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Hydrolysis and oxidation of formamidine disulfide in acidic medium were investigated using high-performance liquid chromatography (HPLC) and mass spectrometry (MS) at 25 C. By controlling the slow reaction rate and choosing appropriate
mobile phase, HPLC provides the unique advantages over other methods (UV-Vis, chemical separation) in species tracking
and kinetic study. In addition to thiourea and formamidine sulfinic acid, two unreported products were also detected in the hydrolysis reaction. Mass spectrometry measurement indicates these two products to be formamidine sulfenic acid and thiocyanogen with mass weights of 92.28 and 116.36, respectively. In the oxidation of formamidine disulfide by hydrogen peroxide,
besides thiourea, formamidine sulfenic acid, formamidine sulfinic acid, thiocyanogen and urea, formamidine sulfonic acid and
sulfate could be detected. The oxidation reaction was found to be first order in both formamidine disulfide and hydrogen peroxide. The rate constants of hydrolysis and oxidation reactions were determined in the pH range of 1.53.0. It was found both
rate constants are increased with the increasing of pH. Experimental curves of different species can be effectively simulated via
a mechanism scheme for formamidine disulfide oxidation, including hydrolysis equilibrium of formamidine disulfide and irreversible hydrolysis of formamidine sulfenic acid.
formamidine disulfide, hydrolysis, oxidation, HPLC-MS
1 Introduction
Oxidation of sulfur(-II) species has attracted considerable
attention in the past decades due to their ability in supporting extremely rich nonlinear phenomena. As a typical sulfur
-containing compound, thiourea (TU, (H2N)2C=S) can react
with various oxidants such as oxyhalogen compounds
[110], hydrogen peroxide [1114] and OH radicals [15], or
via electrochemical methods [16,17]. Investigations during
the past couple of decades indicated that the oxidation processes displayed extremely rich nonlinear phenomena including autocatalysis, autoinhibition, bistability, oligooscillations, oscillations, birhythmicity, chaos, and spatiotemporal pattern [110]. When TU was oxidized on a platinum
electrode, period-doubling bifurcation, two- and three*Corresponding author (email: gaoqy@cumt.edu.cn)
Science China Press and Springer-Verlag Berlin Heidelberg 2011
www.springerlink.com
236
Hu Y, et al.
2 Experimental section
The HPLC analysis was conducted on the Agilent 1100
system (Agilent, Santa Clara, CA), which includes one
Model 1379A degasser, a G1311A quaternary pump, a
G1316 column thermostat, a G1315A multiple wavelength
UV-Vis detector and a 20 L sample loop. The separation
was performed on a Phenomenex Gemini C18 column (5 m,
250 4.6 mm i.d.). In this study, binary mobile phase and
ternary mobile phase were used for HPLC and HPLC-MS,
respectively. For better separation, the mobile phase was
prepared by mixing methanol and 2.5 mM tetrabutylammonium hydroxide (TBAOH) solution in a volumetric ratio of
5:95. Its pH was adjusted by [H3PO4]/[NaH2PO4], and the
flow rate was set to be 0.5 mL/min. When the column effluence was introduced into a mass spectrometer, the mobile
phase composition was changed to methanol, acetonitrile
and buffer (hydrochloric acid) with the volume ratio of
6:26:68. The flow rate was changed to 0.4 mL/min. For
each analysis, the injection volume was 10 L, and the detection wavelengths for five channels were set to be 200,
214, 220, 235 and 254 nm, respectively. In the HPLC-MS
measurement, the LC column effluent was interfaced with
an LCQ Advantage ion-trap mass spectrometer (Thermo
Finnigan, Waltham, MA) equipped with the electrospray
ionization (ESI) ion source. Both positive- and negative-ion
modes were used to detect different substances. The flow
rates of sheath gas and auxiliary/sweep gas were set to be
40 and 10 arbitrary units (arb), respectively, and the temperature of ion-transfer capillary was 300 C. The probe
voltage was kept at +3.0 kV in positive-ion mode and 2.0
kV in negative-ion mode. The reaction was conducted in an
enclosed glass vessel. The reaction temperature was kept at
25 C in a water bath. The pH and ion strength in the mobile
phase were kept the same as the reaction solution. For the
anaerobic reaction, the buffered solutions were bubbled
with nitrogen gas for two hours, followed by sampling under nitrogen atmosphere.
Hydrogen peroxide was titrated with standard potassium
3
3.1
During the oxidation of FDS by hydrogen peroxide, hydrolysis of FDS, which may consume reactant FDS, is expected to occur, and the resulting products would further be
oxidized by H2O2. Therefore, the hydrolysis reaction of
FDS could not be neglected. To evaluate the effect of hydrolysis on the oxidation dynamics, kinetics of FDS hydrolysis was investigated first.
Figure 1 illustrates the chromatograms recorded during
the hydrolysis of FDS, in which the initial concentration of
FDS is 0.5 mM and pH equals 2.0. As shown in Figure 1(a),
10 min after the reaction started, in addition to the reactant
FDS at the retention time of ca. 4.50 min, another peak
with retention time of ca.7.1 min was observed and identified to be TU by comparing its retention time to the standard substance. It was observed that the peak height of TU
significantly increased when the reaction proceeded. As can
be seen in Figure 1(b), three more peaks with characteristic
retention times of 4.80 min, 5.50 min and 6.10 min,
Hu Y, et al.
237
To further confirm the hydrolysis intermediates and assign the two unidentified products mentioned above,
HPLC-MS experiment was conducted as well. Figure 2
presents the total ion chromatograms and mass spectra. As
shown in Figure 2(a) and (b), in the positive-ion mode and
negative-ion mode, five chromatographic species were obtained. The mass spectra indicate that components eluted at
4.76, 5.33, 5.87, 6.25 and 6.95 min should be attributed to
formamidine disulfide ([M + H]+ = 151.34, [M + MeOH +
H]+ = 183.49 and [M + ACN + H]+ = 191.87), formamidine
sulfenic acid (FSEA, H2N(=NH)CSOH, [M + H]+ = 93.28
Figure 2 Total ion chromatogram (TIC) of FDS hydrolysis products and the corresponding mass spectra of HPLC-MS. Sampling time = 150 min, [FDS]0
= 1.0 x 103 mol L1, pH 2.0.
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Hu Y, et al.
kh (s1) (HPLC)
6.00 106
8.07 106
2.83 105
7.17 105
3.25 104
pH
kh (s1) [22]
1.91
2.02
2.43
3.38
3.58 105
3.98 105
9.15 105
6.50 104
Figure 3 Kinetic curves of sulfur species and plots of concentration logarithms versus time during the hydrolysis of FDS. (a) Time series of chromatogram
peak-areas at pH 2.00; (b, c) Integrated rate plots of first order rate equation: ln{[FDS]0/[FDS]t} versus time at pH 1.82 (b) and 2.00 (c). UV detection wavelength = 214 nm and [FDS]0 = 5.0 x 104 M.
Hu Y, et al.
Figure 4 HPLC chromatograms collected at different time of the oxidation reaction between hydrogen peroxide and FDS. Sampling time = 2 min
(a) and 53 min (b). UV detection wavelength = 220 nm, [FDS]0 = 5.0 x
104 M and [FDS]0:[H2O2]0 = 1:20, pH 2.0.
239
Figure 5 Kinetic curves of the reaction and plots of concentration logarithms versus time during the oxidation of FDS. (a) Measured (symbols)
and simulated (lines) time series; (b) integrated rate plots of first order rate
equation: ln{[FDS]0/[FDS]t} versus time. The other conditions are the
same with Figure 4.
k1 (104 s1) b)
1.50
1.82
2.00
2.50
3.00
0.57
1.24
2.14
2.32
2.57
1 1
k0 (102 M s ) b)
0.57
1.24
2.14
2.32
2.57
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Hu Y, et al.
Table 3
The mechanism and rate constant of oxidation of formamidine disulfide by hydrogen peroxide at pH 2.00
No.
Reaction
R1
R2
R5
R6
R7
R8
R9
R10
Several intermediates and the final products during the hydrolysis and oxidation of FDS were identified using HPLC
and MS. The oxidation of FDS by hydrogen peroxide was
accompanied by hydrolysis reaction, which produced TU,
FSIA, FSOA, two previously unreported intermediates,
FSEA and (SCN)2, and finally (H2N)2CO, SO42 and sulfur.
Since the FSEA peak during oxidation was higher than that
during hydrolysis as shown in Figure 4 in contrary to Figure
1, FSEA was produced not only from hydrolysis but also
from oxidation of FDS and TU. In our separate experiments,
we found that the consumption rates of FSOA in decomposition and oxidation by hydrogen peroxide are of the same
when pH < 3, indicating that direct oxidation of FSOA can
be ignored. Table 3 gives the mechanism scheme for oxidation of FDS, where R1 and R2 are hydrolysis equilibrium of
FDS and irreversible hydrolysis of FSEA, respectively. The
scheme does not include R3 and R4 because concentrations
of thiocynogen and FSEA were very low in FDS hydrolysis.
R5, R6, R7 and R8 are oxidation reactions that produce
FSEA, FSIA and FSOA, respectively. R9 is hydrolysis reaction of FSOA followed by sulfite oxidation reaction R10.
The rate constants k1, k-1 and k2 have been fitted. Especially,
our fitted k1 value agrees with the values reported by Rbai
et al. [6] and Gao et al. [13] respectively. k5 and k7 were
reasonably adjusted according to pH difference between this
work (at pH 2.0) and the reference (at pH 1.5). k8 and k9
were determined with HPLC [25]. k10 were obtained from
Rabai et al. [26]. By pre-equilibrium approximation, the
rate constants of hydrolysis reactions (R1, R-1 and R2) were
calculated which are consistent with Table 1.
kh k1 k2/(k1 [FDS]0) = 2.66 105 s1 (pH 2)
Figure 5(a) also shows that the corresponding curves simulated with the mechanism in Table 3 have all the experimental features.
Rate constants
3.30 104 s1
4.80 s1
1.94 104 s1
1.50 102 M1 s1
2.14 102 M1 s1
4.44 102 M1 s1
4.27 104 M1 s1
7.42 106 s1
1.48 105 M1 s1
Conclusion
3
4
9
10
Hu Y, et al.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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