Determination Co2+ in vitamin B12 based on enhancement of 2-(4-substituted-phenyl)-4,5-di(2-furyl) imidazole and H 2O2 chemiluminescence reaction

Article abstract of DOI:10.1016/j.saa.2011.07.025

In this paper, three kinds of imidazole derivatives, 2-(4-methylphenyl)-4, 5-di(2-furyl) imidazole (MDFI), 2-(4-nitrophenyl)-4,5-di(2-furyl) imidazole (NDFI), and 2-(4-tert-butylphenyl)-4,5-di(2-furyl) imidazole (t-BDFI) were synthesized. In an alkaline medium, the chemiluminescence (CL) reaction of imidazole derivatives with H₂O₂ has been investigated. It was also found that MDFI/H₂O₂ and t-BDFI/H ₂O₂ systems gave strong CL. When Co²⁺ was added into the two CL systems, the CL intensity was remarkably enhanced. In the optimum conditions, the CL intensity is linearly related to the logarithm of concentration of Co²⁺. The linear ranges are 5 × 10 ^-9-2.5 × 10^-7 mol/L for MDFI/H₂O ₂ system and 5 × 10^-9-2.5 × 10^-7 mol/L for t-BDFI/H₂O₂ system, and the corresponding detection limits are 1.2 × 10^-9 mol/L and 1.1 × 10 ^-9 mol/L, respectively. The method was applied to the determination of Co²⁺ in vitamin B₁₂ injection. Furthermore, the CL mechanism was also discussed.

Full text of DOI:10.1016/j.saa.2011.07.025

Spectrochimica Acta Part A 82 (2011) 146–152
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Determination Co2+ in vitamin B12 based on enhancement of
2-(4-substituted-phenyl)-4,5-di(2-furyl) imidazole and H2O2 chemiluminescence
reaction
Lu Han, Yumin Zhang, Jing Kang, Jieli Tang, Yihua Zhang∗
College of Chemistry, Jilin University, Changchun 130012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper, three kinds of imidazole derivatives, 2-(4-methylphenyl)-4,5-di(2-furyl) imidazole (MDFI),
2-(4-nitrophenyl)-4,5-di(2-furyl) imidazole (NDFI), and 2-(4-tert-butylphenyl)-4,5-di(2-furyl) imidazole
(t-BDFI) were synthesized. In an alkaline medium, the chemiluminescence (CL) reaction of imidazole
derivatives with H2O2 has been investigated. It was also found that MDFI/H2O2 and t-BDFI/H2O2 systems
gave strong CL. When Co2+ was added into the two CL systems, the CL intensity was remarkably enhanced.
Received 10 December 2010
Received in revised form 1 June 2011
Accepted 7 July 2011
Keywords:
In the optimum conditions, the CL intensity is linearly related to the logarithm of concentration of Co2+
.
Imidazole derivatives
Chemiluminescence
Co2+
The linear ranges are 5 × 10−9–2.5 × 10−7 mol/L for MDFI/H2O2 system and 5 × 10−9–2.5 × 10−7 mol/L for
t-BDFI/H2O2 system, and the corresponding detection limits are 1.2 × 10−9 mol/L and 1.1 × 10−9 mol/L,
respectively. The method was applied to the determination of Co2+ in vitamin B12 injection. Furthermore,
the CL mechanism was also discussed.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Though several kinds of imidazole derivatives were synthesized,
these compounds were rarely used.
The interests in the research of chemiluminescence (CL)
reagents have increased in recent years [1–4]. Imidazole and its
derivatives are a series of significant fluorescent reagents and CL
materials and have been successfully used in CL analysis [5–8].
Lophine (2,4,5-triphenylimindazole) has been used as a kind of CL
reagent since 1877 [9]. The CL reaction of lophine with H2O2 in
alkaline solution has been used for the determination of some trace
metal ions, including Co2+, Cr4+, Cr5+ and Cu2+ [10,11]. The reaction
also can be used for the determination of other inorganic species
such as chlorinated compounds [12]. The range of detection limits
for these substances was 6 × 10−9–1 × 10−6 mol/L. The determina-
tion of ultra trace Co2+ was also developed and the detection limits
were 0.1–10 ng/mL [13]. However, this method was limited due to a
few interferents, which influenced the analysis of practical sample.
In recent years, new lophine derivatives were synthesized and
their properties were determined [14,15]. Nakashima et al. have
synthesized and evaluated the fluorescent and CL properties of a
variety of lophine analogues with 2-pyridyl or 2-furyl group at
both 4- and 5-positions, and 4-dimethylaminophenyl group at 4- or
5-position of 2-(4-substituted) phenyl imidazole derivatives [16].
In the work, we synthesized 2-(4-methylphenyl)-4,5-di(2-furyl)
imidazole (MDFI), 2-(4-nitrophenyl)-4,5-di(2-furyl) imidazole
(NDFI), and 2-(4-tert-butylphenyl)-4,5-di(2-furyl) imidazole (t-
BDFI) based on the method reported by Debus [17]. Their CL
properties were investigated. It was found that MDFI and t-BDFI
can be applied in CL analysis as a kind of new CL reagent. Finally, we
proposed two kinds of new imidazole derivatives/H2O2 CL systems
to determine Co2+ based on its enhancement effect. Compared with
the lophine CL system, when the proposed systems were applied,
the detection limit was lower and the selectivity was higher. Fur-
thermore, this method was applied to the determination of Co2+ in
real injection and the obtained results were satisfactory.
2. Experimental
2.1. Reagents and chemicals
All the reagents were of analytical reagent grade and all solu-
tions were prepared with double-distilled water. NaOH and H2O2
(30%) were purchased from Beijing Chemical Plant in China.
CoCl2·6H2O was purchased from Shanghai Chemical Plant in China.
Vitamin B12 injections were produced by Tianjin Pharmaceutical
Co., Ltd. in China.
∗
The 1.0 × 10−3 mol/L stock solution of MDFI, t-BDFI, NDFI were
prepared by dissolving 0.0074 g of MDFI, 0.0085 g of t-BDFI and
Corresponding author. Tel.: +86 0431 85168352 6.
E-mail address: yihuazh47@yahoo.com.cn (Y. Zhang).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.07.025

L. Han et al. / Spectrochimica Acta Part A 82 (2011) 146–152
147
W
0.0075 g of NDFI with ethanol and then diluting to 25 mL with
ethanol, respectively and the working solution were prepared by
diluting the stock solution with water.
Black box
R
air
(a)
W
BPCL
A
SL1
SL2
P1
2.2. Synthesis of imidazole derivatives
S
W
W
V
PMT
B
air
air
First, diketone was synthesized using furfural by benzoin
condensation and oxidation. And then 2-(4-methylphenyl)-4,5-
di(2-furyl) imidazole (MDFI), 2-(4-nitrophenyl)-4,5-di(2-furyl)
imidazole (NDFI), and 2-(4-tert-butylphenyl)-4,5-di(2-furyl) imi-
dazole (t-BDFI) were prepared by Debus reaction. The synthetic
route is briefly shown in Scheme 1.
PC
P2
Black box
R
(b)
W
BPCL
SL1
A
P1
W
SL2
W
S
W
V
2.3. Characterization of MDFI, t-BDFI and NDFI
PMT
B
air
PC
P2
The MDFI, t-BDFI and NDFI were characterized by melting point,
IR, MS and NMR. The results are described as follows.
Fig. 1. Schematic diagram of the steady-injection CL system. A: H2O2 solution; B:
NaOH solution; P1, P2: peristaltic pump; SL: sample loop; V: eight-way valve; R:
chemifold; S: sample cell; PMT: photomultiplier tube; PC: computer; W: waste. (a)
Loading position. (b) Injection position.
2.3.1. 2-(4-methylphenyl)-4,5-di(2-furyl) imidazole (MDFI)
M.p. 217–219 ◦C. IR (KBr): ꢀ = 3125, 3017, 2917, 2853, 1599,
1530, 1493, 1433, 1374, 1262, 1200, 1021, 912, 823, 732, 590 cm−1
.
1H NMR (300 MHz, CDCl3): ı = 7.79 (d, J = 8.1 Hz, 2H), 7.45 (d,
J = 1.2 Hz, 2H), 7.19 (s, 2H), 6.97 (d, J = 3.2 Hz, 2H), 6.64–6.35 (m,
2H), 2.34 ppm (s, 3H). MS, m/z: (M+H)+ 291.2 (Calcd. 290.3). Anal.
calcd. for C18H14N2O2: C, 74.47; H, 4.86; N, 9.65. Found: C, 74.57;
H, 4.78; N, 9.85.
2.4. Apparatus
The CL analysis was conducted on a laboratory-built steady
injection CL system. As shown in Fig. 1, the system consists of
two peristaltic pumps and a 16-hole eight-way valve. PTFE tube
(0.8 mm i.d.) was used as connection material in the steady sys-
tem. The CL emission was detected by an ultra-weak luminescence
analyzer. The ultra-weak luminescence analyzer is composed of a
weak luminescence measurement room, a photoelectric switch-
box, an electricpulse counterchange electrocircuit, a high pressure
power supply and an intellectualized measurement unit. Data
acquisition and treatment were performed with bioluminescence
and chemiluminescence photon counter (BPCL) software running
under Windows XP.
2.3.2. 2-(4-tert-butylphenyl)-4,5-di(2-furyl) imidazole (t-BDFI)
M.p. 189–191 ◦C. IR (KBr): ꢀ = 3122, 3068, 2962, 2860, 1602,
1528, 1491, 1426, 1364, 1268, 1201, 1080, 971, 886, 839, 732,
593 cm−1 1H NMR (300 MHz, CDCl3), ı = 7.85 (d, J = 8.5 Hz, 2H), 7.49
.
(dd, J = 1.8, 0.7 Hz, 2H), 7.46 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 3.3 Hz, 2H),
6.52 (dd, J = 3.4, 1.8 Hz, 2H), 1.34 ppm (s, 9H). MS m/z: (M++H) 334.2
(Calcd. 332.40). Anal. calcd. for C21
H20N2O2: C, 75.88; H, 6.06; N,
8.43. Found: C, 75.91; H, 6.24; N, 8.67.
The fluorescence spectra were recorded with a RF-5301 spec-
trofluorimeter (Shimadzu, Japan).
2.3.3. 2-(4-nitrophenyl)-4,5-di(2-furyl) imidazole (NDFI)
M.p. 197–198 ◦C. IR (KBr): ꢀ = 3095, 3032, 1609, 1520, 1469,
1345, 1232, 915, 871, 812, 739, 715, 591 cm−1 1H NMR (300 MHz,
.
2.5. Procedure
CDCl3) ı = 8.72 (d, J = 1.9 Hz, 1H), 8.39–8.33 (m, 1H), 8.23 (ddd, J = 8.2,
2.2, 1.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.53 (dd, J = 1.8 0.7 Hz, 2H),
7.05 (d, J = 3.0 Hz, 2H), 6.56 ppm (dd, J = 3.4, 1.8 Hz, 2H). MS m/z:
(M+) 321.9 (Calcd. 321.29). Anal. calcd. for C17H11N3O4: C, 63.55;
H, 3.45; N, 13.08. Found: C, 63.26; H, 3.70; N, 12.87.
The experimental parameters are as follows: sample loop vol-
ume, 200 ␮L; sampling time, 13 s; sample injection time, 13 s; the
photomultiplier tube (PMT) negative voltage, −1000 V; the integral
time of the CL signal, 60 s. Imidazole derivatives solution (200 ␮L)
OH
O
O
O
O
CHO
VitaminB1
Cu(OAc)2
O
O
O
O
CHO
O
O
O
O
N
NH4OAc
HOAc
+
R
N
H
O
R
O
R=
CH3
NO2
C(CH3)3
Scheme 1. The synthesis of 2-(4-substituted-phenyl)-4,5-di(2-furyl) imidazole.

148
L. Han et al. / Spectrochimica Acta Part A 82 (2011) 146–152
450
400
350
300
250
200
150
100
50
a
a
2500
2000
1500
1000
500
0
A
b
c
B
0
10
20
30
40
50
60
0
10
20
30
40
50
60
Time (s)
Time (s)
Fig. 2. Dynamic CL intensity–time profiles. (a) MDFI/H2O2 system; (b) t-BDFI/H2O2
system; (c) NDFI/H2O2 system. Conditions: imidazole derivatives, 2.5 × 10−4 mol/L;
H2O2, 1 mol/L; NaOH, 0.1 mol/L.
b
2000
1500
1000
500
0
A
and sample solution (200 ␮L) were first added into the sample
cell, and then the H2O2 and NaOH solution were simultaneously
injected into the sample cell with the steady injection system.
The operation mode is as follows: (1) Fig. 1(a), pumps P1 and
P2 were activated, and valve V was in the loading position. The
pump P1 was used to deliver H2O2 solution into the sample loop1
(SL1) and the pump P2 was used to deliver NaOH solution into the
sample loop2 (SL2). (2) Fig. 1(b), pumps P1 and P2 were activated,
and valve V was in the injection position. The pumps P1 and P2
were used to deliver the air current. The H2O2 solution and NaOH
solution were simultaneously pumped into chemifold R and mixed.
The mixed solution was carried into sample cell S and reacted with
the mixture of imidazole derivative solution and Co2+ solution in
the sample cell S. The CL signal was measured and recorded. All
experiments were performed in triplicate.
B
0
10
20
30
40
50
60
Time (s)
Fig. 3. CL profiles of MDFI/H2O2 system (a) and t-BDFI/H2O2 system (b) in the pres-
ence (A) and absence (B) of Co2+. Concentration: MDFI, 2.5 × 10−4 mol/L; t-BDFI,
2.5 × 10−4 mol/L; H2O2, 0.05 mol/L; NaOH, 0.25 mol/L; Co2+, 1 × 10−6 mol/L.
The analyte was quantified by measuring the increased CL inten-
sity, ꢁI = Is − I0, where I0 and Is are CL intensity in the absence
and presence of Co2+, respectively. The standard deviation (S) was
obtained by analyzing the blank sample 11 times. The limit of detec-
tion is equal to 3S/k, where k is the slope of the linear calibration
equation.
hemoglobin [18]. Medicinal vitamin B12 injection was used as sam-
ple and analyzed. 1 mL of the injection was added into 50 mL beaker,
and then 5 mL concentrated nitric acid was also added into the
beaker. The beaker was placed on the electric hot plate and heated
until the solution was evaporated to dryness. The step was repeated
until the residue became colorless. Then the residue was cool down
to the room temperature, a little water was added to dissolve
residue. The obtained solution was further diluted with water so
that the concentration of Co2+ was in the working range for analyt-
ical determination.
2.6. Preparation of sample
Vitamin B12 is an organic complex containing cobalt and
its structure is similar to that of pigment-like chlorophyll and
Table 1
Influence of metal ions on the imidazole derivatives/H2O2 systems.
Metal ion (1.0 × 10−7 mol/L)
Relative CL intensity
MDFI/H2O2 system
t-BDFI/H2O2 system
None
Cu2+
Co2+
Cr2+
Pb2+
Fe3+
Fe2+
Mn2+
Cd2+
Ba2+
Ca2+
K+
95
133
1145
111
112
103
151
111
107
99
93
136
1031
126
122
106
123
117
107
107
107
108
107
3. Results and discussion
3.1. Effect of imidazole derivatives
The dynamic CL intensity–time profiles obtained with three
kinds of imidazole derivatives are shown in Fig. 2. As shown in
Fig. 2, MDFI/H2O2 and t-BDFI/H2O2 systems gave stronger CL com-
pared with NDFI/H2O2 CL system. The results indicated that the CL
intensity was related to the structures of imidazole derivatives. The
MDFI/H2O2 and t-BDFI/H2O2 systems were further investigated.
105
106
105
Na+

L. Han et al. / Spectrochimica Acta Part A 82 (2011) 146–152
149
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
550000
500000
450000
400000
350000
300000
250000
200000
150000
100000
b
a
b
a
0.0000
0.0001
0.0002
0.0003
0.0004
0.0005
0.0
0.2
0.4
0.6
0.8
1.0
Concentration of imidazole derivatives (mol/L)
Concentration of NaOH (mol/L)
Fig. 6. Effect of imidazole derivative concentration on the CL intensity. (a)
MDFI/H2O2 system; (b) t-BDFI/H2O2 system. Concentration: NaOH, 0.25 mol/L;
H2O2, 0.05 mol/L; Co2+, 1 × 10−6 mol/L.
Fig. 4. Effect of NaOH concentration on the CL intensity. (a) MDFI/H2O2 system; (b) t-
BDFI/H2O2 system. Concentration: t-BDFI, 2.5 × 10−4 mol/L; MDFI, 1.0 × 10−4 mol/L;
H2O2, 0.05 mol/L; Co2+, 1 × 10−6 mol/L.
the system. The influence of the concentration of NaOH in a carrier
solution was investigated. As shown in Fig. 4, the effect of NaOH
concentration was examined in the ranges of 0.01–1.0 mol/L. The
CL intensity increased first with an increase of NaOH concentra-
tion. Then the CL intensity of MDFI/H2O2 system was unchanged
when the NaOH concentration was higher than 0.5 mol/L and the CL
intensity of the t-BDFI/H2O2 system decreased when the concentra-
tion of NaOH was higher than 0.25 mol/L. When the concentration
of NaOH was too high, Co2+ can react with OH− to form cobalt
hydroxide precipitation and the concentration of Co2+ decreased.
Therefore, the optimum concentrations of NaOH for MDFI/H2O2
and t-BDFI/H2O2 systems were 0.5 mol/L and 0.25 mol/L, respec-
tively.
3.2. CL reaction conditions
3.2.1. CL intensity enhancement by metal ions
The effects of some metal ions on CL of MDFI/H2O2 and t-
BDFI/H2O2 CL systems were examined. The results are listed in
Table 1. The concentrations of all metal ions were 1 ␮mol/L. It is
found from Table 1 that only Co2+, Cu2+ and Fe2+ show enhancement
effect, but the enhancement effect of Co2+ is most remarkable. The
other metal ions have no significant influence on the CL intensity.
The dynamic CL intensity–time profiles for MDFI/H2O2 and t-
BDFI/H2O2 CL systems in the absence and presence of Co2+ are
shown in Fig. 3, respectively. It is seen from Fig. 3, the CL in the
absence of Co2+ is very weak, and the CL intensity of these two CL
systems could be remarkably enhanced in the presence of Co2+. So
in the MDFI/H2O2 and t-BDFI/H2O2 CL systems Co2+ was used as the
enhancer. It was also found that the CL intensity reaches maximum
at 10 s and then the intensity decreases.
3.2.3. Effect of the concentration of H2O2
H2O2 was used as an oxidant in the CL system. So the concen-
tration of H2O2 is an important factor influencing CL intensity. The
influence of H2O2 concentrations ranging from 0.001 to 1 mol/L
on the CL intensity was investigated. As shown in Fig. 5, with the
increase of concentration of H2O2 the CL intensity increased when
the concentration of H2O2 was lower than 0.05 mol/L, and the inten-
sity decreased when the concentration of H2O2 was higher than
0.05 mol/L. When the concentrations of H2O2 were higher or lower
than 0.05 mol/L, the CL intensity decreased dramatically. When
3.2.2. Effect of the concentration of NaOH
The reaction media plays an important role in CL reaction sys-
tem. In the view of the properties of the imidazole derivatives, an
alkaline carrier solution is introduced to improve the sensitivity of
350000
b
300000
250000
a
200000
150000
100000
0.0
0.2
0.4
0.6
0.8
1.0
Concentration of H2O2(mol/L)
Fig. 5. Effect of H2O2 concentration on the CL intensity. (a) MDFI/H2O2 sys-
tem; (b) t-BDFI/H2O2 system. Concentration: t-BDMI, 2.5 × 10−4 mol/L; MDFI,
2.5 × 10−4 mol/L; NaOH, 0.25 mol/L; Co2+, 1 × 10−6 mol/L.
Fig. 7. The linear calibration ranges of the Co2+. (a) MDFI/H2O2 system; (b) t-
BDFI/H2O2 system. Concentration: NaOH, 0.25 mol/L; H2O2, 0.05 mol/L; t-BDMI,
2.5 × 10−4 mol/L; MDFI, 2.5 × 10−4 mol/L.

150
L. Han et al. / Spectrochimica Acta Part A 82 (2011) 146–152
Table 2
Analytical performances for Co2+
.
System
Calibration curve C (nmol/L)
r
Linear range (mol/L)
Detection limit (mol/L)
RSDa (%)
MDFI/H2O2
t-BDFI/H2O2
ꢂI = 90,608lg C − 16292
0.9943
0.9972
5 × 10−9–2.5 × 10−7
5 × 10−9–2.5 × 10−7
1.2 × 10−9
1.1 × 10−9
3.8
3.8
ꢂI = 20,3401lg C − 124618
a
The RSD was estimated at 1 × 10−7 mol/L for Co2+ (n = 11).
Table 3
the concentration was higher than 0.05 mol/L, the H2O2 solution
bubbled and the flow rate of H2O2 was unstable. Therefore, the
optimum concentration of H2O2 was chosen to be 0.05 mol/L and
was used in further experiments.
Determination of Co2+ in vitamin B12 injection sample.
Added (␮g/mL)
Found (␮g/mL)
Recovery (%)
–
RSD (%) (n = 5)
0
19.51
28.59
33.95
1.51
1.15
2.67
8.84
17.68
102
82
3.2.4. Effect of the concentration of MDFI and t-BDFI
The effect of the concentration of MDFI and t-BDFI on the CL
intensity was examined in the range of 1.0 × 10−6–5 × 10−4 mol/L.
The experimental results are shown in Fig. 6. The CL inten-
sity increases with the increase of concentrations of MDFI
and t-BDFI when the concentrations of MDFI and t-BDFI are
lower than 2.5 × 10−4 mol/L. When the concentrations are higher
2.5 × 10−4 mol/L, the CL intensity does not change significantly
with the increase of the concentrations of MDFI and t-BDFI. So
2.5 × 10−4 mol/L was chosen as the optimum reaction concentra-
tion.
25 as much as that of analyte, respectively. It can be known that
imidazole derivatives CL systems have a high selectivity for the
determination of Co2+
.
3.5. Study of the mechanism of the reaction
The fluorescence (FL) emission spectra of MDFI and t-BDFI are
shown in Fig. 8. It can be seen that the characteristic emission
peaks are 425 nm and 427 nm, respectively. Besides, the CL spectra
of these two systems in absence and presence of Co2+ were also
examined, and the results are shown in Fig. 9. The CL emission
peaks are about at 530 nm and 550 nm, respectively. When Co2+
3.3. Determination of Co2+
Under the optimum conditions described above, the calibration
graph was constructed. The increased CL intensity ꢁI versus the
logarithm of concentration of Co2+ was linear. The experimental
results are given in Table 2 and the obtained using two systems
calibration graph for Co2+ is shown in Fig. 7. The linear ranges are
same because the molecular structure of MDFI and t-BDFI are very
similar. The results indicate that the proposed method is satisfac-
tory.
a
260000
A
240000
220000
200000
180000
160000
140000
120000
100000
80000
3.4. Interference study
B
The influence of foreign species was investigated by analyzing a
standard solution of 1.0 × 10−7 mol/L Co2+. The tolerable limit of a
foreign species was taken as a relative error not greater than 5%.
The results showed that no interference could be observed for Na+,
K+, Ba2+, Ca2+, the concentrations of Cd2+, Pb2+, Cr3+, Mn2+, Fe3+
and Cu2+ were tolerable up to a factor of 100, 100, 50, 50, 50 and
60000
400
450
500
550
600
650
Wavelength (nm)
700
a
155000
150000
145000
140000
135000
130000
125000
120000
115000
110000
105000
100000
95000
b
A
600
b
500
400
300
200
100
0
B
90000
400
450
500
550
600
650
300
350
400
450
500
550
600
Wavelength (nm)
Wavelength(nm)
Fig. 9. The CL spectra of MDFI/H2O2 (a) and t-BDFI/H2O2 (b) in presence
Fig. 8. The fluorescence emission spectra of MDFI (a) and t-BDFI (b). Concentration:
t-BDMI, 1 × 10−6 mol/L; MDFI, 1 × 10−6 mol/L.
(A) and absence (B) of Co2+
.
Concentration: t-BDMI, 2.5 × 10−4 mol/L; MDFI,
2.5 × 10−4 mol/L; NaOH, 0.25 mol/L; Co2+, 1 × 10−6 mol/L, H2O2, 0.05 mol/L.

L. Han et al. / Spectrochimica Acta Part A 82 (2011) 146–152
151
Table 4
Determination of Co2+ in vitamin B12 injection with the proposed CL method and the inductively coupled plasma spectrometry (ICP).
Sample
Proposed method
ICP
Relative
error (%)
Content
(␮g/mL)
RSD (%)
1.51
Content
(␮g/mL)
RSD (%)
0.92
Vitamin B12 injection
19.51
19.45
0.31
-
HOO- + H2O
H O +
OH
2
2
OH-
+
Co2+
Co3+ OH*
+
+ H2O2
Co3+ HOO-
+
*
Co2+ HO
+
2
OH*
+
-*
H+ +
+
H2O2
O2
H2O
*
-*
OH-
HO2
+
+
O2
H2O
O
O
O
N
N
N
-*
+ O2
R
R
HO O
N
H
O
O
O
*
O
-
-
N
N
N
O
R
hv
O
O
R
O
N
O
Scheme 2. The mechanism of the CL.
was added into MDFI/H2O2 and t-BDFI/H2O2 systems, it had no
significant influence on the shape of the CL emission peak, but the
CL intensity was remarkably enhanced. The results indicated that
CL was not produced by MDFI and t-BDFI but some intermediate
product.
given in Table 3. The comparison between the proposed CL method
and inductively coupled plasma (ICP) was also carried out. As can
be seen from Table 4, there was good agreement between the two
methods.
The possible mechanism of CL emission was shown in Scheme 2.
H2O2 reacts with OH− to generate HOO−, and Co2+ catalyzes the
radical decomposition of H2O2 and HOO− to produce superoxide
ion (O2−*) in alkaline medium. The O2−* reacts with imidazole
derivatives to form an intermediate product. At last, this interme-
diate product emits light from excitation state [19–22].
As compared above, the order of the CL intensity of the three
imidazole derivatives/H2O2 systems was MDFI > t-BDFI > >NDFI and
nitro imidazole derivatives whose intensity was too weak to mea-
sure. It indicated that nitro had significant influence on imidazole
derivatives CL intensity [23,24]. These results might be due to the
low fluorescence quantum yields of the oxidatively decomposed
FL compounds from CL compounds [16,25]. So the CL intensity
might be related to molecular electron delocalization and steric
hindrance. MDFI and t-BDFI with electron-donating groups –CH3
and –C(CH3)3 at 2-position can give stronger CL than NDFI with
an electron-withdrawing group –NO2. t-BDFI generated a weaker
CL emission than MDFI, which may be due to steric hindrance.
Therefore, MDFI exhibited the maximum emission of light.
4. Conclusions
In this paper, the CL properties of three kinds of imidazole
derivatives were investigated. The influence of various metal ions
on MDFI/H2O2 and t-BDFI/H2O2 CL systems was examined. At last,
a sensitive CL method, based on the enhancive effect of Co2+ on
2-(4-substituted-phenyl)-4,5-di(2-furyl) imidazole and H2O2 CL
reaction, was proposed for determination of Co2+. This method has
been applied to the determination of Co2+ in vitamin B12 injections.
Furthermore, the method could be used to analyze other samples
containing Co2+ and determine vitamin B12 indirectly by determin-
ing the content of Co2+. Furthermore, MDFI and t-BDFI can be used
as CL reagents and widely applied in CL analysis. The extension of
this work is under way.
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