci. 793, 49–74 (2003).
15. Lara, F. J., García-Campaña, A. M. & Aaron, J. J. Analytical applications
of photoinduced chemiluminescence in flow systems-A review. Anal.
Chim. Acta 679, 17–30 (2010).
16. Gámiz-Gracia, L., García-Campaña, A. M., Huertas-Pérez, J. F. & Lara, F.
J. Chemiluminescence detection in liquid chromatography: Applications
to clinical, pharmaceutical, environmental and food analysis-A review.
Anal. Chim. Acta 640, 7–28 (2009).
17. García-Campaña, A. M., Lara, F. J., Gámiz-Gracia, L. & Huertas-Pérez, J.
F. Chemiluminescence detection coupled to capillary electrophoresis.
TrAC - Trends Anal. Chem. 28, 973–986 (2009).
18. Albrecht, S., Brandl, H. & Schönfels, C. Human Oxalate—Really Just an
End‐Product of Metabolism? Angew. Chemie Int. Ed. English 33, 1780–
1781 (1994).
19. Albrecht, S. et al. Determination of serum oxalate using peroxyoxalate
This article is protected by copyright. All rights reserved

chemiluminescence of free oxalic acid. J. Biolumin. Chemilumin. 8, 21–
24 (1993).
20. Cho, H., Lee, S. & Lee, J. H. Roles of CdS quantum dots in 1,1′-
oxalyldiimidazole chemiluminescence. Analyst 137, 5368–5373 (2012).
21. Hadd, A. G.; Seeber, A.; Birks, J. W. No Title. J. Org. Chem. 65, 2675
(2000).
22. Orlovic, M.; Schowen, R. L.; Givens, R. S.; Alvarez, F.; Matuszewski, B.;
Parekh, N. A Simplified Model for the Dynamics of Chemiluminescence in
the Oxalate-Hydrogen Peroxide System: Toward a Reaction Mechanism.
J. Org. Chem. 54, 3606 (1989).
23. Hadd, A. G., Robinson, A. L., Rowlen, K. L. & Birks, J. W. Stopped-Flow
Kinetics Investigation of the Imidazole-Catalyzed Peroxyoxalate
Chemiluminescence Reaction. J. Org. Chem. 63, 3023–3031 (1998).
24. Alvarez, F. J. et al. Multiple Intermediates Generate Fluorophore-Derived
Light in the Oxalate/Peroxide Chemiluminescence System. J. Am. Chem.
Soc. 108, 6435–6437 (1986).
25. Catherall, C. L. R., Palmer, D. T. F. & Cundall, R. B. Chemiluminescence
from Reactions of Bis(pentachlorophenyl)oxalate, Hydrogen Peroxide
and Fluorescent Compounds Kinetics and Mechanism. J. Chem. SOC.
Faraday Trans. 2 80, 823–834 (1984).
26. Neuvonen, H. A T wo - intermediate M odel for I midazole - promoted P
eroxyoxalate C hemiluminescence. J. Biolumin. Chemilumin. 241–248
(1997).
27. Silva, S. M. et al. Kinetic studies on the peroxyoxalate
This article is protected by copyright. All rights reserved

chemiluminescence reaction: Determination of the cyclization rate
constant. Luminescence 17, 313–320 (2002).
28. Stevani, C. V, Lima, D. F., Toscano, V. G. & Baader, W. J. Kinetic studies
on the peroxyoxalate chemiluminescent reaction: Imidazole as a
nucleophilic catalyst. J. Chem. Soc. Trans. 2 989–995 (1996).
29. Baader. W.J., Stevani, C.V., Bastos, E. L. Chemiluminescence of Organic
Peroxides. The Chemistry of Peroxides, Zvi Rappoport (Ed.), Wiley-VCH,
Chichester. 1211-1278. (2006).
30. Stevani, C.V., B. W. J. O sistema quimiluminescente peróxi-oxalato. J.
Quim. Nov. 22, 715 (1999).
31. Boaro, A. & Bartoloni, F. H. Peroxyoxalate High-Energy Intermediate is
Efficiently Decomposed by the Catalyst Imidazole. Photochem. Photobiol.
546–551 (2016). doi:10.1111/php.12608
32. Felipe A. Augusto, Fernando H. Bartoloni, Maidileyvis C. Cabello, Ana
Paula F. dos Santos, W. J. B. Kinetic studies on 2,6-lutidine catalyzed
peroxyoxalate chemiluminescence in organic and aqueous medium:
evidence for general base catalysis. J. Photochem. Photobiol 382,
111967 (2019).
33. Souza, G. A., Lang, A. P. & Baader, W. J. Mechanistic Studies on the
Peroxyoxalate Chemiluminescence Using Sodium Salicylate as Base
Catalyst. Photochem. Photobiol. 93, 1423–1429 (2017).
34. Milofsky, R. E. & Birks, J. W. Laser Photolysis Study of the Kinetics and
Mechanism of Photoinitiated Peroxyoxalate Chemiluminescence. J. Am.
Chem. Soc. 113, 9715–9723 (1991).
This article is protected by copyright. All rights reserved

35. Orosz, G., Givens, R. S. & Schowen, R. L. Chemiluminescence arising
from the interaction of rubrene with intermediates derived from
hydrogen peroxide and bis(2,4-dinitrophenyl) oxalate (DNPO): quantum
yield and carbondioxide yield. Anal. Chim. Acta 266, 219–223 (1992).
36. Mann, B. & Grayeski, M. L. Background Emission from the Peroxyoxalate
Chemiluminescence Reaction in the Absence of Fluorophors. Anal. Chem.
62, 1532–1536 (1990).
37. Lee, J. H., Je, J., Schlautman, M. A. & Carraway, E. R. Fast
peroxyoxalate chemiluminescence for minimized analytical separation
systems. Chem. Commun. 9, 270–271 (2003).
38. Stevani, C. V. & B. W. . Preparation and Characterization of 2,2,2-
triphenyl-2λ 5-1,3,2-dioxastilbolane-4,5-dione as Standard for an
Attempt to Trap 1,2-Dioxetanedione, a Possible High-Energy
Intermediate in Peroxyoxalate Chemiluminescence. J. Chem. Res. 9,
430–432 (2002).
39. Bos, R. et al. Studies on the mechanism of the peroxyoxalate
chemiluminescence reaction. Anal. Chim. Acta 502, 141–147 (2004).
40. Tonkin, S. A. et al. Studies on the mechanism of the peroxyoxalate
chemiluminescence reaction. Part 2. Further identification of
intermediates using 2D EXSY13C nuclear magnetic resonance
spectroscopy. Anal. Chim. Acta 614, 173–181 (2008).
41. Stevani, C. V, Campos, I. P. D. A. & Baader, W. J. Synthesis and
Characterisation
of
an
Intermediate
in
the
Peroxyoxalate
Monoperoxy-
Chemiluminescence:
4-Chlorophenyl
O,O-Hydrogen
This article is protected by copyright. All rights reserved

oxalate. J. Chem. Soc., Perkin Trans. 2 1645–1648 (1996).
42. Stevani, C. V. & Baader, W. J. Kinetic studies on the chemiluminescent
decomposition of an isolated intermediate in the peroxyoxalate reaction.
J. Phys. Org. Chem. 10, 593–599 (1997).
43. Stevani, C. V., Silva, S. M. & Baader, W. J. Studies on the Mechanism of
the Excitation Step in Peroxyoxalate Chemiluminescence. European J.
Org.
Chem.
4037–4046
(2000).
doi:10.1002/1099-
0690(200012)2000:24<4037::AID-EJOC4037>3.0.CO;2-A
44. Ciscato, L. F. M. L., Bartoloni, F. H., Bastos, E. L. & Baader, W. J. Direct
kinetic observation of the chemiexcitation step in peroxyoxalate
chemiluminescence. J. Org. Chem. 74, 8974–8979 (2009).
45. Schuster, G. B. Chemiluminescence of Organic Peroxides. Conversion of
Ground-State Reactants to Excited-State Products by the Chemically
Initiated Electron-Exchange Luminescence Mechanism. Acc. Chem. Res.
12, 366–373 (1979).
46. Schuster, G. B. & Schmidt, S. P. Chemiluminescence of Organic
Compounds. Adv. Phys. Org. Chem. 18, 187–238 (1982).
47. Baader, W. J., Stevani, C. V. & Bechara, E. J. H. ‘Photo’chemistry
Without Light? Rev. Virtual Química 7, 74–102 (2015).
48. Wilson, T. Comments on the mechanisms of chemi- and
bioluminescence. Photochem. Photobiol. 62, 601–606 (1995).
49. Koo, J. A., Schmidt, S. P. & Schuster, G. B. Bioluminescence of the
firefly: key steps in the formation of the electronically excited state for
model systems. Proc. Natl. Acad. Sci. 75, 30–33 (1978).
This article is protected by copyright. All rights reserved

50. Bastos, E. L., Farahani, P., Bechara, E. J. H. & Baader, W. J. Four-
membered cyclic peroxides: Carriers of chemical energy. J. Phys. Org.
Chem. 30, 1–18 (2017).
51. Almeida De Oliveira, M. et al. Revision of singlet quantum yields in the
catalyzed decomposition of cyclic peroxides. J. Org. Chem. 77, 10537–
10544 (2012).
52. Bartoloni, F. H. et al. Chemiluminescence efficiency of catalyzed 1,2-
dioxetanone decomposition determined by steric effects. J. Org. Chem.
80, 3745–3751 (2015).
53. Catherallt, C. L. R. & Palmer, T. F. Determination of Absolute
Chemiluminescence Quantum Yields. J. Biolumin. Chemilumin. 3, 147–
154 (1989).
54. Augusto, F. A., De Souza, G. A., De Souza, S. P., Khalid, M. & Baader,
W. J. Efficiency of electron transfer initiated chemiluminescence.
Photochem. Photobiol. 89, 1299–1317 (2013).
55. Souza, G. A., Peixoto, M. M. M., Santos, A. P. F. & Baader, W. J. General
Acid
and
Base
Catalysis
by
Phosphate
in
Peroxyoxalate
Chemiluminescence. ChemistrySelect 1, 2307–2315 (2016).
56. Mohan, A. G. & Turro, N. J. A facile and effective chemiluminescence
demonstration experiment. J. Chem. Educ. 51, 528 (1974).
57. Cambrea, L. R., Davis, M. C., Groshens, T. J. & Meylemans, H. A. A
soluble,
halogen-free
oxalate
from
methyl
salicylate
for
chemiluminescence demonstrations. J. Chem. Educ. 90, 1253–1254
(2013).
This article is protected by copyright. All rights reserved

58. Cotton, M. L. & Dunford, H. B. Studies on Horseradish Peroxidase. XI.
On the Nature of Compounds I and II as Determined from the Kinetics of
the Oxidation of Ferrocyanide. Can. J. Chem. 51, 582–587 (1973).
This article is protected by copyright. All rights reserved

FIGURE CAPTIONS
Figure 1. Oxalic ester used in the present study of the peroxyoxalate
reaction in aqueous medium.
Figure 2. Dependence of the observed rate constants of the emission
intensity decay (kobs) with [Sal] in its reaction with (A) TCPO (0.10 mmol L-1);
(B) BMePO (0.10 mmol L-1); (C) DMO (0.10 mmol L-1) and H2O2 (10.0 mmol
L-1); using Rhod 6G (0.10 mmol L-1) as ACT in a 1:1 (v/v) DME/H2O mixture
at 25 ºC.
Figure 3. Dependence of observed rate constants adjusted for the emission
decay (kobs) with [H2O2] in its reaction with (A) TCPO (0.10 mmol L-1); (B)
BMePO (0.10 mmol L-1); (C) DMO (0.10 mmol L-1) catalyzed by salicylate
(80.0 mmol L-1); using Rhod 6G (0.10 mmol L-1) as ACT.
Figure 4. Dependence of the singlet quantum yields (S) on [Sal] in the
reactions of (A) TCPO (0.10 mmol L-1; (B) BMePO (0.10 mmol L-1); (C) DMO
(0.10 mmol L-1) with H2O2 (10.0 mmol L-1); using Rhod 6G (0.10 mmol L-1) as
ACT.
Figure 5. Dependence of the singlet quantum yields (S) on [H2O2] in its
reactions with (A) TCPO (0.10 mmol L-1); (B) BMePO (0.10 mmol L-1); (C)
DMO (0.10 mmol L-1); using Rhod 6G (0.10 mmol L-1) as ACT.
This article is protected by copyright. All rights reserved

Figure 6. Dependence of the observed rate constants of the emission
intensity decay (kobs) with [Sal] in its reaction with TCPO (0.10 mmol L-1) or
BMePO (0.10 mmol L-1) and H2O2 (10.0 mmol L-1), using Rhod 6G (0.10 mmol
L-1) as ACT in aqueous borate buffer (100 mmol L-1) at 25 ºC and pH=8.2.
Table 1. Rate constants for the salicylate catalyzed (kSal) and the uncatalyzed
(k0) reaction of TCPO, BMePO and DMO with H2O2, using Rhod 6G as ACT,
obtained from the linear correlation of kobs with [Sal].
Table 2. Perhydrolysis (kper) and hydrolysis (khyd) rate constants obtained
from the linear correlation between kobs and [H2O2] for the sodium salicylate
catalyzed reaction of TCPO, BMePO and DMO with hydrogen peroxide, using
Rhod 6G as ACT.
This article is protected by copyright. All rights reserved

Table 1. Rate constants for the salicylate catalyzed (kSal) and the
uncatalyzed (k0) reaction of TCPO, BMePO and DMO with H2O2, using
Rhod 6G as ACT, obtained from the linear correlation of kobs with [Sal].
TCPO
0.0131 ± 0.0002
0.083 ± 0.002
0.994
BMePO
0.00292 ± 0.00008
0.251 ± 0.007
0.959
DMO
0.00079 ± 0.00008
0.13 ± 0.06
0.993
k0 (s-1)
kSal (L mol-1 s-1)
R2
[oxalic ester] = 0.10 mmol L-1, [H2O2] = 10.0 mmol L-1, [Rhod 6G] = 0.10 mmol L-1 in a binary 1:1
(v/v) DME/H2O mixture. Linear regression performed with kobs = k0 + kSAL [SAL].
Table 2. Perhydrolysis (kper) and hydrolysis (khyd) rate constants obtained
from the linear correlation between kobs and [H2O2] for the sodium
salicylate catalyzed reaction of TCPO, BMePO and DMO with hydrogen
peroxide, using Rhod 6G as ACT.
TCPOa
0.0227 ± 0.0004
0.292 ± 0.007
0.994
BMePOb
0.0214 ± 0.0003
0.167 ± 0.007
0.980
DMOc
-0.003 ± 0.002
0.78 ± 0.07
0.969
khyd (s-1)
kper (L mol-1 s-1)
R2
[oxalic ester] = 0.10 mmol L-1, [SAL] = 80.0 mmol L-1, [Rhod 6G] = 0.10 mmol L-1 in a
binary 1:1 (v/v) DME/H2O mixture. Linear regression performed with kobs = k0 + kper
[H2O2]. Linear regression performed for [H2O2]a from 10.0 to 500.0 mmol L-1 and for
[H2O2]b,c from 10.0 to 160.0 mmol L-1.
This article is protected by copyright. All rights reserved