A Colorimetric and Fluorescent Indicator for Hg2+ Detection Based on Cinnamamide Group-Containing Rhodamine Derivative

Article abstract of DOI:10.1007/s10895-018-2253-0

A colorimetric and fluorescent indicator based on cinnamamide group-containing rhodamine derivative was synthesized for the detection of Hg²⁺. The rhodamine B and cinnamamide were connected via ethylenediamine as a bridging molecule through a condensation reaction to obtain a colorimetric and fluorescent indicator for the detection of Hg²⁺ in H₂O-EtOH (4:1, v/v). The indicator was excellent in the selectivity of Hg²⁺ and was almost unaffected by other common ions such as Na⁺, K⁺, Mg²⁺, Fe³⁺, Cu²⁺, Zn²⁺, Cr³⁺. The Hg²⁺-containing aqueous solution turned from colorless to red within 7?min after the addition of the indicator, and had an absorption peak at 564?nm in UV-vis, which implies a significant colorimetric phenomenon. Their characteristic peaks varied with the Hg²⁺ content, and they reached a linear relationship at low concentrations. The binding stoichiometry proved to be 1:1. The lowest detection limit was 4.1 × 10^?7?mol/L, ranging from acidic to neutral.

Full text of DOI:10.1007/s10895-018-2253-0

Journal of Fluorescence
https://doi.org/10.1007/s10895-018-2253-0
ORIGINAL ARTICLE
A Colorimetric and Fluorescent Indicator for Hg2+ Detection Based
on Cinnamamide Group-Containing Rhodamine Derivative
Zhenglong Yang1 & Kangyu Fu1 & Jing Yu1 & Peiting Zhou2 & Zhihao Cheng2
Received: 20 April 2018 /Accepted: 7 June 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A colorimetric and fluorescent indicator based on cinnamamide group-containing rhodamine derivative was synthesized for the
detection of Hg2+. The rhodamine B and cinnamamide were connected via ethylenediamine as a bridging molecule through a
condensation reaction to obtain a colorimetric and fluorescent indicator for the detection of Hg2+ in H2O-EtOH (4:1, v/v). The
indicator was excellent in the selectivity of Hg2+ and was almost unaffected by other common ions such as Na+, K+, Mg2+, Fe3+,
Cu2+, Zn2+, Cr3+. The Hg2+-containing aqueous solution turned from colorless to red within 7 min after the addition of the
indicator, and had an absorption peak at 564 nm in UV-vis, which implies a significant colorimetric phenomenon. Their charac-
teristic peaks varied with the Hg2+ content, and they reached a linear relationship at low concentrations. The binding stoichiometry
proved to be 1:1. The lowest detection limit was 4.1 × 10−7 mol/L, ranging from acidic to neutral.
2+
.
.
.
.
Keywords Rhodamine derivative Hg detection Colorimetric indicator Fluorescent indicator Aqueous solution
Introduction
In recent years, a large number of colorimetric and fluores-
cent indicators have been reported for the detection of Hg2+
[5–10]. Rhodamine B (RB) as a colorimetric and fluorescent
dye has good photostability, long excitation wavelength and
high colorimetric and fluorescence quantum yield [11, 12].
The spirocyclic structure of lactam in RB has attracted great
interest among researchers. The recognition of functional
groups can achieve synergistic effects through the reaction
of carbonyl groups with some metal ions. The fluorescence
intensity of RB changes from low to high during the opening
and closing of the lactam ring. In this way, certain metal ions
can be selectively recognized.
The reaction of suitable polyamine with benzoic acid,
phthalic anhydride, sulfosalicylic acid, phthalic acid and or-
ganic acid anhydride is based on the typical synthesis of rho-
damine derivatives Hg2+ colorimetric and fluorescent probe
[13–16]. In order to compensate for the lack of selec-
tivity of Hg2+, insufficient hydrophilicity and quantum yield
of RB, it is possible to modify the RB derivatives after the
condensation reaction. Most studies have focused on modify-
ing the main molecular structure to design colorimetric and
fluorescent probes, which focus on the detection of certain
metal ions, particularly in all aspects where Hg2+ detection
capability is prominent [17–21]. However, there are still prob-
lems with probe selectivity and sensitivity or environmental
friendliness.
Heavy-metal pollution has become a serious problem in the
world. Mercury (Hg) is derived from chemical industries such
as chloro-alkali industry and the synthesis of mercuric com-
pounds, and it has been confirmed that mercury has certain
damage to the nervous system, immune system and internal
organs [1, 2]. For this reason, scientists have provided several
Hg2+ detection methods, usually including atomic absorption
spectroscopy (AAS) [3], inductively coupled plasma mass
spectrometry (ICP-MS) [4], etc. Despite the high selectivity
and accuracy, these large devices cannot overcome the re-
quired special staff and time-consuming disadvantages.
Therefore, there is an urgent need to find a quick, simple and
economical method to detect mercury in the environment.
* Zhenglong Yang
yangzhenglong@tongji.edu.cn
1
College of Transportation Engineering, Key Laboratory of road and
TRAFFIC Engineering of Ministry of Education, Tongji University,
Shanghai 201804, China
2
Broadvision Engineering Consultants, National Engineering
Laboratory for Land Transport Meteorological Disaster Control
Technology, Kunming 650041, China

J Fluoresc
In this paper, a colorimetric and fluorescent probe based on
cinnamamide containing RB has been synthesized to detect
Hg2+. The structures, colorimetric and fluorescence capabili-
ties were characterized by 1H-NMR, UV-vis and fluo-
rescent spectra. The effects of pH value, binding mode
and external ion interference on its selectivity performance
were discussed in detail.
after the flask was cooled to room temperature. The light
brown solid was extracted with dichloromethane (30 mL)
for 3 times. The organic phase was dried over anhydrous so-
dium sulfate for 12 h, and then an orange solid can be obtained
after rotary evaporation. The RB- ethylenediamine de-
rivative (intermediate B) can be prepared by chromatog-
raphy using methanol / dichloromethane (1:10, v/v) as the
developing agent. Intermediate B was stored under dry and
dark conditions.
Experimental
Synthesis of RB - Ethylenediamine - Cinnamamide
Derivative (Probe 1)
Materials and Instruments
1H-NMR spectra were recorded on a Bruker DMX500 with
CDCl3 as the solvent and TMS as an internal standard record
under 400 MHz. FT-IR spectra were obtained on a Bruker
HYPERION 2000. The fluorescent spectra were re-
ceived from Hitachi F-2500 with an excitation wave-
length of 500 nm. UV-vis spectra were measured on a
Hitachi U-3100 at a scan speed of 300 nm/min and a range
from 500 nm to 800 nm.
Rhodamine B (AR), ethylenediamine (AR), methanol
(AR), dichloromethane (AR), cinnamic acid (AR), thionyl
chloride (AR), N,N-Dimethylformamide (AR), triethylamine
(AR), sodium bicarbonate (AR), absolute ethanol (AR), cop-
per (II) sulfate pentahydrate (AR), iron (III) chloride hexahy-
drate (AR), sodium chloride (AR), potassium nitrate (AR),
magnesium nitrate hexahydrate (AR) and zinc nitrate (AR)
were all purchased from Sinopharm Chemical Reagent Co.,
Ltd. Different stock solutions of Na+, K+, Mg2+, Fe3+, Cu2+,
Zn2+ were prepared by dissolving the corresponding salts. The
above materials were utilized without further treatment.
The standard solution of Hg2+ was purchased from
Keli Technology Company in Analysis and Test Center,
Guangzhou.
Intermediate B (2.0 g) was dissolved in ethanol (50 mL) in a
250 mL reaction flask. Subsequently, triethylamine (1.0 mL)
and sodium bicarbonate (0.5 g) were added while Intermediate
A (1.0 g) dissolved in ethanol (10 mL) was added dropwise to
the 250 mL reaction flask. After the reaction was allowed to
proceed at room temperature under a nitrogen atmosphere for
24 h, the organic solvent was evaporated, and the product was
extracted twice with dichloromethane (50 mL). The organic
phase was washed three times with water and combined.
Then, the organic phase was dried over anhydrous sodium
sulfate for 5 h (Fig. 1).
After removing anhydrous sodium sulfate by filtra-
tion, the resulting yellow product was washed for three
times with cold ethanol, and then a brown solid named Probe
1 was prepared by chromatography using methanol / dichlo-
romethane (1:15, v/v) as developing agent. The product was
stored in dry, dark and sealed conditions.
The 1H-NMR spectra of Probe 1 is given in Fig. 2: δ 7.83
(s, 1H, imine), 7.60 (s, 1H, benzene), 7.37 (t, 2H, benzene),
7.02 (s, 1H, benzene), 6.30 (d, 2H, methyne), 6.28 (m, 5H,
benzene), 6.24 (d, 4H, benzene), 3.18 (d, 8H, methylene),
2.87 (dd, 4H, methylene), 2.32 (s, 2H, benzene), 1.12(t,
12H, methyl).
Synthesis of Cinnamoyl Chloride (Intermediate a)
The multiple states appearing at 2.87 correspond to the
hydrogen n, o on the methylene of ethylenediamine, while
the multiple peaks at 7.83 correspond to hydrogen a
on the imine group, and the ratio of hydrogen to methyl
is 1:12. It shows that RB has reacted with ethylenediamine,
and the terminal amino group has then reacted with
cinnamoyl chloride, which confirms the structure of the RB
derivative.
The FT-IR spectra of Probe 1 are shown in Fig. 3(a). The
characteristic absorption peaks of RB can be clearly observed
at wavenumbers of 1600 cm−1 and 1250 cm−1. Typically, the
characteristic absorption peaks of the diamines are generally
around 3300 cm−1, 2900 cm−1 and 900 cm−1. However, due to
the large number and high intensity of the absorption peaks of
the rhodamine itself, the diamine’s peaks have been covered to
some extent. Therefore, another characterization should be
performed to prove the generation of Probe 1.
Cinnamic acid (1 g) was dissolved in thionyl chloride (10 mL)
in a 50 mL reaction flask. Subsequently, DMF (0.1 mL) was
added dropwise to the solution. The mixture was heated to
flux for 2 h and then thionyl chloride was removed by vacuum
distillation. A light yellow powder was obtained and named as
cinnamoyl chloride (Intermediate A). The product was sealed
and stored under dry and cool condition.
Synthesis of RB - Ethylenediamine Derivative
(Intermediate B)
RB (5 g) was dissolved in ethanol (50 mL) in a 250 mL reac-
tion flask. Ethylenediamine (10 mL) was dropped into the
solution with stirring. Under nitrogen protection, the reaction
was carried at 75–85 °C for 4 h. The solvent was removed

J Fluoresc
Fig. 1 Synthesis routine of (a)
Intermediate A; (b) Intermediate;
(c) Probe 1
The fluorescent spectra of Probe 1 and RB are given
in Fig. 3(b). Compared to RB, Probe 1 does not show a fluo-
rescent peak at 580 nm in the excitation wavelength.
Probe 1 did not fluoresce under irradiation with a fluo-
rescent lamp, indicating that a lactam structure was generated
in Probe 1.
metal ion was 0.5 mmol/L). At the same time, Intermediate B
was also dropped into the above solution with various ions for
comparative experiments. From the results, there was no sig-
nificant change in color or fluorescence after Intermediate B
was mixed with common metal ions. When Probe 1 was mixed
with Hg2+, the solution containing Hg2+ turned intense pink.
However, the rest did not show any significant changes. The
result is shown in Fig. 4(a).
Results and Discussions
By naked-eye observation, we also performed UV-vis
spectra analysis using all of the above samples to determine
if each metal ion had a combination effect with Probe 1. The
results are given in Fig. 4(b), and it was found that in the Hg2+-
containing solution, the peak intensity of Probe 1 at 564 nm
increased significantly, which represents a strong connection
between Probe 1 and Hg2+. In other samples, although the
High Selectivity for Hg2+
To test whether Probe 1 has any response to external ions, it is
added to a standard solution with ordinary metal ions (Na+, K+,
Mg2+, Fe3+, Cu2+, Zn2+, Cr3+, Hg2+, the concentration of each
Fig. 2 1H-NMR spectra of
Probe 1

J Fluoresc
Fig. 3 a FT-IR and (b)
Fluorescent spectra of Probe 1
peak intensity at 564 nm also increased, their peak intensity
wad 90% lower than that generated by Hg2+. It can reflect the
results of the color, while those containing Hg2+ can be easily
identified by observation. The UV-vis results indicate that the
strong combination between Probe 1 and Hg2+ allows Probe 1
to be used for sensitive Hg2+ detection without interference
from other common metal ions.
are shown in Fig. 5(a), from which we can learn that the
highest intensity reached 7 min after the addition of Probe 1,
and then there was a little change. This proves that Probe 1 has
a relatively long detecting time and is suitable for non-real-
time monitor.
As for pH response, 10 mL of Probe 1 and 10 parts each
containing 2 mL Hg2+ solution with different pH values
(pH = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) were mixed and the
fluorescence intensity was measured after 10 min. From Fig.
5(b), it can be concluded that when the pH of the system is in
the range of 2 to 7, the solution has better absorption intensity
than the sample with pH greater than 8. This means that Probe
1 has a predominant binding nature to Hg2+ under neutral and
acidic conditions, mainly because Hg2+ is not stable under
alkaline conditions. The above results indicate that the
Time and pH Response
In order to measure the sensitivity of Probe 1, 0.5 mL of a
solution in a certain concentration of Hg2+ was prepared and
mixed with 10 mL of Probe 1 by ultrasonic vibration. The
intensity of the characteristic UV-vis peak was measured at
0–10 min intervals after the addition of Probe 1. The results

J Fluoresc
Fig. 4 a Photograph of Probe 1
(0.1 mmol/L) mixed in
H2O-EtOH (4:1, v/v, 0.1 mmol),
b UV-vis spectra of Probe 1 with
A-H, and (c) UV-vis spectra of
Probe 1 only with Hg2+. A-H:
Na+, K+, Mg2+, Fe3+, Cu2+
,
Zn2+, Cr3+, Hg2+
ꢀ
ꢁ
detection of Hg2+ by Probe 1 is suitable for acidic or neutral
conditions.
1
a
1
¼
∙
þ 1
ð1Þ
A−A0 a−b K½MŠ
Binding Form
In Formula (1), K represents the complexation constant
between the fluorescent probe and the metal ion; A0 represents
the UV adsorption of Probe 1 without the metal ion, and A
represents the UV adsorption of Probe 1 with the metal ion.
[M] represents the concentration of added metal ion, where a
The binding stoichiometry of Probe 1 with Hg2+ can be stud-
ied using the Benesi-Hildebrand equation and Job’s plot
model [22–24].

J Fluoresc
Fig. 6 Fluorescent intensity of Probe 1 vs. Hg2+ concentration in H2O-
EtOH (4:1, v/v) and the proposed binding form
that with the increase of Hg2+, the absorbance at 564 nm be-
comes stronger due to the ring-opening effect of Hg2+ in Probe
1 caused by the lactam structure. It means the combination of
Probe 1 and Hg2+. The specific data is given in Table 1. In
order to determine the stoichiometry, several combination
models were tried. Finally, 1:1 in stoichiometry was accepted
for the fitting. From the data in Table 1, it can be seen that the
fitted line is very close to the linear regression equation, and
the correlation coefficient is 0.99971.
The fitted curve of the fluorescence intensity of Probe 1 and
Hg2+ is shown in Fig. 6. The total concentration of Probe 1
and Hg2+ was maintained at 2.0 × 10−4 mol/L. It can be seen
that when the concentration of Hg2+ is equal to 0.5 equiv.,
there is a maximum fluorescence intensity at 564 nm, which
also indicates that the binding form of Hg2+ and Probe 1 fol-
lows a stoichiometric ratio of 1:1. The proposed binding form
is also given.
Fig. 5 a UV-Vis spectra and (b) fluorescent intensity of Probe 1 with Hg2+
and b are constants. The complexation constant can be calcu-
lated by fitting a curve from 1/(A-A0) to 1/[M].
Probe 1 was added to a solution containing various con-
centrations of Hg2+. As shown in Fig. 4(c), it can be observed
Table 1 The absorbance of Probe 1 (0.1 mmol/L) with Hg2+ (0.5–4
equiv.) in solution (H2O-EtOH, 4:1, v/v)
No.
Relative content of Hg2+
Maximum absorption intensity
1
0.05
0.1
0.2
0.3
0.4
0.5
0.8
1
0.007236
0.014652
0.028896
0.041004
0.058068
0.073116
0.096324
0.140592
0.220092
0.279972
0.351156
0.407820
0.495216
0.561852
2
3
4
5
6
7
8
9
1.5
2
10
11
12
13
14
2.5
3
Fig. 7 UV-vis absorbance of Probe 1 mixed with various metal ions (Na+,
K+, Mg2+, Fe3+, Cu2+, Zn2+, Cr3+) in H2O-EtOH (4:1, v/v) (wavelength:
564 nm)
3.5
4

J Fluoresc
4. Rahaman GMM, Fahrenholz T, Kingston HM (2009) J. Anal. At.
Spectromery 24:83–92
Detection Limit and Anti-Interference
5. Shan F, Lai J, Sun H, Zhang P, Luo C, He Y, Feng H (2016) A
facile, fast responsive and highly selective mercury(ii) probe char-
acterized by the fluorescence quenching of 2,9-dimethyl-1,10-
phenanthroline and two new metal–organic frameworks. RSC
Adv 6:66215–66223
6. Zhang Y, Li W, Wang Q, Zhang R, Xiong Q, Shen X, Guo J, Chen
X (2013) A Benzoxazole-Based Fluorescent Probe for Visual
Detection of Hg2+. Acta Chim Sin 71:1496–1499
To find the detection limit (DL) of Probe 1, the excess Probe 1
was added to the solution with low concentration of Hg2+.
According to the formula DL = 3S/ρ [17], where S represents
the standard deviation of the blank test, ρ represents the slope
of the line (ratio of relative intensity to sample concentration),
and the detection limit can be calculated to be 4.13 × 10−7 mol/
L, which means that Probe 1 is still valid in very low concen-
trations of Hg2+ treated water samples.
7. Zhang X, Huang X, Zhu Z (2013) A reversible Hg(ii)-selective
fluorescent chemosensor based on a thioether linked bis-rhoda-
mine. RSC Adv 3:24891–24895
The specific procedure for determining whether other metal
ions interfere with the detection of Probe 1 is as follows: 8 parts
(2 mL each) solution (H2O-EtOH, 4:1, v/v) were mixed with
Hg2+ (1 mmol/L) with X (1 mmol/L, X can be Na+, K+, Mg2+,
Fe3+, Cu2+, Zn2+, Cr3+, respectively), respectively, then Probe 1
(2 mL, 0.1 mmol/L) was added to the solution. Their absorbance
in the UV-vis spectra after 10 min were compared. As shown in
Fig. 7, it can be inferred that the absorbance becomes stronger in
the presence of other metal ions. All samples containing Hg2+
and foreign ions have stronger absorption than samples contain-
ing only foreign ions, indicating that Probe 1 can be used to
detect Hg2+ in samples containing various other ions.
8. Huang J, Gao X, Jia J, Kim J, Li Z (2014) Graphene Oxide-Based
Amplified Fluorescent Biosensor for Hg2+Detection through
Hybridization Chain Reactions. Anal Chem 86:3209–3215
9. Zhu X, Yuan Y, Li L, Du Y, Li F (2017) Identification of interfacial
transition zone in asphalt concrete based on nano-scale metrology
techniques. Mater Des 129:91–102
10. Zhao F, Qian J, Quan F, Wu C, Zheng Y, Zhou L (2017) Aconitic
acid derived carbon dots as recyclable Bon–off–on^ fluorescent
nanoprobes for sensitive detection of mercury(ii) ions, cysteine
and cellular imaging. RSC Adv 7:44178–44185
11. Fang Q, Liu Q, Song X (2015) An aqueous fluorescent probe for
Hg2+detection with high selectivity and sensitivity. Luminescence
30:1280–1284
12. Zhang J, Qiu T, Yuan H, Chin XL (2013) The synthesis of Ag-
polypyrrole nanocomposite coated latex particles and their applica-
tion as a fluorescent quenching agent. J Polym Sci 31:434–443
13. Lin W, Yuan L, Cao X, Tan W, Feng Y (2008) A Coumarin-Based
Chromogenic Sensor for Transition-Metal Ions Showing Ion-
Dependent Bathochromic Shift. Eur J Org Chem 2008:4981–4987
14. Köhn F, Hofkens J, De Schryver FC (2000) Emission of the contact
ion pair of rhodamine dyes observed by single molecule spectros-
copy. Chem Phys Lett 321:372–378
15. Sun D, Lu T, Xiao F, Zhu X, Sun G (2017) Formulation and aging
resistance of modified bio-asphalt containing high percentage of
waste cooking oil residues. J Clean Prod 161:1203–1214
16. Renge I (2001) A Model of Inhomogeneous Broadening and
Pressure Induced Hole Shifts in the Optical Spectra of Organic
Chromophores in Glasses. J Phys Chem A 105:9094–9103
17. Pevenage D, Auweraer M, Schryver F (1999) Influence of the
Molecular Structure on the Lateral Distribution of Xanthene Dyes
in Langmuir−Blodgett Films. Langmuir 15:8465–8473
18. Zhang D, Li M, Jiang Y, Wang C, Wang Z, Ye Y, Zhao Y (2013) A
new sensitive and selective chromogenic and fluorescent
chemodosimeter for Hg(II) in aqueous media and its application
in live cell imaging. Dyes Pigments 99:607–612
Conclusions
In summary, we have synthesized a colorimetric and fluores-
cent indicator based on rhodamine derivatives containing
cinnamamide group that can be used to detect Hg2+ in aqueous
solutions. The detection limit of this indicator is 4.13 ×
10−7 mol/L, which can respond within 7 min. It has excellent
anti-interference performance to other common ions (Na+, K+,
Mg2+, Fe3+, Cu2+, Zn2+, Cr3+) in H2O-EtOH solution (1/4, v/v).
We assume that these studies on RB- ethylenediamine -
cinnamamide derivatives provide a promising prospect for the
detection of Hg2+ in aqueous solutions.
Acknowledgements This study was funded by National Natural Science
Foundation of China (No. 51622805 and U1633116) and the opening fund
for the subject of Transportation Engineering in Tongji University
(2016 J012306). The authors are grateful to these financial supports. The
authors declare that they have no conflict of interest.
19. Sun D, Sun G, Zhu X, Ye F, Xu J (2018) Intrinsic temperature
sensitive self-healing character of asphalt binders based on molec-
ular dynamics simulations. Fuel 211:609–620
20. Hu J, Hu Z, Cui Y, Zhang X, Gao H, Uvdal K (2014) Sensor Actuat.
B-Chem 203:452–458
21. Tong S, Zhang L, Bing L, Yi L (2014) Sensor Actuat. B-Chem 203:
157–164
References
22. Mahato P, Saha S, Suresh E, Liddo RD, Parnigotto PP, Conconi
MT, Kesharwani MK, Ganguly B, Das A (2012) Ratiometric
Detection of Cr3+and Hg2+by a Naphthalimide-Rhodamine
Based Fluorescent Probe. Inorg Chem 51:1769–1777
23. Liu Y, Zhao XL, Chen M, Liu J, Wang P, Guo W (2012) A
naphthalimide–rhodamine ratiometric fluorescent probe for Hg2+
based on fluorescence resonance energy transfer. Dyes Pigments
92:909–915
1. Kaur N, Kumar S (2011) Colorimetric metal ion sensors.
Tetrahedron 67:9233–9264
2. Kuar K, Saini R, Kumar A, Luxami V, Kaur N, Singh P, Kumar S
(2012) Coordination Chem. Rev 256:1992–2028
3. Ghaedi M, Fathi MR, Shokrollahi A, Shajarat F (2006) Highly
Selective and Sensitive Preconcentration of Mercury Ion and
Determination by Cold Vapor Atomic Absorption Spectroscopy.
Anal Lett 39:1171–1185
24. Sun Y, Liu Y, Guo W (2009) Sensor Actuat. B-Chem 143:171–176