Vol.3, No.1, 88-100 (2012) Journal of Biophysical Chemistry
http://dx.doi.org/10.4236/jbpc.2012.31011
Mechanism of chemiluminescence in Fenton reaction
Irina Pavlovna Ivanova1, Svetlana Vladimirovna Trofimova1, Igor Mihailovich Piskarev2*,
Nat alia Alekseevna Aristova3, Olga Evgenevna Burhina4, Oksana Olegovna Soshnikova4
1Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia
2Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia;
*Corresponding Author:
3Nizhny Tagil Technological Institute Branch of Ural Federal University, Nizhny Tagil, Russia
4Lobachevsky State University, Nizhny Novgorod, Russia
Received 23 October 2011; revised 4 December 2011; accepted 16 December 2011
ABSTRACT
A scheme of the processes in Fenton solution
with various substances is offered, and the ch-
annels of light formation registered by the
luminometer are analyzed. Under the proposed
scheme we discuss the possibilities of studying
the properties of antioxidants and prooxidants.
Oxidation of alanine, albumin and sodium oxa-
late have been taken as an example. The proper-
ties of ascorbic acid and the mechanism of
display of its oxidant and prooxidant properties
are analyzed herewith. Methodical questions of
the chemiluminescence research in Fenton solu-
tion such as the selection of reagents concen-
tration, water preparation and the effect of the
background radiation have been considered in
this study as well.
Keywords: Fenton Reaction; Chemiluminesce nce;
Oxidation Scheme; Antioxidant; Prooxidant
1. INTRODUCTION
Fenton reaction is widely used in the practice of scien-
tific research as a source of hydroxyl radicals. Fenton
reaction leads to the light radiation detected by modern
luminometer. Secondary reactions initiated by the Fenton
reagent are also often a source of photons. Recording
these photons, we can obtain information about bio-
physical processes. A great number of papers have been
written about bioluminescence and chemiluminescence
[1-3]. Luminescence is enhanced with introduction of
luminol, therefore the mechanism of luminol lumines-
cence has been researched in detail [4]. With introduction
of the Fenton reagent a flash of light is observed, that
may last for several seconds to tens of minutes, although
the duration of the Fenton reaction for the concentration
of Fe2+ and H2O2, which are commonly used, can range
from a few hours to many days [5]. New bursts of radia-
tion are observed with a re-introduction into a solution of
a fresh Fenton reagent. No research on formation of the
mechanism of short bursts has been yet made.
The Fenton reaction is used to determine the antioxi-
dant capacity of substances. One variant of the method is
described here [6]. They measure the light sum of chemi-
luminescence of the Fenton reaction in the absence of the
test substance S0 and the light sum in the presence of the
test substance S. If S0 = S, then we say that there are no
antioxidant properties. If S < S0, then the antioxidant
properties are present. In this case, the anti-oxidant
properties mean the ability of the test substance to be
oxidized by hydroxyl radicals, to absorb them and
thereby reduce the rate of hydroxyl radical reactions with
other substances. The case when S > S0 is also possible.
Then we can say that the substance is prooxidant and
enhances radical reactions.
It is interesting to investigate the general characteris-
tics of reactions initiated by the Fenton reagent and
leading to the formation of radiation in order to study the
oxidative capability of various substances including me-
dications. The processes occurring in a liquid environ-
ment are described by a great number of reactions be-
tween active particles and substances introduced to the
solution. An analytical solution of the equations of
chemical kinetics in this case is possible only if some
strong simplifying assumptions with a highly restricted
set of reactions are made. The solution is found by the
method of stationary concentrations. This method gives a
result related to a limited range of conditions and does
not always have a general meaning. Therefore, we used
numerical methods. We compiled a reactions scheme
involving the formation of light radiation with a pure
Fenton reagent, as well as the addition of luminol, inor-
ganic and organic substances, one oxidation channel of
which represents a chain reaction.

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00 89
2. EXPERIMENTAL METHODS
2.1. Registration of Light
A light emission was recorded by a chemiluminometer
BCh-06 (N. Novgorod, Russia). Spectral range sensitiv-
ity of the photomultiplier (PM) is 400 - 700 nm. A cali-
bration of the device was performed via the reference
light source of the known intensity. When recording the
light from the sample, the measurement of the noise of
the registering device, that was made automatically, was
performed directly before and immediately after each
measurement. The lower detection limit of the light ra-
diation is determined by fluctuations of the noise. The
measurements showed that the mean-square fluctuations
of the noise ranged from 200 to 500 pulses per second
depending on the device condition. Thus the minimum
detectable effect was about 200 photons per second. The
sample volume was 1.0 ml. The composition of the sam-
ple was as follows: 0.4 ml of FeSO4, 0.4 ml of the test
substance, 0.2 ml of hydrogen peroxide. The reactant
concentration was [FeSO4] = 10–3 mol/l, [H2O2] = 10–4
mol/l. A cuvette containing the sample was located very
closely to the PM photocathode ensuring high detection
efficiency (solid angle  = ). The cuvette was firstly
injected with a solution of ferrous iron, the substance
under investigation, and last of all peroxide. A solution of
hydrogen peroxide of a necessary concentration for the
Fenton reagent had been prepared in advance, while a
solution of FeSO4—right before the commencement of
the experiment. The registration of radiation began in 0.5 -
1 second after mixing the sample solution containing
FeSO4 with peroxide. This time is required to add the
peroxide and switch the device to the mode of measuring
the intensity of chemiluminescence. Analytical grade
reagents, double-distilled and only distilled water (pH 6)
were used for the experiment.
2.2. Preparation of Water
Preparation of water for these experiments is of great
importance. First of all the sameness of water contents is
crucial. Even the water Milli-Q, Milli-pore, standing in
the air, absorbs carbon dioxide. Its properties will depend
on how much time the water was the subject to contact
with the air after the preparation. Accumulated in the
water under absorption of carbon dioxide hydrocarbons
react with the hydroxyl radicals generated in the Fenton
reaction, thereby reducing their concentration. The cal-
culation in the scope of the model used in this study
shows that the concentration [3] = 10–7 mol/l does
not affect the consumption of OH· radicals. However,
when [3]  10–6 mol/l hydrocarbons absorb a sig-
nificant amount of hydroxyl radicals, which affect the
results of the experiment. The calculation results have
been confirmed by the experiment. Therefore we used
only freshly prepared distilled water. To monitor the sta-
bility of the water composition the chemiluminescence
light sum for the pure Fenton solution prepared with this
water was measured.
HCO
HCO
The chemiluminescence light sum was registered dur-
ing 30 seconds. Every point was measured in 10 - 12
repetitions. Cleanliness of the solutions, the composition
of the reaction products were controlled at various stages
by monitoring the UV spectra with the spectrophotometer
Fluorat-02 Panorama. The spectral characteristics of the
light radiation from water was qualitatively evaluated
using color filters of blue and red plastic films with a
thickness of 0.5 mm. The transmission spectrum of the
films was measured by the spectrophotometer Fluorat-02
Panorama. The bandwidth of the blue filter at least 10%
of the maximum transparency was ranging from 410 to
590 nm, red—from 590 to 750 nm. The films that do not
give the secondary radiation in the visible spectrum were
sorted out.
3. RESULTS AND DISCUSSION
3.1. Kinetic Model of the Process
The model of the process included the Fenton reaction,
the interaction of radicals, the products of this reaction,
the formation and de-excitation of the singlet oxygen.
The scheme of the reaction is presented in Ta bl e 1 . The
reaction rate constants are given in the references [7].
The concentrations of ions OH и H+ (pH of solution) are
set as coefficients. The model includes the interaction of
ferrous iron with hydrogen peroxide and subsequent
formation of radicals OH, 2, 2 and singlet oxy-
gen, the dissociation of hydrogen peroxide H2O2 
2
HOO
HO

+ H+, pKa = 11.5 (reactions 12, 13) and the equi-
librium 2
HO

 H+ + 2 O

, pKa = 4.8 (the 7, 8). It was
assumed that in the neutral and alkaline medium ferrous
and ferric irons precipitate (reactions 15, 16, Table 1.)
Air usually contains carbon dioxide and it is absorbed by
water. The existence of hydrocarbons was taken into ac-
count (reactions 18 - 20). The external ionizing radiation
(background and cosmic rays) caused radiolysis of water.
The yield of the radiolysis products and their reactions
are known [8]. These reactions are included in the calcu-
lation scheme (Table 1). The system of equations of
chemical kinetics was solved using a package MathCad
14. The purpose of the numerical simulation was to
evaluate the contribution of specific mechanisms and to
analyze the concentration of active species (radicals and
molecular products). These data are necessary for the
quantitative analysis of free radical products formed in
biological systems during the development of pathologi-
cal processes and after the action of physical and chemi-
cal factors.

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90
Table 1. The rate constants for reactions in Fenton solution.
No Reaction k, l/(mols), [7]
1. Fe2+ + H2O2 → Fe3+ + OH + OH k1 = 56
2. OH + H2O2 → + H2O
2
HOk2 = 3 × 107
3. 2
HO + → H2O2 + O2 +
O2(a1g)
2
HO
k3 = 8.3 × 105
4. Fe2+ + OH → Fe3+ + OH k4 = 3 × 108
5. OH + OH → H2O + 1
2(О2 + O2(a1g)) k5 = 5.5 × 109
6. OH + → H2O + O2 + O2(a1g)
2
HOk6 = 7.1 × 109
7. 2
HO → H+ +
2
Ok7 = 7.5 × 106, pKa = 4.8
8. H+ + →
2
O
2
HOk8 = 5.1 × 1010
9. 2
HO + → + O2
2
O
2
HOk9 = 9.7 × 107
10. 2
HO + OH → + H2O
2
Ok10 = 1010
11. 2
O + Fe3+ → Fe2+ + O2 k11 = 1.9 × 109
12.H2O2 → + H+
2
HOk12 = 2 × 10-2
13. 2
HO + H+ → H2O2 k13 = 1010, pKa = 11.5
14.OH + → + OH
2
HO
2
HOk14 = 7.5 × 109
15.Fe3+ + 3OH → Fe(OH)3 k15 = 106, pH = 12
16.Fe2+ + 2OH → Fe(OH)2 k16 = 106, pH = 12
17.H2O2 + → OH + OH + O2
2
Ok17 = 16
18. 3
HCO + OH → + H2O
3
CO k18 = 4 × 107
19. 3
CO  + OH → CO2 +
2
HOk19 = 3 × 109
20. 3
CO  + H2O2 → +
3
HCO
2
HO

k20 = 8 × 105
The rate constant for each reaction is known to have
some errors. Various sources provide different values.
With a large number of reactions that simultaneously
take place in the system, changes in the reaction con-
stants over a wide range do not lead to noticeable changes
in results. This is due to the fact that in the chain of the
sequential and parallel transformation the variation in the
reaction constants make the concentration of the inter-
mediate products also change. If the rate of consumption
of the intermediate product is reduced, its concentration
increases. As a result, the rate of the final product forma-
tion may not change at all.
3.2. The Mechanism of Luminescence of
Fenton Solution
The reactions in the water involving reactive oxygen
species, initiated by the Fenton reagent, lead to lumines-
cence registered by BCh-06. The spectral composition of
the light radiation was estimated with red and blue filters.
We have established that the blue filter reduces the emis-
sion by more than 10 times, while the red filter does not
change the light intensity.
A dimer of singlet oxygen might be used for lumines-
cence in this spectral region. The formation and decay
scheme of the singlet oxygen is shown in Table 2.
Luminescence is recorded if the sample contains the
reaction products in an excited state. Almost all of the
reaction products that are listed in Tables 1 and 2 can
form excited states displayed in the UV spectrum (wave-
length below 400 nm). But its radiation is out of spectral
sensitivity of the PM photocathode. In the red region of
the spectrum only a dimer of the singlet oxygen can be
displayed (
 = 480, 535 and 580 nm) [1-3].
The mechanisms of the singlet oxygen formation were
analyzed. The spin selection rules allow the formation
both a singlet and a triplet oxygen in the reactions (3, 5,
6. Tab le 1 ). Ratio of probabilities of the singlet and trip-
let states production is determined by the rules of quan-
tum mechanics and is equal to 1:3. Only singlet oxygen
can be formed in the reactions (21, 22, Table 2). The
calculation showed that the yield of O2(a1g) in the reac-
tions (3, 5, 6) is small because the concentration of radi-
cals 2
HO

and OH is small.
Therefore, reactions with a radical 2 (21, 22) are
the basic mechanism of the singlet oxygen formation.
The source of 2
O
O

 is radicals 2 (reactions 7 and
10). The final luminescent product is a singlet oxygen
(more definitely, a dimer of the singlet oxygen). A dimer
decomposes almost instantaneously, so the intensity of
emission is determined by the rate of its formation. This
is not a rate of one single reaction, but a total (resultant)
rate of the whole sequence (chain) of reactions, which
ends with the formation of the singlet oxygen dimer. In
case of a Fenton reagent ferric ion, formed in solution,
reacts with 2
HO
O

 (reaction 11, Table 1), which signifi-
cantly reduces the concentration of these particles. With
decreasing the concentration of 2
O the yield of the
singlet oxygen decreases, so in Fenton solution a short
burst of radiation is always observed immediately after
mixing the reagents, the duration of which significantly
smaller than the total reaction time at these concentra-

Table 2. Formation and decay of singlet oxygen.
NoReaction k, (mols)–1 [9]
21. 2
O


+ OH + H+ → H2O + O2(a1g) k21 = 1010
22. 2
O


+ H+ → 1
2H2O2 + 1 2O2(a1g) k22 = 1010
23. Decay O2(a1g) 
1/2 = 2.9 × 10–4 с
24. O2(a1g) + O2(a1g) → 2O2 +  k24 = 0.1
25. O2(a1g) + O2(a1g) → products k25 = 1011

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00
91
tions of reagents [5]. When there are no ferric ions in
solution, ion-radicals 2 accumulate, and the number
of the generated singlet oxygen molecules increases. The
lifetime of the singlet oxygen in water with low concen-
trations of impurities is approximately 40 minutes. The
probability of the dimer formation is about 1000 times
higher than the spontaneous decay [9]. The calculation
showed that the quenching of the singlet oxygen on the
vessel walls and in the reactions with other particles is
negligible. With the increasing concentration of O2(a1g)
the reaction 25 (Ta b le 2 ), the products of which do not
give the radiation in the visible light spectrum, plays a
decisive role in their deaths. The kinetics of changes in
the concentration of radicals after addition of a Fenton
reagent is described [5].
O
that the optimal time of the registration light emission in
terms of high stability is 30 seconds. It is reasonable to
choose the concentration of reactants to make the time of
the Fenton reaction also about 30 sec. According to the
study results [5], when [Fe2+] = 103 mol/l and [H2O2] =
104 mol/l the time of the reducing reaction rate up to 1%
of its initial value is 60 seconds. In practice, the concen-
trations of [Fe2+] = 103 mol/l and [H2O2] = 10–1 mol/l
are commonly used [10]. We compare these two concen-
trations. First of all, the concentration of hydrogen per-
oxide under other equal conditions affects the duration
and amplitude of the leading edge of the light flash. The
calculated values of the light sum S on the time in the
range of 0.01 to 30 seconds for the concentration of
[H2O2] = 101 and 104 mol/l are presented in Figure 1. A
step on the time axis is 0.01 seconds. We see that when
[H2O2] = 101 mol/l at the beginning of the reaction there
is a short light flash with a duration of less than 0.1 sec-
onds and a large amplitude and almost all the photons are
emitted. During the time from 0.1 to 30 seconds, the light
sum increases slowly. The loss of photons emitted for 0 -
0.5 seconds (time delay of registration) reduces the reg-
istered light sum by 100 - 1000 times. At [H2O2] = 104
mol/l the light sum increases slowly, the loss of radiation
at t < 0.5 seconds has a little effect on the outcome. Since
it is impossible to begin the registration of the light ra-
diation in less than 0.5 seconds after the last injection of
the reagent, when [H2O2] = 101 mol/l the largest and
uncontrollable part of the radiation remains unregistered.
The situation is aggravated by the fact that the time for
mixing the solution after the introduction of peroxide at
the final stage is not equal to zero. The introduced per-
oxide has a concentration of up to 5 times greater than
To sum up, immediately after mixing the components
of the Fenton reagent no ferric iron has yet been formed
in the solution, and all nascent 2 radicals are con-
sumed on the singlet oxygen production. Therefore, im-
mediately after mixing a maximum light emission is ob-
served. Ferric iron that is being formed begins to con-
sume radicals 2, luminescence is decreased, and stops
if the concentration of Fe3+ obtains high concentrations,
capturing all 2 radicals. Thus, the light flash duration
is not determined by the consumption of the reagents in
the Fenton reaction, but it is determined by the time that
is sufficient to form a large number of Fe3+ ions. Lumi-
nescence in this case is in the red region of the light
spectrum.
O
O
O
3.3. The Selection of Peroxide
Concentration
The peculiarity of the chemiluminometer BCh-06 is
Figure 1. Time dependence of the total number of emitted photons, the light sum
S, on the time since mixing the reagents for the concentration of [Fe2+] = 10–3
mol/l and [H2O2] = 10–1 mol/l (1) and 10–4 mol/l (2).
OPEN A CCESS

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00
92
that averaged by the volume, and the reaction starts im-
mediately after the contact with the tested solution. In
this case the front of the light flash will be even shorter.
The homogeneity of the solution is of high importance.
When the concentration of [H2O2] = 104 mol/l the light
flash front is significantly longer.
Therefore, the delay of start registration, the quality of
mixing the tested solution and the diffusion time of the
peroxide from the introduced drop into the full volume
affect the result significantly less. An uncontrolled sys-
tematic error of the measurement will be significantly
less than that in the previous case.
The use of the Fenton reagent components concentra-
tions [Fe2+] = 103 mol/l and [H2O2] = 104 mol/l pro-
vides more reliable results than those with a high con-
centration of peroxide, therefore we selected these con-
centrations.
3.4. The Yield of Water Radiolysis Products
Created by the Background Radiation
The power of the dose produced by the background
radiation is known to be determined by cosmic radiation
and other factors. It ranges from 0.05 to 0.3 µSv/h with
an average value of 0.12 µSv/h. In water the radiation
creates products of radiolysis. The yields of the primary
products of radiolysis at pH 7 are presented in Table 3.
The yield of radicals are calculated for an average ra-
diation background of 0.12 Sv/h. This corresponds to
the energy released in 1 liter of water, 2.06 × 108 eV/s.
Formed in the radiolysis the particles with reducing
properties (hydrated electron and atomic hydrogen) in-
teract with the oxygen dissolved in water, forming radi-
cals 2
O

 and 2
HO

[8].
3.5. Stationary Concentration of the
Radiolysis Products
Under the constant influence of the radiation back-
ground the radiolysis products accumulate and establish
their steady-state concentration. A steady-state concen-
tration depends on the level of the background radiation.
Table 4 shows the calculated steady-state concentrations
of the short-lived products and the time required to es-
tablish steady-state concentrations. The time for estab-
lishing a steady-state concentration depends on the level
of the background radiation. Active particles cumulated
under the action of the background radiation cause the
solution chemiluminescence. In Table 4 the calculated
values of the photon yield for 30 seconds are also pre-
sented.
Under the action of the background radiation hydrogen
peroxide accumulates both through the direct formation
by radiation (Ta bl e 3 ) and in reactions with radicals (re-
action 3 and 19). In the reaction of the bicarbonate with
hydroxyl radicals ion-radicals 3 are initially formed.
Then they again react with hydroxyl radicals, forming a
product of dissociation of hydrogen peroxide, 2
CO
HO

and
carbon dioxide. Thus, the bicarbonates influence the
concentration of hydrogen peroxide. A stationary con-
centration of peroxide at different levels of the back-
ground radiation and different concentrations of the bi-
carbonate are presented in Ta bl e 5. The presence of fer-
rous iron in the solution does not affect the steady-state
concentration of peroxide up to [Fe2+] = 108 mol/l. At
higher concentrations of iron the concentration of perox-
Table 3. The yield of the primary products of radiolysis at pH 7 [8].
Product aq e H OH H2O2 H
2
Yield at 100 eV 2.8 0.5 2.8 0.7 0.45
Converted into radicals 2
O 2 HO

- - -
Yield mol/(l·s) 9.5 × 10–18 1.7 × 10–18 9.5 × 10–18 2.4 × 10–18 1.53 × 10–18
Table 4. The stationary concentration of the active particles formed in the water under the influence of background radiation (calcu-
lated).
Concentration, mol/l at different levels of background radiation
The product of radiolysis
0.012 µSv/h 0.12 µSv/h 1.2 µSv/h
The time for establishing steady-state concentration, s
2
O 1 × 10–16 1.4 × 10–15 1.9 × 10–14 100 - 200
O2(a1g) 7 × 10–16 2.4 × 10–15 8 × 10–15 1000 - 1500
OH 10–24 10–23 10–22 1
HO2 2·10–26 3 × 10–25 3 × 10–24 1
Luminescence for 30 sec 2 × 10–16 mol/l 4.7 × 10–16 mol/l 2.1 × 10–15 mol/l Not less than 20 minutes

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00 93
Table 5. The stationary concentration of hydrogen peroxide
formed in the water under the influence of background radia-
tion (calculated). The estimated time for the steady-state con-
centration is 107 seconds.
[H2O2] (mol/l) at various levels of background
radiation
[] (mol/l)
3
HCO
0.012 µSv/h 0.12 µSv/h 1.2 µSv/h
10–2 2.5 × 10–11 2.3 × 10–10 2.5 × 10–9
10–3 2.5 × 10–11 2.3 × 10–10 2.3 × 10–9
0 1.1 × 10–11 8 × 10–11 6.2 × 10–10
ide decreases.
3.6. Scheme of the Oxidation by Radicals
Generated in the Fenton Reaction with
the Formation of Luminous Products
The mechanisms of luminescence of Fenton solution
in a neutral solution (pH ~ 7) are analyzed for the fol-
lowing cases:
1) solution of Fenton (no other chemicals are intro-
duced);
2) addition of luminol;
3) ideal antioxidant M (a substance capable of oxida-
tion);
4) addition of organic substance RH, which can de-
velop a chain reaction of oxidation:
a) a substance capable of forming radicals 2
RO

(with
availability of oxygen);
b) a substance that generates only radicals R (with a
lack of oxygen). Channels reactions are conditionally
presented in Scheme 1 and numbered.
Case 1. Pure Fenton solution. Scheme 1, channel 1.
The reaction products are ferric ions and hydroxyl radi-
cals ОН. A luminescence mechanism has already been
shown (see Tables 1 and 2). The sequence of reactions of
radicals can be represented as follows: OH → 2 HO

→
2 → O2(a1g). A luminiscence of the dimer of singlet
oxygen is in the red spectrum (
 = 480, 535 and 580 nm).
Luminescence duration is not determined by spending
the reagents in the Fenton reaction, but by the time re-
quired to attain a sufficiently large number of ions Fe3+
that use radicals and thus cease to glow.
O
2
Case 2. Luminescence with luminol in a neutral
medium. Scheme 1, channel 2. Luminol Lum reacts
with a hydroxyl radical, forming radical L. This radical
reacts with superoxide radicals 2, and after a chain of
reactions, conditionally presented in the scheme by two
crosses, luminescence of a quantum in the blue spectral
region appears [3,4]. This is the case as long as there is
no ferric iron. When ions Fe3+ appears, the radical 2
O
O
O


is consumed in the reaction with Fe3+. In this case the
blue glow decreases, and fully stops with high concen-
trations of Fe3+. But with low concentrations of ferric
iron the luminescence covers the blue spectral region
(luminol) and the red one (dimer of singlet oxygen),
since luminol does not intercept all the produced hy-
droxyl radicals. The luminol-dependent luminescence of
Fenton solution in a neutral solution is considered in
more detail in [5].
Case 3. An ideal antiox idant M is the substance oxi-
dized by radicals. The reaction products are low-active
radicals not capable to participate in further conversions
with a considerable rate. In this case, the hydroxyl radi-
cals are consumed in its oxidation. If the reaction rate M
+ OH is much greater than that of in channel 1, no light
emission will occur. When the rate of consumption of
ОН radicals in the processes 1 and 3 are comparable, the
luminescence will be observed in the red spectrum. With
increasing concentration of substance M luminescence
will diminish until it completely disappears. The de-
pendence of the light sum for 30 seconds on the concen-
tration of introduced material M is shown in Figure 2.
Figure 2(a) represents the results of the calculation and
(a)
(b)
Figure 2. Chemiluminescence of ideal antioxidant M
in the oxidation by the Fenton reagent, [Fe2+] = 10–3
mol/l, [H2O2] = 10–4 mol/l. Along the ordinate axis:
the ratio of the light sum for 30 seconds at a given
concentration of test substance S to the light sum for
30 seconds for a pure Fenton solution S0, S/S0. (a)
The calculation of the conditional material M. [M] -
concentration of substance, mol/l; (b) The experiment-
tal data on the chemiluminescence of alanine in Fen-
ton solution. [Al]—the alanine concentration, mg/l.

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00
94
Scheme 1. Reactions in Fenton solution, leading to light emission. Numerals indicate the following cases: 1. Only
Fenton solution; 2. Luminol is added in Fenton solution; 3. Fenton solution with ideal antioxidant M; 4. Oxidation
of organic matter RH in Fenton solution: a) in the presence of dissolved oxygen b) with a deficiency or complete
absence of oxygen.
Figure 2(b)—the experimental data for alanine, an ideal
antioxidant. The reaction constant M + ОН is taken
equal to 108 (mol·s)1.
Case 4. Oxidation of organic substances. A distinc-
tive feature of the organic substances is the ability to
continue the chain reactions. A typical scheme of oxida-
tion of organic matter RH in water solution is presented
in Table 6. The first active product of oxidation is radical
R (reaction 26). Further, in the absence of oxygen, these
radicals can be lost in interactions with each other (reac-
tion 31). No luminescence will occur if the concentration
of the substance introduced is high and it catches all the
hydroxyl radicals. In the intermediate case there can be
the situation considered for case 3 when not all hydroxyl
radicals are used by RH substance and luminescence in
channel 1 is preserved. This channel will be active in
case of the lack of oxygen if [R] >> [O2] (channel 4b,
Scheme 1). If there is enough oxygen, then radical 2
RO

(reaction 27) is formed leading to singlet oxygen emer-
gence (reaction 32). There is no interdiction implied on

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00 95
Tab le 6. Reactions of organic matter RH initiated by hydroxyl
radicals in an aqueous solution.
No Reaction k, (mol·s)–1 [11]
26. RH + OH → R + H2O 108
27. R + O2 →
2
RO104
28. 2
RO + RH → ROOH + R 106
29. ROOH + Fe2+  Fe3+ + OH– + RO 100
30. RO + RO  ROOR 105
31. R + R  R-R 106
32. 2
RO +  ROOR + O2 + O2(a1g)
2
RO107
the formation of oxygen in a particular spin state for re-
action 32. The ratio of the population probability for
triplet and singlet states is determined by the rules of
quantum mechanics and is 3:1. A dimer of singlet oxygen
irradiates in the red spectrum (channel 4a, Scheme 1).
The spectral composition of radiation has been tested
with blue and red filters. The dependence of lumines-
cence on the concentration of injected substances into
channels 4a and 4b will be different.
Case 4b. If there is no substance, [RH] = 0, the solu-
tion luminescence will be determined by channel 1 (pure
Fenton solution). With the increase of RH concentration
more radicals OH are consumed and luminescence di-
minishes until it completely disappears. The dependence
of luminescence on the concentration of introduced sub-
stances will be similar to that of shown in Figure 2.
Case 4a. The addition of RH in low concentrations in
Fenton solution has no effect on chemiluminescence. As
[RH] increases, the luminescence caused by the reaction
of the radicals 2 becomes more intense. The total
luminosity can increase tens times as large and reach
maximum intensity. With the further increase of [RH],
reaction 2 + RH (reaction 28, Table 6 ) consuming
radical 2 begins to play its role and the emission de-
creases. When [RH] >> [2
RO
RO
RO
RO

], the interaction of the
radicals 2 with each other (reaction 32, Tab l e 6 ) is
very unlikely and the emission stops.
RO
The results of calculation of S/S0 from the concentra-
tion of RH at different oxygen concentrations in solution
are shown in Figure 3(a). The flash of luminescence is
seen to decrease with the decrease of oxygen concentra-
tion and in the full absence of oxygen RH substance be-
comes almost a perfect anti-oxidant (compare Figure 2
(a)). The substantiality of calculation is confirmed by the
experimental data for albumin (Figure 3(b)), when at a
certain concentration of albumin luminescence reaches
its maximum and then decreases.
3.7. Individual Substance
There may be cases, especially for low-molecular or-
(a)
(b)
Figure 3. Chemiluminescence of organic matter RH in
the oxidation by the Fenton reagent, [Fe2+] = 10–3 mol/l,
[H2O2] = 10–4 mol/l. The ordinate axis: the ratio of the
light sum for 30 seconds at a given concentration of the
test substance to the light sum S for 30 seconds clear
solution Fenton S0, S/S0. (a) The calculation for the
conditional substance RH. [RH]—concentration of sub-
stance, mol/l. The rate constants are given in Tab le 6.
The concentration of dissolved oxygen [O2]: 1) 10–4
mol/l, 2) 10–5 mol/l, 3) [O2] = 0; (b) The experimental
data on the chemiluminescence of albumin in Fenton
solution. [A]—concentration of albumin, mg/l. The
nonmonotoneness of presented dependence is associ-
ated with experimental errors.
ganic substances, when oxidation cannot be described by
the scheme shown in Ta bl e 6 . In this case the character-
istics of the substance are to be taken into account. The
oxidation of oxalic acid by hydroxyl radicals was studied
in detail [12,13]. A simplified scheme of reactions con-
tributing to the chemiluminescence is given in Table 7.
The particularity of this case is the formation of the
radical 2
HO

(reaction 34) instead of 2 and regen-
eration of oxalic acid (reaction 35). Radicals 2
RO
HO

do
not decompose oxalic acid at an appreciable rate, so with
the increase of acid concentration the luminescence is
not reduced. The calculated dependence of the chemilu-
minescence of oxalic acid on its concentration is pre-
sented in Figure 4(a). With the increase of oxalic acid
concentration the luminescence is seen to increase. With

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00
96
Table 7. The reactions of oxalic acid affecting the chemiluminescence in Fenton solution.
Reaction k (mol·s)–1 [13]
33. (COOH)2 + OH → HOOC-COO + H2O 1.4 × 106
34. HOOC-COO + O2 → HO2 + 2CO2 1 × 108
35. HOOC-COO + HOOC-COO → (COOH)2 + 2CO2 7.7 × 106
OPEN A CCESS
(a)
(b)
Figure 4. Chemiluminescence of oxalic acid in the
course of oxidation by the Fenton reagent, [Fe2+] =
10–3 mol/l, [H2O2] = 10–4 mol/l. S/S0—see Figure 2.
(a) The calculation is according to the reaction scheme
shown in Table 7. [Oxalic]—concentration of oxalic
acid, mol/l. 1) The concentration of dissolved oxygen
[O2] = 10–4 mol/l. 2) [O2] = 10–6 mol/l. 3) [O2] = 0 (no
oxygen). 4) Chemiluminescence of Fenton solution
due to the external background radiation. (b) The ex-
perimental data for sodium oxalate, [oks]—concentra-
tion of sodium oxalate, mg/l. The nonmonotoneness of
presented data is related to experimental errors.
decreasing concentration of oxygen the intensity of che-
miluminescence decreases and in the absence of oxygen
oxalic acid behaves as an ideal antioxidant. If oxalic acid
concentration is high in the absence of oxygen not only
radicals OH generated in the Fenton reaction are con-
sumed but also those generated by external radiation
background. Figure 4(a) shows that at concentrations of
[Oxalic] = 10–2 mol/l chemiluminescence yield (curve 3)
is significantly below the level set by the background ra-
diation (line 4).
The calculated dependence is qualitatively confirmed
by the experiment with sodium oxalate (Figure 4(b)).
The main difference from the reaction schemes presented
in Tab le 6 is that with increasing concentration of oxalic
acid luminescence increases without passing through its
maximum and decreases no further as shown in Figure 3.
A higher yield of chemiluminescence compared to a pure
Fenton solution is due to the fact that in Fenton solution
most OH radicals disappear during interactions with
each other failing to transform into radicals 2
HO

. When
oxalic acid is added in a sufficiently high concentration,
so that [Oxalic] > [OH], a major part of ОН radicals
interacts with it and the radicals fully transform into
radicals 2
HO

. As the concentration of oxalic acid in-
creases, the part of radicals OH transformed into 2
HO

also grows.
3.8. The Form of Chemiluminescence Light
Impulse
Three cases are possible.
1a. Fenton solution. The maximum intensity is ob-
served at first when the reaction rate is at its maximum.
Taking into account the delay in the registration system
the front duration can be 0.1 - 0.2 seconds. The ferric
iron that is formed consumes 2 radicals and weakens
the luminescence. When 20% - 30% of the initial Fe2+ is
converted to Fe3+, the luminescence practically stops.
O
1b. Luminol in neutral medium (pH 6 - 7). The
situation is similar to a pure solution of Fenton. In Fen-
ton solution with luminol the maximum light emission
intensity is observed immediately after mixing the re-
agents. With increasing concentration of Fe2+ and H2O2
reaction rate increases and the amplitude of the light
flash grows. The characteristic forms of impulse for this
case are described [5].
2. Simple substance absorbing the hydroxyl radi-
cals (ideal antioxidant M). At first it can absorb practi-
cally all the hydroxyl radicals, so there will be no flash
of light. Noticeable luminescence appears when the part
of the substance is oxidized by radicals. The leading
front of the pulse will be longer and can take 1 - 2 sec-
onds.
3. Organic matter RH. If it absorbs not all the hy-
droxyl radicals, the luminescence of Fenton solution it-
self will be seen. As the chain reaction starts, the oxida-
tion products (radicals 2
RO

) are produced, and their in-

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00 97
teraction leads to the production of an additional singlet
oxygen. It gives a new outbreak of light radiation, which
is superimposed on the luminescence of Fenton solution.
The delayed appearance of a new light impulse is deter-
mined by reaction rates 26 - 32 (Table 6) and can last 10
- 30 seconds, the flash duration being a few minutes. The
amplitude of the second light flash can exceed by far the
luminescence of Fenton solution. Calculated under the
above-stated model luminosity of the mixture of Fenton
solution with the RH substance is given in Figure 5. The
graph shows the luminescence of a pure Fenton solution
(peak 1) and luminescence formed in reactions 26 - 32
(peak 2).
The form of the light radiation impulse, as shown in
Figure 5, was really observed in the studies of chemilu-
minescence [2,3]. The emission maximum 1 and 2 can be
interpreted as follows:
Maximum 1—glow caused by reactive oxygen spe-
cies.
Maximum 2—the glow caused by free-radical proc-
esses in the sample (the formation of ).
2
Not all organic substances during oxidation pass
through a stage of formation of 2 type compounds.
In this case maximum 2 is not observed. For example, in
oxalic acid oxidation 2 is formed instead of radical
2 (see Table 7). Radical 2 plays a minor part in
the oxidation of ascorbic acid. Thus, the fact of appear-
ance or absence of maximum 2 (Figure 5) enables to
draw conclusions about the reaction mechanism. In gen-
eral case, the oxidation scheme of the test substance is
necessary to be considered for a detailed analysis of the
results of the chemiluminescence oxidation.
RO
RO

HO
RORO
3.9. Evaluation of Properties of a Certain
Antioxidant with the Fenton Reaction
Ascorbic acid can be considered as an example. Re-
searchers repeatedly note that ascorbic acid possesses
both antioxidant and prooxidant properties [14-16]. The
scheme of ascorbic acid oxidation is presented in Table 8.
The characteristics of reactions are taken from ref. [15,
17]. Ascorbic acid in an aqueous solution dissociates in
two steps (reactions 36 and 37, Ta bl e 8). At pH values
typical for biological objects (pH 6 to 7.5), ascorbic acid
remains in solution as ions AscH (more than 99%).
The primary oxidation product of AscH is radical
Asc (reaction 38, Table 8). A feature of ascorbic acid is
that neither the ion AscH nor radical Asc interacts with
oxygen [15]. Only ion Asc2 interacts with oxygen (reac-
tion 40), forming an ion-radical 2. Two radicals of
Asc transform to initial ion and DHA (dehydroascorbic
acid, reaction 39). DHA is further oxidized by oxygen to
form various compounds, including a prooxidant such as
O
Table 8. Oxidation reaction of ascorbic acid AscH2. AscH– and
Asc2 are the products of the first and second stages of disso-
ciation of ascorbic acid. Asc is ascorbate ion-radical, DHA -
dehydroascorbic acid.
NoReaction The equilibrium constant, The
rate constant [15,17]
36. AscH2 ↔ AscH– + H+ pKa1 = 4.1
37. AscH– ↔ Asc2– + H+ pKa2 = 11.8
38.AscH– + OH → Asc + H2Ok3 = 1.1 × 1010 (mol·s)–1
39.2Asc + H+ → AscH– + DHAk4 = 1.4 × 105 (mol·s)–1
40.Asc2– + O2 → Asc + 2
O


k5 = 100 (mol·s)–1
Figure 5. The intensity of chemiluminescence P of organic matter RH in Fenton solution.
[Fe2+] = 10–3 mol/l, [H2O2] = 10–4 mol/l, [RH] = 10–4 mol/l. The figures denote: 1) The flash
of chemiluminescence due to reactions in a pure Fenton solution by oxygen active forms; 2)
Chemiluminescence of singlet oxygen formed in reaction 32 (Table 6) due to free radicals.

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00
98
oxalic acid.
Calculated dependence of S/S0 on concentrations of
ascorbic acid without regard to the products of DHA
transformation is presented in Figure 6 for two cases: in
oxygen medium and in the one without. It is evident that
without oxygen S/S0 monotonically decreases with in-
creasing [Asc] (curve 2), and in the presence of oxygen
for [Asc] > 103 mol/l ratio S/S0 begins to increase
(curve 1). The increase is due to formation of superoxide
ion-radical 2. In the course of reactions in Fenton so-
lution, this radical can, although with a low probability,
transform into a hydroxyl radical OH. In this case,
ascorbic acid will absorb secondary radicals initiated by
its oxidation. At the concentration of [Asc] up to 1 mol/l
the antioxidant effect will occur, although the probability
of suppression of radicals with increasing concentration
of acid in solution, saturated by air, will decrease.
O
The experimentally obtained dependence S/S0 on a
wide range of concentrations of ascorbic acid [Asc] is
shown in Figure 7. Measurements were performed for
two cases: 1) the solution contains oxygen dissolved in
natural conditions, 2) solution is depleted of oxygen by
adding 0.1 ml of Na2SO3 solution at a concentration of
3.2 g/l. In a separate experiment it has been found that
the introduction of Na2SO3 has no influence on lumines-
cence of Fenton solution. With the help of color filters it
has been identified that chemiluminescence occurs in the
red spectrum.
In the presence of oxygen (curve 1) and at a low con-
centration of [Asc] value S/S0 equals to 1, which may be
primarily due to the small contribution of secondary re-
actions (initiated by the interaction AscH + OH) to the
general flow of radicals generated in the Fenton reaction.
With increasing of [Asc] ratio S/S0 exceeds 1, then it
decreases with increasing [Asc] and becomes less than 1.
When S/S0 < 1, we can see the marked antioxidant prop-
erties of the substance introduced in Fenton solution.
Value S/S0 > 1 indicates the number of secondary radi-
cals formed in reactions with introduced substance to
exceed the number of primary ones, i.e. chain reaction
takes place. In case of lack of oxygen (curve 2) the S/S0
Figure 6. The ratio dependence of the light sum during 30 sec-
onds for Fenton solution with ascorbic acid S to the light sum
for a pure Fenton solution S0 (S/S0) on the concentration of
ascorbic acid, [Asc], mol/l. 1) solution, saturated by oxygen,
[O2] = 2 × 10–4 mol/l (7 mg/l), 2) without oxygen. When [Asc]
< 103 mol/l curves 1 and 2 are the same.
Figure 7. Chemiluminescence of ascorbic acid in the oxidation by the Fenton reagent,
[Fe2+] = 10–3 mol/l, [H2O2] = 10–4 mol/l. S/S0—see Figure 6. [Asc]—concentration
of ascorbic acid, mol/l. Errors of ratio S/S0 do not exceed 5%. 1) There is oxygen dis-
solved in vivo in the solution; 2) Solution is depleted of oxygen by the addition of 0.1
ml [Na2SO3] = 3.2 g/l.

I. P. Ivanova et al. / Journal of Biophysical Chemistr y 3 (2012) 88-1 00 99
is less than 1 for all concentrations of ascorbic acid,
which means that the ascorbic acid itself is an antioxi-
dant.
Prooxidant properties were observed in [14,15] at the
concentration of ascorbic acid of approximately 103
mol/l, which is close to the concentration at which the
maximum S/S0 is reached in Figure 7 for the solution
containing oxygen. The authors of [14,15] report ascor-
bic acid at concentrations of 103 mol/l to have different
properties compared to larger concentrations. In terms of
Scheme 1 the properties of the acid do not change with
concentration, but the ratio of individual channels of re-
actions occurring in course of Asc oxidation is changed.
According to the mechanism of its oxidation, ascorbic
acid itself cannot be prooxidant, which is confirmed by
other studies [18]. The results of the present study sug-
gest that the prooxidant properties do not appertain to the
ascorbic acid, but to products oxidized by DHA oxygen,
among which can be oxalic acid. Figure 4 shows that
oxalic acid in the presence of oxygen has prooxidant
properties. Reduction of the prooxidant activity observed
experimentally in [Asc] > 103 mol/l in the presence of
oxygen can be attributed to the expenditure of dissolved
oxygen in reaction 40, and perhaps its shortage as the
concentration of oxygen in the water is about 104 mol/l,
while [Asc] is more than 103 mol/l.
The form of the impulse also helps to draw the con-
clusion on the composition of the secondary radicals. If
the secondary radicals are of complex sedentary nature
2, which are discharged into reaction 32 (Table 6)
with the formation of singlet oxygen, a flash of light
should have the form shown in Figure 5, i.e. the delay in
appearance of light—tens of seconds, and duration of the
light pulse—a few minutes. If the secondary radicals are
highly movable (2,2), the flash of light will be
short, less than 30 seconds. Such flash is observed in
oxalic acid and at all concentrations of ascorbic acid.
Such radicals as 2 might yet form during the de-
composition of DHA. However, their formation is ex-
tended in time, is not bound by the time of introducing
the components of the Fenton reagent, and cannot be
detected by the applied technique of chemiluminescence.
Thus, the analysis of the properties of antioxidants in
Scheme 1 allows revealing the features of the tested sub-
stances.
RO
HO
RO
O
4. CONCLUSIONS
1) If 30 seconds have been selected as radiation regis-
tration time, the preferred concentration of Fenton solu-
tion reagent is [Fe2+] = 103 mol/l, [H2O2] = 104 mol/l.
2) The radiation background produced by cosmic rays
and other sources induce luminescence which is regis-
tered by luminometer and is to be taken into account.
3) The main radiant product in the solution of Fenton
is a dimer of singlet oxygen. The glow of the solution is
stopped when the ferric iron that is formed absorbs al-
most all radicals 2
O

. The same mechanism of lumi-
nescence quenching operates for luminol in a neutral
medium.
4) The luminescence of organic substances relates to
radicals 2 RO

. No luminescence appears in a non-oxy-
gen solution. In the presence of oxygen with an increas-
ing concentration of RH the reaction RH + 2
RO

→ R
+ ROOH begins to dominate and the emission stops.
5) Depending on the mechanism of the process, che-
miluminescence is grouped in different time intervals
after the injection of all the substances. In the period
from 0 to 30 seconds the glow is caused by reactive
oxygen species. During the time period from 30 seconds
to several miutes it is due to the emission of free-radical
reactions occurring in the sample.
6) Observed for a single substance oxidant and prooxi-
dant properties are caused by the same reaction mecha-
nism. At high concentration the intermediate radicals, the
reaction products are absorbed by the initial substance
and antioxidant properties are observed. At low concen-
trations of introduced substances intermediate radicals
are preserved and prooxidant properties are observed.
7) Prooxidant properties of ascorbic acid observed in
some cases are associated with the DHA oxidation prod-
ucts with oxygen. In the absence of oxygen the prooxi-
dant effect does not occur.
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Abbreviations
Lum—luminol
L—luminol’s radical
M—ideal antioxidant
S0—light sum of chemiluminescence for pure Fenton
solution for 30 seconds
S—light sum of chemiluminescence for 30 seconds at a
given concentration of test substance in Fenton solution
Al—alanine
A—albumin
Oxalic—oxalic acid
Oks—oksalat
Asc—ascorbic asid