Spectrofluorimetric determination of both hydrogen peroxide and -O-O-H in polyethylene glycols (PEGs) using 2-hydroxy-1-naphthaldehyde thiosemicarbazon (HNT) as the substrate for horseradish peroxidase (HRP)

Article abstract of DOI:10.1016/S1386-1425(03)00107-0

2-Hydroxy-1-naphthaldehyde thiosemicarbazon (HNT) had been synthesized and used as a new kind of substrate for horseradish peroxidase (HRP) in spectrofluorimetric determination of hydrogen peroxide (H₂O ₂). The oxidation reaction of HNT with H₂O₂ under the catalysis of HRP was studied in detail. The possible reaction mechanism was discussed. Under optimum experimental conditions, the oxidized product of HNT had excitation and emission maxima at 260 and 450 nm, respectively. The linear range of this method was 1.30 × 10^-9 -1.25 × 10^-5 mol 1^-1 with a detection limit of 3.89 × 10^-10 mol 1^-1. The effect of interferences, surfactants and organic solvents on the determination of H₂O ₂ had been investigated. A study to prove the existence of -O-O-H in PEGs was carried out. The proposed method was successfully applied to the determination of -O-O-H in polyethylene glycols.

Full text of DOI:10.1016/S1386-1425(03)00107-0

Spectrochimica Acta Part A 59 (2003) 2867ꢂ2874
/
www.elsevier.com/locate/saa
Spectrofluorimetric determination of both hydrogen peroxide
H in polyethylene glycols (PEGs) using 2-hydroxy-
-naphthaldehyde thiosemicarbazon (HNT) as the substrate
and Ã
/
OÃ
/
OÃ
/
1
for horseradish peroxidase (HRP)
Bo Tang *, Yan Wang
Department of Chemistry, Shandong Normal University, Jinan 250014, China
Received 14 October 2002; received in revised form 26 January 2003; accepted 10 February 2003
Abstract
2
-Hydroxy-1-naphthaldehyde thiosemicarbazon (HNT) had been synthesized and used as a new kind of substrate for
horseradish peroxidase (HRP) in spectrofluorimetric determination of hydrogen peroxide (H O ). The oxidation
2
2
reaction of HNT with H O under the catalysis of HRP was studied in detail. The possible reaction mechanism was
2
2
discussed. Under optimum experimental conditions, the oxidized product of HNT had excitation and emission maxima
ꢁ
9
ꢁ5
ꢁ1
at 260 and 450 nm, respectively. The linear range of this method was 1.30ꢀ
/
10
mol l . The effect of interferences, surfactants and organic solvents on the determination of
had been investigated. A study to prove the existence of ÃOÃOÃH in PEGs was carried out. The proposed
method was successfully applied to the determination of ÃOÃOÃH in polyethylene glycols.
2003 Elsevier B.V. All rights reserved.
ꢂ
/
1.25ꢀ
/
10 mol l with a detection
ꢁ
10
ꢁ1
limit of 3.89ꢀ10
/
H
2
O
2
/
/
/
/
/
/
#
Keywords: Hydrogen peroxide determination; HNT; HRP; Catalytic spectrofluorimetry; Polyethylene glycols
1
. Introduction
enzymatic reactions in bioanalytical chemistry [1ꢂ
].
The characteristics of the enzyme were system-
atically studied with H O as oxidizing agent and
/
4
In recent years, various methods for hydrogen
peroxide (H O ) determination have been pro-
2
2
2
2
posed, including the use of enzymatic assays,
which have been widely used in analytical bio-
chemistry because of their rapidity and high
selectivity. The horseradish peroxidase (HRP)-
catalyzed reaction is one of the most widely used
various substances as fluorogenic substrates.
Based on these catalytic reactions, various highly
sensitive spectrofluorimetric methods for determi-
nation of H O have been developed [5ꢂ14]. For
/
2
2
example, homovanillic acid, hydroxyphenylacetic
acid, tyrosine and tyramine have been used as the
fluorescent substrates of HRP in H O determina-
2
2
* Corresponding author.
E-mail address: toplet@jn-public.sd.cninfo.net (B. Tang).
tion by Guilbault et al. [5,6]. Zaitsu et al. [7] found
1386-1425/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S1386-1425(03)00107-0

2868
B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ2874
/
that a series of p-hydroxy-phenyl group com-
pounds can be oxidized to fluorescent dimers by
2. Experimental
H O under the catalysis of HRP. Ci et al. [9ꢂ
/13]
2.1. Apparatus
2
2
had systematically studied the reactive character-
istics of a series of substrates such as tyrosine and
chavicol. They researched the reaction mechanism
and proposed the best structure of substrate.
Based on fluorescence quenching of scopoletin in
the reaction with H O catalyzed by HRP, the
All fluorescence measurements were carried out
on a PerkinꢂElmer (Norwalk, CT, USA) LS-5
spectrofluorimeter, equipped with a xenon lamp,
1.0 cm quartz cells and a PerkinꢂElmer model 561
recorder. All pH measurements were made with a
pHS-3C digital pH meter (Shanghai Lei Ci Device
Works, Shanghai, China) with a combined glass-
calomel electrode. The elemental analysis was
carried out on a Yanaco (Japan) model MF-3
elemental analyzer.
/
/
2
2
fluorimetric HRP-scopoletin method was chosen
for H O determination by Holm et al. [14].
2
2
2
-Hydroxy-1-naphthaldehyde
thiosemicarba-
zone (HNT) was used to manganese determination
in coffee by Perez-Bendito et al. in 1984 [15], they
found that HNT can be oxidized to form fluor-
2
ꢃ
2.2. Reagents
escent by H O under the catalysis of Mn
.
was
2
2
2
ꢃ
Based on the reaction, 7.5ꢂ
/
48 ng of Mn
All chemicals used were of analytical-reagent or
higher grade. De-ionized water was used through-
spectrofluorimetricly determined.
In our studies, we found that the oxidization of
HNT with H O could be catalyzed by HRP, the
product of HRP-catalyzed reaction was same as
ꢁ
1
out. A 10 mg ml
stock solution of HRP was
2
2
prepared by dissolving an appropriate amount of
HRP (Shanghai Dongfeng Boichemical Technol-
ogy, China) in distilled deionized water. H O
2
ꢃ
that of Mn -catalyzed reaction. Therefore, in
this paper, HNT have been synthesized and used
as a new kind of substrate for HRP in spectro-
fluorimetric determination of H O with high
2
2
solution was prepared by dilution of a 30%
solution with distilled deionized water (standar-
dized by titration with potassium permanganate).
2
2
sensitivity. The effects of surfactants and organic
solvents were studied. The possible mechanism of
the reaction was discussed.
The boric acidꢂ
/
sodium hydroxide (H BO ꢂ
/
3
3
ꢁ
1
NaOH) buffer (0.2 mol l , pH 11.5) was used.
ꢁ
4
ꢁ1
HNT solution (8.0ꢀ
/10
mol l ) was prepared
Polyethylene glycols (PEGs) is a kind of widely
used polymer in medicine manufacture, whose
by dissolving an appropriate amount of the
reagent (synthesized following the method in the
literature [16], mp 245 8C, anal. found (calc.): C,
molecular formula is expressed as CH Ã
/
(CH2Ã
/
3
58.82; (58.75), H, 4.58;(4.52)) in dimethylforma-
CH2Ã
/
OÃ
/
) Ã
/
OH. Because of its special linear
OH)
n
mide (DMF). All polyethylene glycols solutions
were prepared to 10% solutions with distilled
deionized water.
molecular structure, the hydroxyl group (Ã
/
in PEGs can be easily activated to form hydro-
peroxide (ROOH). Moreover, ether chains in
PEGs having low degree of polymerization can
2.3. Determination of H O
2 2
be oxidized to form hydroperoxyl group (Ã
/
OÃ
/
OÃ
/
H) in the air. The higher the content of ÃOÃ
/
/
OÃ
/
H
In a 10 ml color comparison tube were placed
in PEGs, the lower the degree of polymerization
and pureness of PEGs. Therefore, the pureness
and degree of polymerization of PEGs can be
2
1
.0 ml of buffer solution (pH 11.5), 0.4 ml of 8.0ꢀ
/
ꢁ4
ꢁ1
ꢁ1
0
mol l HNT solution, 0.5 ml of 10 mg ml
HRP solution and different amount of H O
2
2
detected by determining the Ã
PEGs. In this work, a study to prove the existence
of ÃOÃOÃH in PEGs was carried out. The
proposed method was applied to the determina-
tion of ÃOÃOÃH in polyethylene glycols with
satisfactory results.
/
OÃ
/
OÃ
/
H content in
standard solutions. After dilution to volume with
deionized water, the mixture was equilibrated at
room temperature for 10 min, then the fluores-
cence intensity of the solution was measured at 450
nm with excitation at 260 nm against a reagent
blank. A standard addition method was used to
/
/
/
/
/
/

B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ
/2874
2869
determine amount of Ã
/
OÃ
/
OÃ/H in polyethylene
NaOH buffer (pH 11.5). Peaks: 1 and 1?, HNT; 2
and 2?, oxidized product of HNT.
glycols.
3.2. Preliminary discussion of reaction mechanism
3
. Results and discussion
The reaction mechanism of H O with 2-hy-
2
2
droxy-1-phenylaldehyde
(
They considered that the CÄ
broken off to form a cyclic fluorescent product in
the oxidation reaction with H O .
thiosemicarbazone
HBTS) have been studied by Morene et al. [17].
3
.1. Excitation and emission spectra
/
N bond of HBTS was
In order to determine the optimum working
wavelength, the spectral characteristics of the
oxidized product of HNT by H O were studied
2
at various pH values. The corrected excitation and
emission spectra (Fig. 1) showed that the wave-
lengths of maximum excitation and emission of the
oxidized product of HNT at pH 11.5 were 260 and
2
2
In the experiments, we studied the spectral
characteristics of HNT and its oxidized product
by H O . The results (Fig. 1) showed that the
2
2
2
fluorescence response (l /lem) of HNT and its
ex
oxidized product were 410/475 and 260/450 nm,
respectively. So we inferred that the CÄ
HNT was broken off in the HRP-catalyzed reac-
tion with H as oxidizing agent, just as that of
HBTS having similar structure to HNT. The
/
N bond of
4
50 nm, respectively.
The concentrations of HNT, HRP and H O
2
2
ꢁ
5
ꢁ1
ꢁ1
were 3.2ꢀ
/
10
ꢁ1
mol l , 0.5 mg ml
and 3.8ꢀ
/
O
2 2
ꢁ
6
ꢁ1
1
0
mol l , respectively, in 0.2 mol l H BO ꢂ
/
3
3
2 2
Fig. 1. Excitation (a) and emission (b) spectra of HNT and its oxidized product by H O .

2870
B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ2874
/
possible mechanism of the reaction was as follows:
Table 1
Effect of interferences on the determination of H
2
O
2
(3.8ꢀ
/
ꢁ
6
ꢁ1
1
0
mol l
)
Interferent
Additions (mg)
Relative error (%)
2
4
ꢁ
SO
500
0.2
100
3
ꢁ7
/
2
ꢃ
Ca
ꢁ
ꢁ5
/
I
Fe
ꢁ6
/
3
ꢃ
ꢁ6
/
3
.3. Optimization of experimental variables
ꢁ
F
V
12
15
5
ꢁ5
/
B6
ꢃ6
/
The pH of the medium had an important effect
Vc
V
B2
ꢃ5
/
on the fluorescence intensity of the reaction
system. The experimental results showed that the
optimum pH range was between 11.0 and 12.0.
Therefore, a pH of 11.5 was fixed with the use of
1
ꢃ/5
Phenylalanine
Tyrosine
3
ꢃ5
/
2
ꢃ4
/
ꢁ
NO
3
2ꢃ
500
1
0
Zn
Mn
ꢁ3
/
2
ꢃ
H BO ꢂ
/NaOH buffer.
1
ꢃ
/4
3
3
2
ꢃ
As the volume of the buffer added (from 1.0 to
.0 ml) had little effect on the fluorescence
Cu
Cl
Tryptophane
2
0
ꢁ
1000
10
200
4
ꢁ2
/
3
ꢃ5
/
intensity, 2.0 ml was used in subsequent experi-
ments.
2ꢁ
3
CO
V
B1
ꢁ3
/
ꢁ/2
The influence of HNT and HRP concentrations
were studied. The results showed that the optimum
Sucrose
Glucose
1000
900
ꢃ5
ꢃ/5
/
ꢁ
5
concentrations of HNT and HRP were 3.2ꢀ10
/
ꢁ
1
ꢁ1
range of 1.30ꢀ
10ꢁ
9
ꢂ
/
1.25ꢀ
10ꢁ5 mol lꢁ1 with a
correlation coefficient of 0.9983. The regression
/
mol l
and 0.5 mg ml , respectively.
/
The effect of reaction time was discussed. The
results showed that the oxidation reaction was
7
ꢁ1
equation was R ꢄ
The R.S.D. was 2.3% obtained from a series of 12
/
2.16ꢀ
/
10 C (mol l )ꢁ0.169.
/
IF
completed within 7ꢂ10 min at room temperature;
/
ꢁ
6
ꢁ1
the fluorescence intensity reached a maximum 10
min after the regents had been added and
remained constant for at least 1 h. Hence, after
all the oxidation reactions were carried out for 10
min, the subsequent fluorescence measurements
were made at room temperature within 1 h.
standards, each containing 3.8ꢀ
. The standard deviation of the fluorescence
measurements was 0.0028 obtained from a series
of 11 blank solutions. The limits of detection (kꢄ
) and of determination (kꢄ10) of the method
were established according to the IUPAC defini-
tions (c ꢄkS /s, where c is the limit of detection,
/10
mol l
H O
2 2
/
3
/
/
1
0
1
S0 is the standard error of blank determination, s
3
.4. Effect of interferences
is the slope of the standard curve and k the
constant related to the confidence interval) [18]
A systematic study of the interferences by
foreign substances in determination of H O
ꢁ10
and the values found were 3.89ꢀ
/
10
mol l , respectively. In Table 2,
and
ꢁ9
ꢁ1
2
2
1.30ꢀ/10
ꢁ
6
ꢁ1
(
3.8ꢀ
/
10
mol l ) was carried out. The result
characteristics of the method are compared with
those of similar published fluorescence methods
for H O determination, which showed that the
in Table 1 showed that the anions had little effect
on the fluorescence intensity, but most of the
cations had quenching effect on reaction system.
2
2
proposed method offered higher sensitivity than
other methods.
3
.5. Analytical characteristics
3.6. Effect of surfactants
Under the optimum experimental conditions,
there was a linear relationship between the fluo-
rescence intensity and H O concentration in the
A study of the effect of surfactants on reaction
system was carried out. The results are given in
2
2

B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ
/2874
2871
Table 2
Comparison of the characteristics of the proposed method with those published previously
Sensitivity (nmol lꢁ
)
1
Reference
Substrate
lex (nm)
lem (nm)
Homovanillic acid
p-Hydroxyphenylacetic acid
Tyrosine
315
320
316
325
312
260
425
400
412
430
410
450
0.01
20
[5]
[8]
169
[11]
[7]
p-Hydroxyphenylpropionic acid
Chavico
HNT
0.71
0.527
0.389
[12]
This work
At the same time, HRP could be denatured in
organic solvents so that part of HRP had been
activeless [20], which caused the fluorescence
quenching of the system. As a result, all of the
absolute values of F were not very large, which
indicated that the general effect of organic solvents
on the fluorescence intensity of system was not
very noticeable.
Table 3
(3.8ꢀ
10ꢁ
/
6
Effect of surfactants on the determination of H
ꢁ
2
O
2
1
mol l
)
a
Surfactant
Concentration (%)
R
IF
F (%)
ꢂ/
ꢂ/
97.8
24.0
77.4
45.8
51.4
44.6
58.8
0
Tween-80
CTMAB
PVA
0.1
0.1
0.5
0.1
0.1
ꢁ
/
75.7
20.9
53.2
47.4
54.4
39.9
ꢁ
/
ꢁ
/
Triton X-100
SDS
ꢁ
/
3
.8. Existence of Ã
/
OÃ
/
OÃ
/
H in PEGs
ꢁ
/
10ꢁ
3
ꢁ/
b-CD
2.0ꢀ
/
In order to prove the existence of Ã
/
OÃ
/
OÃ
/
H in
PEGs, a series of study was carried out as follows.
a
CTMAB, bromocetyltrimethyl amine; SDS, sodium dode-
cyl sulfate; PVA, polyvinyl alcohol; b-CD, b-cyclodextrin.
3.8.1. Oxidizing property of PEGs
Tyrosine (Tyr) could be oxidized to fluorescent
dimer by H O under the catalysis of HRP. Under
Table 3. At first, the effective factor (F) was
2
2
defined as follows: Fꢄ
/
[IRFꢁ(I ) ]/(I ) , where
/
RF 0 RF 0
(
I ) is the relative fluorescence intensity of the
RF 0
reaction system when surfactant had not been
added. When F ꢀ0, the surfactant has sensibiliza-
tion effect on the system. When F B0, that means
/
/
the surfactant has an restraint effect. Therefore,
according to the values of F in Table 3, we found
that almost all of the surfactants added have
restraint effect on the system. The possible reason
is that HNT and its oxidized product can not be
solubilized in or on the micelle of surfactants
because they are insoluble in water [19].
3
.7. Influence of organic solvents
The influence of organic solvents was discussed.
Fig. 2. Emission spectra of Tyr after oxidation by H
ꢁ
2
O
2
(a) and
After adding organic solvents to the reaction
system, the reaction regents and products were
solubilized in organic solvent, which could
strengthen the fluorescence intensity of system.
1
PEGs (b), catalyzed by HRP. Tyr, 0.04 mg ml ; HRP, 0.1 mg
ꢁ1
ꢁ4
ꢁ1
ml ; H
a) HRPꢃ
TyrꢃPEGs600; (d) regent blank.
2
O
2
, 3ꢀ
/
10
mol l ; PEGs400, 15%; PEGs600, 15%.
(
/
Tyrꢃ
/
H
O
2 2
; (b) HRPꢃTyrꢃPEGs400; (c) HRPꢃ
/
/
/
/

2872
B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ2874
/
the same experimental conditions, Ã
/
OÃ
/
OÃ
/
H in
PEGs also oxidized Tyr to fluorescence dimer. The
emission spectra of Tyr after oxidation by H O
2
2
and by Ã
/
OÃ
/
OÃ/H in PEGs were given in Fig. 2(a)
and (b), respectively. The result in Fig. 2 showed
that the oxidized product of Tyr by H O had a
2
2
maximum emission at 408 nm, whose emission
spectra was similar to that of the oxidized product
by Ã
/
OÃ
/
OÃ
/
H in PEGs, which indicated that the
oxidized product by H O was same as that by Ã
/
2
2
OÃ
/
OÃ
/
H in PEGs. So we inferred that the mechan-
ism of the catalytic oxidation of Tyr by H O was
2
2
same as that by Ã
/
OÃ
/
OÃ
/
H. Thus, Ã
/
OÃ
/
OÃ
/
H in
PEGs had oxidizing property.
Fig. 4. Absorbance spectra of I
2
generated by the reaction of
H was destroyed. PEGs400
10 mol l ; heating time: (a) 0 min; (b) 10
3
.8.2. Reaction of KI with PEGs
H O could oxidize KI to I , which had two
KI with PEGs, in which ÃOÃOÃ
/
/
/
,
ꢁ
3
ꢁ1
2
2
2
15%; KI, 1.125ꢀ
/
absorption peaks at 288 and 353 nm (Fig. 3(a)).
Under the same experimental conditions, after KI
was treated with PEGs400, the position of the two
peaks of I2 did not shift (Fig. 3(b)), which
indicated that the reaction product and mechan-
ism of KI with H O was same as those of KI with
min; (c) 20 min; (d) 40 min.
The solution of PEGs400 was deaerated with
pure nitrogen when it was heated to boiling for 40
min in order to decompose the Ã
After cooling to room temperature, the solution
was treated with KI, then the absorbance spectrum
was detected. The results (Fig. 4) showed that the
/
OÃ
/
OÃ/H in PEGs.
2
2
PEGs.
absorption peaks of I disappeared after Ã
/
OÃ
/
OÃ
/
2
H in PEGs was destroyed.
Liquid PEGs having low degree of polymeriza-
tion (degree of polymerization B400) can easily
be oxidized to form ÃOÃOÃH in the air, but solid
PEGs (degree of polymerization ꢀ600) can not be
/
/
/
/
/
easily to. So the solid PEGs was solved in water,
after passing pure oxygen through the solution for
different times, the solution was reacted with KI.
The result showed that the height of peaks of I2
increased with the increasing of oxygen supplied to
the solution, which indicated that the content of Ã
OÃOÃH in PEGs was increased after pure oxygen
was supplied.
/
/
/
3.8.3. Thin-layer chromatographic spectrum
analysis of PEGs
The silicic acid H chromatographic plate was
used. The development agent was the mixture
solution of acetoacetate, acetic acid and water
(70:16:15). The saturated solution of KI was used
Fig. 3. Absorbance spectra of I
2
generated by the reaction of
KI with H (a) and PEGs (b). PEGs400, 15%; KI, 1.125ꢀ
2
O
2
/
ꢁ
3
mol lꢁ1; H
ꢁ4
10
O
2 2
, 3ꢀ
/
10 %. (a) KIꢃ
/H
2
O
2
(b) KIꢃ
/
PEGs400 (c) regent blank.

B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ
/2874
2873
Table 4
Determination of Ã
/
OÃ
/
OÃ
/
H in polyethylene glycols (Pꢄ
/
0.95, nꢄ
/6)
ꢁ
1
)
Mean found (mmol lꢁ1)
Recovery value (mmol lꢁ1)
Sample
H
2
O
2
added (mmol l
Mean recovery (%)
PEGs400
0
3.719
5.809
7.659
2.599
4.559
6.669
1.769
3.709
5.849
/
0.01
0.03
0.04
0.01
0.03
0.02
0.01
0.02
0.03
2
4
0
2
4
0
2
4
.00
.00
/
2.099
/
0.02
105.0
99.0
/
3.9490.03
/
PEGs600
PEGs800
/
.00
.00
/
1.969
/
0.02
98.0
102.0
/
4.0790.01
/
/
.00
.00
/
1.949
/
0.01
97.0
102.0
/
4.0890.02
/
as color-developing agent. The result showed that
the existence of Ã
/
OÃ
/
OÃ
/
H in PEGs was carried out
there was only one color-developing point (R ꢄ
/
in this work. The proposed method was applied to
H in polyethylene
f
0
.312), which indicated that there was no impurity
the determination of Ã
glycols with satisfactory results.
/
OÃ
/
OÃ
/
having oxidizing property except for Ã
/
OÃ
/
OÃ/H in
PEGs.
3
.9. Sample analysis
In the experiment, we found that Ã
Acknowledgements
/
OÃ
/
OÃ/H in
This work was supported by the Natural Science
Foundation of China (29975016) and Natural
Science Foundation of Shandong Province, China
PEGs having different degree of polymerization
can oxidize HNT under the catalysis of HRP. The
oxidized product of HNT by PEGs was same as
that by H O . The working solutions of PEGs
400,
(No. Z2000B03).
2
2
PEGs600, PEGs800 were prepared by diluting to
00, 200, 250 times of 10% stock solutions of
5
References
PEGs400, PEGs600, PEGs800, respectively. To 1.00
ml of the working solution of PEGs were added
various amounts of standard H O solutions, then
[
[
1] P. George, Nature 169 (1952) 612.
2] G.G. Guilbault, P.J. Brignac, Jr., M. Zimmer, Anal.
Chem. 40 (1968) 190.
2
2
the Ã
/
OÃ
/
OÃ
/
H amount of PEGs was determined
spectrofluorimetrically by the proposed method
for the determination of H O . The standard
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2
2
[
[
[
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that the results obtained were satisfactory.
3
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4
[
[
[
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4
. Conclusions
In this work, HNT has been successfully used as
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1.
(
the new kind of substrate for HRP in highly
sensitive spectroflourimetric determination of
H O . Compared with other substrates used in
[
8
2
2
[
[
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the published methods [5ꢂ/14], HNT offers higher
sensitivity. (Table 2) Moreover, a study to prove

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B. Tang, Y. Wang / Spectrochimica Acta Part A 59 (2003) 2867ꢂ2874
/
[
[
[
[
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(
(
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1
[20] G.Z. Cheng, X.Z. Huang, Progression in Fluorimetric
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