A High Selective Chemiluminescent Probe Derived from Iso-luminol Enabling High Sensitive Determination of Ferrous Ions in the Environmental Waters

Article abstract of DOI:10.1002/cjoc.202100057

A new chemiluminescence (CL) reagent named 4-amino-5-thiocyanato-phthalyl-hydrazine (iso-luminol-SCN) is synthesized by the bromide-mediated substitution reaction of iso-luminol with sodium thiocyanate in dimethyl formamide (DMF) at room temperature. Stro

Full text of DOI:10.1002/cjoc.202100057

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Title: A High Selective Chemiluminescent Probe Derived from Iso-luminol Enabling
High Sensitive Determination of Ferrous Ions in the Environmental Waters
Authors: Shenghai Zhang,* Yalin Shi, Jiawang Deng, Jidong Zhang, Mengqi Cheng,
and Geting Yu
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Cite this paper: Chin. J. Chem. 2021, 39, XXX—XXX. DOI: 10.1002/cjoc.202100XXX
A High Selective Chemiluminescent Probe Derived from
Iso-luminol Enabling High Sensitive Determination of Ferrous
Ions in the Environmental Waters
Shenghai Zhang,*, a , b Yalin Shi,a Jiawang Deng,a Jidong Zhang,a Mengqi Cheng,b and Geting Yub
a Quality Supervision and Inspection Centre of Se-enriched Food of Shaanxi Province, School of Chemistry & Chemical Engineering, Ankang
Univerisity, An'kang 725000, P.R. China
b Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi
Normal University, Xi'an 710119, P.R. China
Keywords
4-amino-5-thiocyanato-phthalylhydrazine | Chemiluminescence | Ferrous ions | Determination| Environmental Waters
Main observation and conclusion
A new chemiluminescence (CL) reagent named 4-amino-5-thiocyanato-phthalyl-hydrazine (iso-luminol-SCN) is synthesized by the bro-
mide-mediated substitution reaction of iso-luminol with sodium thiocyanate in dimethyl formamide (DMF) at room temperature. Strong
CL is observed for iso-luminol-SCN oxidized by hydrogen peroxide (H2O2) in the presence of Fe2+, with the CL intensity strongly depending
on the concentration of Fe2+ and hardly interfering by other common metal ions. Based on this fact, a flow injection CL method is estab-
lished for the high sensitive and selective determination of Fe2+. The CL intensity was linearly correlated with the concentration of Fe2+ in
the range of 1.0×10-8 mol/L to 1.0×10-5 mol/L following the equations to CLnet=166.3C+57.7 (C, ×10-6 mol/L; n=7; R2= 0.9988). The detec-
tion limit is calculated to be 3.2×10-9 mol/L Fe2+ based on the 3S0/K principle. The relative standard deviation (RSD) was 3.7% for 1.0×10-6
mol/L Fe2+ (n=11), a support for the precision of Fe2+ measurement. The recoveries of Fe2+ were obtained in the range of 98.8–101.6% in
three real water samples, showing the applicability and reliability by the proposed method. The possible CL mechanism is proposed
based on the kinetic characteristic of the CL reaction and the CL spectrum.
Comprehensive Graphic Content
* Corresponding authors. E-mail: zhshhai512@163.com (S. Zhang)
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Supporting Information
Chin. J. Chem. 2021, 39, XXX－XXX
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O
O
O
Background and Originality Content
H2N
NCS
H2N
KSCN/Br2
DMF, rt,
NH
NH
NH
NH
As the most abundant transition metal for both plants and
animals,[1] iron is essential for sustaining life and ubiquitous in the
environment and in biology,[2] plays crucial roles in many physio-
logical and pathological processes such as oxygen transportation,[3]
participates in DNA and RNA synthesis,[4] increases the synthesis
rates of some proteins,[5] acts as the cofactor in many enzymatic
reactions,[6] etc. Previous studies have shown that the
one-electron transfer between the two principal oxidation states
(ferrous ions and ferric ions, Fe2+ and Fe3+) of iron is one of the
important redox pairs[7] and that it is easily accomplished in bio-
logical systems.[8] The total iron content in a well-nourished people
is about 4 g (70% in Hgb, 25% in storage),[9] with either iron defi-
6h
O
90.2 %
iso-luminol-SCN
iso-luminol
O
H2N
NCS
O-
O-
H
2O2
+
v
h
Fe2+
O
4-APH-SCN
ciency or overload leading to the occurrence of many diseases[10]
.
The decreased synthesis of hemoglobin and the consequent cellu-
lar oxygen deficiency are the major effects of iron deficiency in
animals, known as the most common cause of anemia.[11] The
accretion of free iron ions, particularly Fe2+ has been observed in a
Scheme 1 The key technical step of iso-luminol-SCN synthesis and it is used
as the chemiluminescence probe for ferrous ion detection.
number of serious central nervous system diseases such as Park- Results and Discussion
inson's, Huntington's and Alzheimer's diseases,[9,12] with the high
levels of iron associated with an increased risk for arthritis, diabe-
CL selectivity and responsiveness study
tes, liver disease, heart disease and cancer, etc.[13]
Drinking water is an important source of iron for the human
body.[14] A security limit of iron in a sanitary standard for drinking
water was restricted to 2.0 mg/L (3.57×10-5 mol/L) by World
Health Organization (WHO), and European Legislation has estab-
lished a maximum contaminant level (MCL) at 200 µg/L for iron
(3.6×10-6 mol/L).[13] In addition to its roles in physiological and
pathological processes mentioned above, evidence shows that
iron can also control the mobility, bioavailability and toxicity of
other trace metals in the natural water system.[15] Therefore, to
develop rapid, sensitive and efficient methods for measuring the
amount of iron in environmental waters is of great importance to
assure the public health.
CL selectivity for ferrous ions Considering the complexity of
the actual samples, the iso-luminol-SCN the selective CL response
for analyte is of top importance. We examined the CL response of
iso-luminol-SCN to Fe2+ against common metal ions. The CL meas-
urements were conducted with the flow setups as illustrated in
Figure 3. It can be found in Figure 1 that Fe2+ takes overwhelming
CL response against the rest metal ions. That is, the obtained
iso-luminol-SCN is specific to Fe2+.
Metabolic iron is Fe2+,[5] with many methods established, such
as colorimetry,[9,16] spectrophotometry,[15,17] atomic absorption
spectrometry (AAS),[18] high performance liquld chromatog-
raphy,[19] ion chromatography,[20] inductively coupled plasma-mass
spectrometry (ICP–MS),[21] inductively coupled plasma optical
emission spectrometry (ICP-OES),[22] fluorimetry,[1, 2a, 4, 23] chemi-
luminescence[24] and electrochemical,[25] etc., which have been
developed for Fe2+ determination. However, most of the methods
mentioned above either lack sufficient sensitivity or require the
complex procedure and expensive analytical equipment, a limit to
their potential application. The chemiluminescence method such
as luminol-based and other chemiluminescence methods with the
advantages of simple equipment, low cost, high sensitivity, fast
analysis and easily automated detection gives its full play for the
Fe2+ determination in water samples[24], but the selectivity for Fe2+
measuring over other metal ions still needs to be improved.
In this paper, we synthesized a new chemiluminescence (CL)
reagent named 4-amino-5-thiocyanato-phthalylhydrazine
(iso-luminol-SCN), thus enriching the CL reagent family members.
Iso-luminol-SCN oxidized by hydrogen peroxide (H2O2) in the
presence of Fe2+, and a strong Fe2+ concentration-dependent CL
was observed. However, other common metal ions can hardly
enhance the CL reaction of iso-luminol-SCN and H2O2. Based on
this, a new, rapid and simple method with high selectivity and high
sensitivity is proposed for the determination of Fe2+. Satisfactory
results were obtained by using this method for determining Fe2+ in
environmental water samples. Furthermore, the CL reaction
mechanism is discussed for iso-luminol-SCN oxidized by H2O2 in
the presence of Fe2+.
Figure 1 Chemiluminescence selectivity of iso-luminol-SCN for Fe2+ meas-
uring. 1.0×10-3 mol/L H2O2 and 5.0×10-6 mol/L iso-luminol-SCN (in 0.1
mol/L pH 7.4 PBS); Metal ions (nitrate, aqueous solution), left to right-
blank, 1.0×10-4 mol/L of NH4 , Li+, K+ , Ag+, Mg2+ , Al3+, Ca2+, Ba2+ , Fe3+
,
+
Fe2+ , Co2+ , Ni2+ , Cu2+ , Zn2+ , Cr3+ , Sn2+ , Pb2+ , Cd2+, and Hg2+; PMT voltage,
_800V.
Kinetic characteristic of CL reaction Kinetic characteris-
tics of the CL reactions were examined. The CL intensity–time
curve is shown in Figure 2. When H2O2 solution was injected into
the mixture of iso-luminol-SCN and Fe2+ (0.1 mL), a strong CL reac-
tion was initiated immediately. The maximum CL intensity was
obtained at about 27 s after H2O2 addition. Thereafter, the CL
signal declined to the baseline in 300 s approximately. So, the
iso-luminol-SCN~ H2O2~ Fe2+ CL reaction is a fast CL reaction and
our experiment results suggest iso-luminol-SCN a potential appli-
cation for Fe2+ detection.
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Chin. J. Chem. 2018, XX, 1－X

Running title
Chin. J. Chem.
Figure 4 Effect of flow rate of pump on CL intensity. H2O2, 1.0×10-3 mol/L
(in 0.1 mol/L pH 7.4 PBS); Fe2+, 8.0×10-5 mol/L (aqueous solution);
iso-luminol-SCN, 5.0×10-6 mol/L (in 0.1 mol/L pH 7.4 PBS); PMT voltage,
_600V.
Figure 2 CL intensity–time curve. 0.1 mL 1.0×10-2 mol/L H2O2 (in 0.1 mol/L
pH 7.4 PBS) was injected into the mixture of 0.8 mL 1.0×10-4 mol/L
iso-luminol-SCN (in 0.1 mol/L pH 7.4 PBS) and 0.1 mL 1.0×10-3 mol/L of Fe2+
(aqueous solution); PMT voltage, _800V.
Effect of concentration of hydrogen peroxide H2O2 reacts with
Fe2+ to produce ·OH, acting as the oxidant in the CL reaction, and
hence the concentration of H2O2 influences the CL signal. The
effect of H2O2 concentration on the CL intensity was investigated
in the range of 1.0×10-4 mol/L to 5.0×10-3 mol/L. Experiments
showed that the maximal CL signal could be obtained with
1.0×10-3 mol/L H2O2 (Figure 5).
Optimization of CL conditions
The initial test showed that a high CL selectivity to Fe2+ was
observed in 0.1 mol/L pH 7.4 PBS; therefore, 0.1 mol/L pH 7.4 PBS
was used to control the reaction medium in the following experi-
ments. In order to obtain the optimum analytical conditions of
Fe2+, the parameters for flow system, flow rate, iso-luminol-SCN
concentration and H2O2 concentration were optimized using the
single factor rotation method.
Effect of flow systems In order to obtain the maximal CL signal,
several flow systems were designed. When Fe2+ solution is carried
by air to react with the mixing stream of iso-luminol-SCN and H2O2,
the weak CL signal was detected (Fig. S2). When employing the
system shown schematically in Figure 3 and Fe2+ is carried by H2O2
solution to react with iso-luminol-SCN, no CL signal was detected.
Instead, a strong CL signal with good repeatability was captured
immediately by adopting iso-luminol-SCN to carry Fe2+ solution to
react with H2O2. So, the flow system shown in Figure 3 was se-
lected.
Figure 5 Effect of H2O2 concentration on CL intensity. iso-luminol-SCN,
5.0×10-6 mol/L (in 0.1 mol/L pH 7.4 PBS); Fe2+, 80.0 µM (aqueous solution);
H2O2 , varying from 0.1 to 5.0×10-3 mol/L (in 0.1 mol/L pH 7.4 PBS); PMT
voltage, _600V.
Effect of concentration of iso-luminol-SCN The concentration
of CL reagent has an effect on the CL intensity, and therefore, the
effect was investigated of iso-luminol-SCN concentration on the
CL intensity in the range of 1.0 µM to 0.1 mM. The results showed
that the CL signal increased with the increasing of iso-luminol-SCN
till its concentration reached 25.0 µM with the concentration
selected to obtain the maximal CL signal (Figure 6).
Figure 3 Schematic diagram of CL flow system: (a) H2O2 solution; (b) Fe2+
standard or sample solution; (c) iso-luminol-SCN solution. P: peristaltic
pump; V: injection valve; F: flow cell; W: waste; HV: high voltage; PMT:
photomultiplier tube; PC: personal computer.
Effect of flow rate on CL intensity The flow rate influences the
analytical sensitivity in adopting the flow-injection chemilumines-
cence analysis system shown schematically in Figure 3. The effect
of the flow rate of pump on CL intensity was examined in the
range of 1.5~4.5 mL/min. The results showed that the CL signal
increased with the increasing of flow rate due to this rapid CL
reaction. Accordingly, the maximal flow rate 4.5 mL/min was se-
lected in the following experiments (Figure4).
Figure 6 Effect of iso-luminol-SCN concentration on CL intensity. H2O2, 1.0
×10-3 mol/L (in 0.1 mol/L pH 7.4 PBS); Fe2+, 8.0×10-5 mol/L (aqueous solu-
Chin. J. Chem. 2021, 39, XXX－XXX
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Shenghai Zhang et al.
Report
tion); iso-luminol-SCN, varying from 1.0×10-6 mol/L to 1.0×10-4 mol/L (in
0.1 mol/L pH 7.4 PBS); PMT voltage, _500V.
River, tap water from our laboratory, and commercial available
mineral spring water. The sample solutions were prepared in Sec-
tion 4.4 and analyzed in Section 4.3. To examine the accuracy, the
recovery experiments were carried out with all samples spiked
with iron standard solutions in two levels. The obtained recoveries
were reasonable for Fe2+ in samples in a range of 95%~104%. The
results of the determination for sample solution are shown in
Table 1.
Dynamic range, detection limit and RSD
In the optimized conditions, the CL intensity was linear to
Fe2+ concentration in the range of 1.0×10-8 mol/L to 10.0 µM fol-
lowing equations to CLnet=166.3C+57.7 (C,×10-6 mol/L; n=7; R2=
0.9988) (Figure 7). The detection limit is calculated to be 3.2×10-9
mol/L Fe2+ according to the IUPAC principle. The RSD was found to
be 3.7% by 11 replicate determinations of 1.0×10-6 mol/L Fe2+.
Tab1 Results for determination of Fe2+ in water samples
Sample
Fe2+ added(µg/L)
Found(µg/L)
89.7 ± 1.9
188.9 ± 1.8
290.7 ± 2.3
68.1 ± 1.5
Recovery(%)
__
99.1 %
101.1 %
__
0
Hanjiang River
watera
100
200
0
Tap water
100
200
0
100
200
168.7 ± 1.7
269.2 ± 2.1
163.7 ± 2.2
265.7 ± 1.6
361.7 ± 2.4
100.9 %
101.6 %
__
101.2 %
98.8 %
Mineral spring
water
a (Collected from Hanyin County, An’kang city.) b (Mean value ± standard
deviation (n = 3) and 95% confidence limit.)
Mechanism speculation
Figure 7 Linear relationship between reaction of CL intensity and Fe2+
(1.0×10-8 mol/L ~1.0×10-5 mol/L) (aqueous solution). H2O2, 1.0×10-3 mol/L
(in 0.1 mol/L pH 7.4 PBS); iso-luminol-SCN, 2.5×10-5 mol/L (in 0.1 mol/L pH
7.4 PBS); PMT voltage, _900V.
When H2O2 was injected into the mixture of iso-luminol-SCN
and Fe2+, a strong CL signal was observed; however, no CL signal
was observed in the absence of Fe2+. It has been confirmed that
the inorganic SCN– and isomers of –SCN can be fast oxidized by
H2O2 in producing a series of complex intermediates.[26] We spec-
ulated that –SCN substituent also could be reacted with H2O2 in
generating the corresponding intermediate, with the possible
mechanism of iso-luminol-SCN~H2O2~Fe2+ CL reaction proposed in
the following. H2O2 reacted with Fe2+ to produce hydroxyl radical
(·OH), which cooperated with excess H2O2 to oxidize
iso-luminol-SCN to produce the excited state 4-amino-5-thio- cy-
anatophthalate (4-APH-SCN*). 4-APH-SCN* came back to the
ground state and released its energy in the form of light radiation
(λmax=448 nm). The mechanism was assumed to be synergistical-
ly oxidized by ·OH and H2O2, which could be expressed simply as
follows (Supporting information, Fig. S15 and S18):
Interference study
The influence of foreign species was investigated by measur-
ing a standard solution to 1.0×10-6 mol/L Fe2+ to which increasing
amounts of adscititious species were added. A substance was
considered to be of no interference for the variation of the CL
intensity to be within ±5%. It was found that the tolerable con-
centration ratios of foreign species to 1.0 µM of Fe2+ were over
20-fold for Cu2+, 60-fold for Co2+, Hg2+, 100-fold Fe3+,
+
Ca2+ , >200-fold for NH4 , Li+, Ag+, Mg2+, Ba2+, Ni2+, Zn2+, Sn2+, Pb2+,
-
-
Cd2+, Cr3+, Al3+; 10-fold for S2-, 25-fold SO32-, 20-fold NO3 , NO2 ,
CO32-, and >100-fold for F-, Cl-, Br-, I-, HCO3 , SO42-. Therefore, the
-
(1) H2O2 + Fe2+ → Fe3+ + OH- +·OH
proposed method has adequate selectivity for the determination
(2)·OH + iso-luminol-SCN+ 2H2O2→4-APH-SCN* + 2H2O+ N2
(3) 4-APH-SCN* →4-APH-SCN + hν (λmax = 448 nm)
of Fe2+ in real water samples.
Conclusions
In this work, a novel CL reagent iso-luminol-SCN was synthe-
sized, enriching the CL reagent family members. Based on its CL
properties, a good selectivity and high sensitivity method for the
determination of Fe2+ was established. The method was satisfac-
torily applied to detect the Fe2+ in environmental water samples.
Furthermore, the mechanism for the iso-luminol-SCN~H2O2~Fe2+
CL reaction was briefly discussed.
Experimental
Apparatus
Figure 8 Influence of coexisting ions on CL intensity. H2O2, 1.0×10-3 mol/L
(in 0.1 mol/L pH 7.4 PBS); iso-luminol-SCN, 2.5×10-5 mol/L (in 0.1 mol/L pH
7.4 PBS); Fe2+, 1.0×10-6 mol/L (aqueous solution); PMT voltage, _900V.
The flow injection CL analyzer (IFFM-E, Xi'an Remex analysis
Instrument Co., Ltd., China) was equipped with an automatic in-
jection system and a detection system. The flow cell was a coil of
glass tube positioned in front of the detection window of the PMT.
CL dynamic profile and brightness are measured with the
above-mentioned CL analyzer; CL spectra were measured with a
fluorescence spectrometer (RF-5301PC, Shimadzu Co., Ltd., Ja-
pan).
Analytical application
In order to evaluate the applicability and reliability by the
proposed methodology, it was applied to detect Fe2+ in the envi-
ronmental water samples. Three water samples from Hanjiang
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Chin. J. Chem. 2021, 39, XXX－XXX

Running title
Chin. J. Chem.
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Unless otherwise stated, all the chemical reagents are used
directly without further purification. Hydrogen peroxide (H2O2,
30%) is purchased from Tianjin Tianli Chemical Reagents Ltd.
(Tianjin, China), sodium hypochlorite(NaClO, active chlorine≥10%)
is purchased from Tianjin Fuchen Reagents Ltd. (Tianjin, China),
and All other AR grade chemicals, including sodium hydrox-
ide(NaOH), disodium hydrogen phosphate(Na2HPO4), sodium di-
hydrogen phosphate dihydrate(NaH2PO4·2H2O), ferrous sulfate
heptahydrate (FeSO4·7H2O), etc. are purchased from Sinopharm
Chemical Reagent Co., Ltd. (Xi’an, China).
The standard solution to Fe2+ (1.0×10-2 mol/L) was prepared
by dissolving 0.278 g FeSO4·7H2O in water and diluting to 100 mL.
The 1.0×10-4 mol/L stock solution to iso-luminol-SCN was prepared
by dissolving 23.5 mg iso-luminol-SCN (The synthetic processes
are detailed in the Supporting information) in 20 mL 1.0×10-2
mol/L NaOH solution, and then by diluting them to 1000 mL with
0.1 mol/L pH 7.4 PBS. The 1.0×10-3 mol/L H2O2 solution was pre-
pared by diluting the H2O2 with 0.1 mol/L pH 7.4 PBS directly.
Water is purified by using a Millipore water purification sys-
tem (18.2 MΩ·cm, Direct-Q 3UV, Millipore, FR) and used
throughout the whole experiment.
Sample preparation
All samples including the river water collected from Hanjiang
River, tap water obtained from our laboratory and bottled mineral
water purchased from local market were filtered through 0.22 µm
filter paper and saturated with nitrogen at room temperature in
order to minimize the influence of the dissolved oxygen in the
samples, and they are used for the chemiluminescence determi-
nation as soon as possible.
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CL signal measurement
Investigations of CL behaviors were performed using the sys-
tem shown schematically in Figure 1. Flow tubes (a, b and c) were
connected with H2O2, Fe2+ standard or sample solution and
iso-luminol-SCN, respectively. The sample or Fe2+ standard solu-
tion was injected into iso-luminol-SCN solution by a six-way injec-
tion valve, and then merged with H2O2 solution through a Y-piece
to produce CL in the flow cell. The CL intensity produced by H2O2
solution was considered as the blank. The concentration of Fe2+
was quantified via the peak height (CL intensity), which was ob-
tained by subtracting the blank CL intensity from that of the sam-
ple or Fe2+ standard solution.
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Supporting Information
The supporting information for this article is available on the
WWW under https://doi.org/10.1002/cjoc.2021xxxxx or from the
author.
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The authors sincerely thank the Special Funds for Scientific
Research of High-level Talents of Ankang University (No.2020
AKQDZR04), the Open Foundation of the Key Laboratory of Ana-
lytical Chemistry for Life Science of Shaanxi Province (No.2017001)
and the Shaanxi Provincial Innovation Experiment Program for
University Students (No.S202011397059) for financial support.
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Manuscript revised: XXXX, 2021
Manuscript accepted: XXXX, 2021
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Entry for the Table of Contents
A High Selective Chemiluminescent Probe Derived from Iso-luminol Enabling High Sensitive Determination of Ferrous Ions in the Environmen-
tal Waters
Shenghai Zhang,*,1 Yalin Shi,1 Jiawang Deng,1 Jidong Zhang,1 Mengqi Cheng,2 and Geting Yu2
Chin. J. Chem. 2021, 39, XXX—XXX. DOI: 10.1002/cjoc.202100XXX
A New CL reagent family member termed iso-luminol-SCN with high oxidation efficiency by the produced .OH from Fenton Reaction results in superb
selective and sensitive detection for Fe2+.
a Quality Supervision and Inspection Centre of Se-enriched Food of c High School of Hanyin County, An'kang 725100, P.R. China
Shaanxi Province, School of Chemistry & Chemical Engineering, E-mail: 171854406@qq.com
Ankang Univerisity, An'kang 725000, P.R. China
E-mail: zhshhai512@163.com
b Key Laboratory of Analytical Chemistry for Life Science of Shaanxi
Province, School of Chemistry and Chemical Engineering, Shaanxi
Normal University, Xi'an 710119, P.R. China
E-mail: 510186170@qq.com
Chin. J. Chem. 2018, template
© 2018 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim