Colorimetric and fluorescent detection of hydrazine with high sensitivity and excellent selectivity

Article abstract of DOI:10.1016/j.saa.2017.07.003

It is critical to develop probes for rapid, selective, and sensitive detection of the highly toxic hydrazine in both environmental and biological science. In this work, under mild condition, a novel colorimetric and off-on fluorescent probe was synthesized for rapid recognition of hydrazine with excellent selectivity over other various species including some biological species, metal ions and anions. The limit of quantification (LOQ) value was 1.5?×?10^??4?M–3.2?×?10^??3?M (colorimetric method) and 1.5?×?10^??4?M???3.2?×?10^??3?M (fluorescent method) with as low as detection limit of 46.2?μM.

Full text of DOI:10.1016/j.saa.2017.07.003

Accepted Manuscript
Colorimetric and fluorescent detection of hydrazine with high
sensitivity and excellent selectivity
Bingjie Shi, Sujie Qi, Mingming Yu, Chunxia Liu, Zhanxian Li,
Liuhe Wei, Zhonghai Ni
PII:
DOI:
Reference:
S1386-1425(17)30552-8
doi: 10.1016/j.saa.2017.07.003
SAA 15290
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
30 November 2016
28 June 2017
6 July 2017
Please cite this article as: Bingjie Shi, Sujie Qi, Mingming Yu, Chunxia Liu, Zhanxian
Li, Liuhe Wei, Zhonghai Ni , Colorimetric and fluorescent detection of hydrazine with
high sensitivity and excellent selectivity, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.07.003
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ACCEPTED MANUSCRIPT
Colorimetric and fluorescent detection of hydrazine with high sensitivity
and excellent selectivity
Bingjie Shia,b, Sujie Qib, Mingming Yub, Chunxia Liub, Zhanxian Lib, Liuhe Weib,
Zhonghai Nia,*
a
School of Chemical Engineering and Technology, China University of Mining and
Technology, Xuzhou 221116, Jiangsu Province, China.
b
College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou
450001, China.
* Corresponding author.
E-mail address: nizhonghai@cumt.edu.cn.

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2
Abstract
It is critical to develop probes for rapid, selective, and sensitive detection of the highly
toxic hydrazine in both environmental and biological science. In this work, under mild
condition, a novel colorimetric and off-on fluorescent probe was synthesized for rapid
recognition of hydrazine with excellent selectivity over other various species including
some biological species, metal ions and anions. The limit of quantification (LOQ) value
was 1.5×10−4 M–3.2×10−3 M (colorimetric method) and 1.5×10−4 M−3.2×10−3
(fluorescent method) with as low as detection limit of 46.2 μM.
M
Keywords: Colorimetric; Off-on fluorescent probe, Quantitative detection; Hydrazine;
Naked-eye detection

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Introduction
As a highly reactive base and reducing agent, hydrazine plays crucial roles in many
fields such as the chemical, pharmaceutical and agricultural industries [1−3]. Hydrazine is
also a high-energy fuel for rocket-propulsion systems and missile systems due to its
detonable characteristics [4,5]. However, as a class of highly toxic and pollutant
compound, hydrazine can potentially lead to serious environmental contamination during
its manufacture, use, transport and disposal. In addition, as a neurotoxin, hydrazine has
several mutagenic effects, which can cause damage to the liver, lungs, kidneys and human
central nervous system [6,7]. As a result, hydrazine has been classified as a probable
human carcinogen by the U.S. Environmental Protection Agency and has a low threshold
limit value of 10 ppb [8]. Therefore, an efficient and simple method for determining
hydrazine level is therefore urgently needed in both environmental and biological science.
In recent years, the development of small molecular fluorescent probes toward metal
ions, anions and biomolecules has attracted great attention due to their simplicity, high
sensitivity, and instantaneous response [9−15]. However, available fluorescent probes for
hydrazine are still very limited [16−20].
In general, fluorescent probes depend on emission intensity or position power and
detector sensitivity, while color changes based on absorption properties of the ground
state are more suitable for direct observation with the naked eye [21,22], and considerable
efforts have been devoted to designing colorimetric probes toward hydrazine during the
past few decades [23−31]. Therefore, it is still meaningful to prepare colorimetric and
fluorescent probes for hydrazine.

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Using 1,8-naphthalimide unit as useful fluorophores, we have designed and
synthesized several 1,8-naphthalimide-based colorimetric and fluorescent probes
because the photophysical properties are easily influenced by reagents [32,33]
.
Based on photo-induced electron transfer (PET) mechanism, a few 4-substituted
1,8-naphthalimide-based fluorescent off-on probes have been recently reported
[34−36]. These observations encouraged us to make use of such PET process to
design hydrazine-responsive fluorescent probes based on 1,8-naphthalimide
derivatives.
In this paper, based on 1,8-naphthalimide, selective colorimetric and fluorescent probe
1 (Scheme S1) was synthesized. Probe 1 can quantitatively detect hydrazine with both
great color and fluorescence change in solution. The detection limit on UV-vis response
of the sensor can be as low as 74.5 nM. Further studies demonstrate that this probe has
good sensitivity and selectivity.
Experimental
Methods and materials
4-Bromo-1,8-naphthalic anhydride, monoethanolamine, glacial acetic acid, ethanol,
2-methoxyethanol, 80% hydrazine hydrate, triethylamine, phthalic anhydride, KCl, NaCl,
MgCl2, Al(NO3)3, CaCl2, Cd(NO3)2, MnCl2, Co(NO3)2, Ni(NO3)2, ZnCl2, Hg(NO3)2,
AgNO3, Fe(NO3)3, CuCl2, and PbCl2, NaBr, NaCl, NaNO3, Na2SO3, Na2SO4, NaClO3,
NaClO4, NaHSO3, NaHSO4, NaH2PO4, Cys, Phe, Gly, His, Tyr, Pro, Ser, Thr, Val, Trp,
Asp, Glu, and SOCl2. All chemicals and solvents were analytical grade except
2-methoxyethanol (chemical grade) and used without further purification. Compounds 3
and 2 and 1 were synthesized by following Scheme S1. Compounds 3 and 2 have ever

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been synthesized and reported by us and here were synthesized according to our reported
method [33].
Measurements
1H NMR and 13C NMR spectra were recorded on a Bruker 400 NMR spectrometer.
Chemical shifts are reported in parts per million using tetramethylsilane (TMS) as the
internal standard. Elemental analysis data were obtained on Automatic Element Analyzer.
All spectral characterizations were carried out in HPLC-grade solvents at 20°C within
a 10 mm quartz cell. UV-vis absorption spectra were measured with a TU-1901
double-beam UV-vis spectrophotometer. Fluorescence spectroscopy was determined on a
Hitachi F-4600 spectrometer.
Synthesis of Probe 1
Compound 2 (0.2713 g, 1 mmol) and phthalic anhydride (0.1852 g, 1.25 mmol) were
dissolved in 50 mL glacial acetic acid. The mixture was refluxed for 5 h, and then cooled
to room temperature. After filtration of the above mixture, the crude product was obtained
as yellow solid. The final product (296.3 mg, 73.0%) was obtained by washing the crude
product with glacial acetic acid three times. 1H NMR (400 MHz, DMSO-d6, TMS): δH
4.29 (t, 4H), 7.02 (d, 1H), 7.89 (t, 1H), 8.03 (m, 4H), 8.26 (d, 1H), 8.52 (d, 1H), 8.72 (d,
1H), 10.21 (s, 1H). 13C NMR (100 MHz, DMSO-d6): δC 170.8, 166.3, 164.2, 163.43,
148.8, 135.8, 133.7, 131.6, 130.0, 129.3, 128.8, 126.3, 124.4, 122.5, 119.6, 113.1, 106.4,
61.5, 21.0. Elemental analysis calcd. for C22H15N3O5, C: 65.83%; N: 10.47%; H: 3.77%;
found, C: 65.374%; N: 9.982%; H: 3.738%.

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Results and Discussion
Synthesis of probe 1
In this work, the probe 1 was synthesized via three-step synthetic procedure starting
from
4-bromo-1,8-naphthalic
anhydride.
The
reaction
of
compound
4-bromo-1,8-naphthalic anhydride with monoethanolamine in ethanol gave the product 3
in a 88.8% yield. Compound 3 reacted with 80% hydrazine hydrate in 2-methoxyethanol
and gave compound 2 in an 89.7% yield. The probe 1 was obtained in a 73.0% yield by
the reaction of compound 2 with phthalic anhydride in glacial acetic acid. The chemical
structures were confirmed by 1H, 13C NMR, and elemental analysis.
Hydrazine hydrate colorimetric sensing properties of probe 1 in solution
To investigate the changes in absorption spectrum of probe 1 upon exposure to
hydrazine hydrate, UV-vis titration experiments were conducted with hydrazine hydrate
in solution of 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) (Fig. 1a). As is evident from
inspection of Fig. 1a, probe 1 exhibits no absorption at 520 nm in solution in the
absence of hydrazine hydrate. However, upon addition of hydrazine hydrate, a
significant increase in the absorption peak at 520 nm was very quickly (several seconds)
observed with the original peak at 400 nm decreasing. Meanwhile, two clear isosbestic
points were observed at 358 and 444 nm respectively, indicating that only one product
was generated from 1 upon the reaction to hydrazine. Fig. S1 (Supporting Information)
indicates the relationship between the absorbance ratio A520/A400 (A520 and A400 mean the
absorbance at 520 nm and 400 nm respectively) and the concentration of hydrazine
hydrate. In the concentration range of 1.5×10–4 M and 13.2×10–3 M, the absorbance ratio
A520/A400 of 1 is in good linear relationship with hydrazine hydrate concentration (Fig.
1b), implying that hydrazine hydrate can be quantitatively detected in a wider

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concentration range. At the same time, the solution color changed from colorless to pink
(Fig. 1c), implying that probe 1 can be a naked-eye probe toward hydrazine hydrate.
These results led us to conclude that 1 could be an effective colorimetric probe for
hydrazine hydrate. The proposed sensing mechanism was shown in Scheme 1.
(Insert: Fig. 1)
(Insert: Scheme 1)
Hydrazine hydrate fluorescent sensing properties of 1 in solution
After standing for 24 hours, the color of the above prepared solutions of probe 1
containing different concentration of hydrazine (tested in colorimetric sensing
experiments) changed colorless, indicating new chemical change happened in the solution.
Upon excitation at 440 nm on these solutions, the emission intensity at 540 nm gradually
increased with increased hydrazine concentration (Fig 2a). Fig. S2 (Supporting
Information) indicates the relationship between the fluorescence intensity at 540 nm and
the concentration of hydrazine hydrate. In the range between 1.5×10−4 M and 3.2×10−3 M,
a good linear relationship between the fluorescence peak intensity with hydrazine
concentration was obtained (Fig. 2b), implying that hydrazine can be quantitatively
detected in a wider concentration range both with UV-vis and fluorescence spectroscopy.
From the linear calibration graph with the fluorescence titration experiment, the detection
limit of probe 1 for hydrazine was found to be about 46.2 μM based on signal-to-noise
ratio (S/N) = 3 [36, 37], which was sufficiently low for the detection of hydrazine. Fig 2c
demonstrates the fluorescence of the above solutions change. These results led us to
conclude that 1 could be an effective colorimetric and fluorescent off-on probe for
hydrazine.
(Insert: Fig. 2)

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Sensing mechanism
As illustrated in Fig. 1, upon addition of hydrazine hydrate and within several seconds,
a new absorption band centered at 520 nm appeared and increased with the original peak
at 400 nm decreasing, and then the peak at 520 nm gradually disappeared. This process is
so fast that it is difficult to study this colorimetric sensing mechanism, and we propose
that such absorption spectra change may be ascribed to the formation of compound 4
(Scheme 1). The fluorescent sensing mechanism was studied by Mass spectra. Whilst on
reaction of hydrazine, one peak appeared at m/z = 227 corresponding to the species
[2+3H]. Such Mass spectra data confirm that compound 2 was formed through the
reaction of hydrazine with probe 1 (Scheme 1).
Selectivity sensing of 1 toward hydrazine
To evaluate the selectivity of probe 1 for hydrazine, various species including
environmentally and biologically relevant biomolecules, metal ions and anions were
tested. As shown in Figures 3, 4, 5, and 6, only the introduction of hydrazine to the probe
1 solution induced a significant enhancement in the absorption at 520 nm, a great color
change from colorless to pink, and a great fluorescence intensity increase at 540 nm. In
the same condition, other tested species mentioned above did not induce any UV-vis
and fluorescence spectroscopic change to the probe 1 solution, indicating that probe 1 as a
probe have high selectivity toward hydrazine over many common organic amine
molecules, biomolecules, metal ions and anions.
(Insert: Fig. 3)
(Insert: Fig. 4)
(Insert: Fig. 5)

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(Insert: Fig. 6)
Conclusion
Based on 1,8-naphthalimide, a novel colorimetric and off-on fluorescent probe for
hydrazine has been developed with high selectivity over various environmentally and
biologically relevant molecules, metal ions and anions. With colorimetric method, the
probe can detect hydrazine with a distinct color change from colorless to pink in very fast
response time (<1 min), and can detect hydrazine quantitatively in the concentration
range from 1.5×10−4 M to 3.2×10−3 M. In addition, the probe can also detect hydrazine
quantitatively in the concentration range from 1.5×10−4 M to 3.2×10−3 M with a LOD of
46.2 μM with an off-on fluorescent mode.
Acknowledgements
This work was supported by “the Fundamental Research Funds for the Central
Universities (2015XKZD08)”.

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Figure Captions
Scheme 1 Proposed sensing mechanism of probe 1 toward hydrazine.
Fig. 1 a) Absorption spectra change of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v)
upon addition of hydrazine hydrate (0.01 M, water). b) Absorption spectra response of
probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) as a linear dependence with the
concentration of hydrazine hydrate. A520 and A400 represent the absorbance at 520 nm and
400 nm respectively. c) photographs of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in
the presence of hydrazine hydrate (the concentrations from left to right are 0, 3.0×10−5 M,
9.0×10−5 M, 1.5×10−4 M, 2.1×10−4 M, 3.0×10−4 M, 4.5×10−4 M, 6.0×10−4 M, 7.5×10−4 M,
9.0×10−4 M, 1.05×10−3 M, 1.2×10−3 M, 1.35×10−3 M, 1.65×10−3 M, 1.8×10−3 M) in
daylight.
Fig. 2 a) Emission spectra change of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v)
upon addition of hydrazine hydrate (0.1 M, water). b) Emission spectra response of probe
1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) as a linear dependence with the concentration of
hydrazine hydrate. I540 represent the emission intensity at 540 nm. c) photographs of
probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in the presence of hydrazine hydrate (the
concentrations from left to right are 0, 3.0×10−5 M, 9.0×10−5 M, 1.5×10−4 M, 2.1×10−4 M,
3.0×10−4 M, 4.5×10−4 M, 6.0×10−4 M, 7.5×10−4 M, 9.0×10−4 M, 1.05×10−3 M, 1.2×10−3
M, 1.35×10−3 M, 1.5×10−3 M, 1.8×10−3 M) under a UV lamp (365 nm). The reaction time
is 24 hours.
Fig. 3 Absorption (a) and emission (b) spectra response of probe 1 (3.0×10−5 M,
DMSO/PBS = 9:1, v/v) in the presence of different organic amine molecules (1.8×10−3
M). The organic amine molecules used were methylamine, 1,2-ethylenediamine, aniline,
toluidine, trimethylamine, and 1,4-diaminobenzene. The excitation wavelength was 440 nm

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and the reaction time was 24 hours.
Fig. 4 Absorption (a) and emission (b) spectra response of probe 1 (3.0×10−5 M,
DMSO/PBS = 9:1, v/v) in the presence of different biomolecules (1.8×10−3 M). The
biomolecules used were Cys, Gly, Met, Tyr, Pro, Ser, Thr, Val, His, Glc, Trp, and Asp.
The excitation wavelength was 440 nm and the reaction time was 24 hours. photographs
of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in daylight (c) and under a UV lamp
(365 nm) (d) in the presence of 60 eq different species (From left to right: only probe 1,
hydrazine hydrate, Cys, Gly, Met, Tyr, Pro, Ser, Thr, Val, His, Glc, Trp, and Asp.
Fig. 5 (a) absorption spectra response of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v)
in the presence of different metal ions (1.8×10−3 M). The metal ions used were Ca2+, Cd2+,
Co2+, K+, Mg2+, Mn2+, Na+, Ni2+, Zn2+, Hg2+, Ag+, and Pb2+. (b) Emission spectra response
of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in the presence of different metal ions
(1.8×10−3 M). The metal ions used were Ca2+, Cd2+, Co2+, K+, Mg2+, Mn2+, Na+, Ni2+,
Zn2+, Hg2+, Ag+, Fe3+, Cu2+, and Pb2+. The reaction time was 24 hours and the excitation
wavelength was 440 nm. photographs of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in
daylight (c) and under a UV lamp (365 nm) (d) in the presence of 60 equiv. different
species (From left to right: only probe 1, hydrazine hydrate, Ca2+, Cd2+, Co2+, K+, Mg2+,
Mn2+, Na+, Ni2+, and Zn2+).
Fig. 6 Absorption (a) and emission (b) spectra response of probe 1 (3.0×10−5 M,
DMSO/PBS = 9:1, v/v) in the presence of different anions (1.8×10–3 M). The anions used
−
2−
−
−
−
−
were Br−, Cl−, NO3 , SO32−, SO4 , ClO3 , H2PO4 , HSO3 , and HSO4 . The reaction time
was 24 hours and the excitation wavelength was 440 nm. Photographs of probe 1
(3.0×10−5 M, DMSO/PBS = 9:1, v/v) in daylight (c) and under a UV lamp (365 nm) (d) in
the presence of 60 eq different species (From left to right: only probe 1, hydrazine hydrate,

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−
2−
2−
−
−
−
−
Br−, Cl−, NO3 , SO3 , SO4 , ClO3 , H2PO4 , HSO3 , and HSO4 ).

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Scheme 1 Proposed sensing mechanism of probe 1 toward hydrazine.

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Fig. 1 a) Absorption spectra change of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) upon
addition of hydrazine hydrate (0.01 M, water). b) Absorption spectra response of probe 1
(3.0×10−5 M, DMSO/PBS = 9:1, v/v) as a linear dependence with the concentration of
hydrazine hydrate. A520 and A400 represent the absorbance at 520 nm and 400 nm
respectively. c) photographs of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in the
presence of hydrazine hydrate (the concentrations from left to right are 0, 3.0×10−5 M,
9.0×10−5 M, 1.5×10−4 M, 2.1×10−4 M, 3.0×10−4 M, 4.5×10−4 M, 6.0×10−4 M, 7.5×10−4 M,
9.0×10−4 M, 1.05×10−3 M, 1.2×10−3 M, 1.35×10−3 M, 1.65×10−3 M, 1.8×10−3 M) in
daylight.

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Fig. 2 a) Emission spectra change of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) upon
addition of hydrazine hydrate (0.1 M, water) with the excitation at 440 nm. b) Emission
spectra response of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) as a linear dependence
with the concentration of hydrazine hydrate. I540 represent the emission intensity at 540
nm. c) photographs of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in the presence of
hydrazine hydrate (the concentrations from left to right are 0, 3.0×10−5 M, 9.0×10−5 M,
1.5×10−4 M, 2.1×10−4 M, 3.0×10−4 M, 4.5×10−4 M, 6.0×10−4 M, 7.5×10−4 M, 9.0×10−4 M,
1.05×10−3 M, 1.2×10−3 M, 1.35×10−3 M, 1.5×10−3 M, 1.8×10−3 M) under a UV lamp (365
nm). The reaction time is 24 hours.

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Fig. 3 Absorption (a) and emission (b) spectra response of probe 1 (3.0×10−5 M,
DMSO/PBS = 9:1, v/v) in the presence of different organic amine molecules (1.8×10−3
M). The organic amine molecules used were methylamine, 1,2-ethylenediamine, aniline,
toluidine, trimethylamine, and 1,4-diaminobenzene. The excitation wavelength was 440 nm
and the reaction time was 24 hours.

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Fig. 4 Absorption (a) and emission (b) spectra response of probe 1 (3.0×10−5 M,
DMSO/PBS = 9:1, v/v) in the presence of different biomolecules (1.8×10−3 M). The
biomolecules used were Cys, Gly, Met, Tyr, Pro, Ser, Thr, Val, His, Glc, Trp, and Asp.
The excitation wavelength was 440 nm and the reaction time was 24 hours. photographs
of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in daylight (c) and under a UV lamp (365
nm) (d) in the presence of 60 eq different species (From left to right: only probe 1,
hydrazine hydrate, Cys, Gly, Met, Tyr, Pro, Ser, Thr, Val, His, Glc, Trp, and Asp.

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Fig. 5 (a) absorption spectra response of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in
the presence of different metal ions (1.8×10−3 M). The metal ions used were Ca2+, Cd2+,
Co2+, K+, Mg2+, Mn2+, Na+, Ni2+, Zn2+ Hg2+, Ag+, and Pb2+. (b) Emission spectra response
of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in the presence of different metal ions
(1.8×10−3 M). The metal ions used were Ca2+, Cd2+, Co2+, K+, Mg2+, Mn2+, Na+, Ni2+,
Zn2+ Hg2+, Ag+, Fe3+, Cu2+, and Pb2+. The reaction time was 24 hours and the excitation
wavelength was 440 nm. photographs of probe 1 (3.0×10−5 M, DMSO/PBS = 9:1, v/v) in
daylight (c) and under a UV lamp (365 nm) (d) in the presence of 60 equiv. different
species (From left to right: only probe 1, hydrazine hydrate, Ca2+, Cd2+, Co2+, K+, Mg2+,
Mn2+, Na+, Ni2+, and Zn2+).

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Fig. 6 Absorption (a) and emission (b) spectra response of probe 1 (3.0×10−5 M,
DMSO/PBS = 9:1, v/v) in the presence of different anions (1.8×10–3 M). The anions used
−
2−
2−
−
−
−
−
were Br−, Cl−, NO3 , SO3 , SO4 , ClO3 , H2PO4 , HSO3 , and HSO4 . The reaction time
was 24 hours and the excitation wavelength was 440 nm. Photographs of probe 1
(3.0×10−5 M, DMSO/PBS = 9:1, v/v) in daylight (c) and under a UV lamp (365 nm) (d) in
the presence of 60 eq different species (From left to right: only probe 1, hydrazine hydrate,
−
2−
2−
−
−
−
−
Br−, Cl−, NO3 , SO3 , SO4 , ClO3 , H2PO4 , HSO3 , and HSO4 ).

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Graphical Abstract
Based on 1,8-naphthalimide, a novel colorimetric and off-on fluorescent probe for
hydrazine has been developed with high selectivity over various environmentally and
biologically relevant molecules, metal ions and anions.

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Highlights
►A hydrazine-responsive colorimetric and fluorescent probe has been developed.
►The probe can quantitatively detect hydrazine with great color and fluorescence change.
►The probe showed high selectivity and sensitivity toward hydrazine.