Fluorescence “Turn On–Off” Sensing of Copper (II) Ions Utilizing Coumarin–Based Chemosensor: Experimental Study, Theoretical Calculation, Mineral and Drinking Water Analysis

Article abstract of DOI:10.1007/s10895-020-02503-4

Herein, we report the preparation of a fluorescent sensor based on coumarin derivative for copper (II) ion sensing in CH₃CN/HEPES media. 6,7–dihydroxy–3–(4–(trifluoro)methylphenyl)coumarin (HMAC) sensor was fabricated and analyzed by spectroscopic techniques. The sensor demonstrates “turn on–off” fluorescence quenching in the presence of copper (II) ions at 458?nm. A clear complex between the chemosensor HMAC and copper (II) ions was characterized by ESI–MS as well as the Job’s method. Also, the limit of detection (LOD, 3σ/k) value was determined as 24.5?nM in CH₃CN/HEPES (95/5, v/v) buffer media (pH = 7.0). This value is lower than the admissible level of copper (II) ions in drinking water (maximum 31.5?μM) reported by EU Water Framework Directive (WFD) and World Health Organization (WHO) guidelines. The theoretical calculations (density functional theory, DFT) have been performed for the geometric optimized structures. As a final stage, real sample analyses have successfully been performed by using HMAC, as well as ICP–OES method. The relative standard deviation for copper (II) in mineral and drinking water samples has been determined to be below 0.15% and recovery values are in the range of 95.48–109.20%.

Full text of DOI:10.1007/s10895-020-02503-4

Journal of Fluorescence
https://doi.org/10.1007/s10895-020-02503-4
ORIGINAL ARTICLE
Fluorescence “Turn On–Off” Sensing of Copper (II) Ions Utilizing
Coumarin–Based Chemosensor: Experimental Study, Theoretical
Calculation, Mineral and Drinking Water Analysis
Fatma Nur Arslan1,2 & Gonul Akin Geyik1 & Kenan Koran3 & Furkan Ozen4 & Duygu Aydin1 & Şükriye Nihan Karuk Elmas1
Ahmet Orhan Gorgulu3 & Ibrahim Yilmaz1
&
Received: 13 January 2020 /Accepted: 27 January 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Herein, we report the preparation of a fluorescent sensor based on coumarin derivative for copper (II) ion sensing in CH3CN/
HEPES media. 6,7–dihydroxy–3–(4–(trifluoro)methylphenyl)coumarin (HMAC) sensor was fabricated and analyzed by spec-
troscopic techniques. The sensor demonstrates “turn on–off” fluorescence quenching in the presence of copper (II) ions at 458 nm.
A clear complex between the chemosensor HMAC and copper (II) ions was characterized by ESI–MS as well as the Job’s
method. Also, the limit of detection (LOD, 3σ/k) value was determined as 24.5 nM in CH3CN/HEPES (95/5, v/v) buffer media
(pH = 7.0). This value is lower than the admissible level of copper (II) ions in drinking water (maximum 31.5 μM) reported by EU
Water Framework Directive (WFD) and World Health Organization (WHO) guidelines. The theoretical calculations (density
functional theory, DFT) have been performed for the geometric optimized structures. As a final stage, real sample analyses have
successfully been performed by using HMAC, as well as ICP–OES method. The relative standard deviation for copper (II) in
mineral and drinking water samples has been determined to be below 0.15% and recovery values are in the range of 95.48–
109.20%.
.
.
.
.
.
Keywords Fluorescence sensor Copper Coumarin Drinking water Mineral water DFT
Introduction
many fundamental physiological and biological processes, for
instance, mitochondrial electron transport, enzyme functions,
producing red blood cells, acting as a catalyst for the production
of melanin, and also serving to be neurotransmitters [1–3]. The
Copper ion is the most abundant d–block metal ion in the
human body behind iron and zinc ions. It is a crucial agent in
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-020-02503-4) contains supplementary
material, which is available to authorized users.
* Ibrahim Yilmaz
Şükriye Nihan Karuk Elmas
snihankaruk@gmail.com
iyilmaz@kmu.edu.tr
Fatma Nur Arslan
arslanfatmanur@gmail.com
Ahmet Orhan Gorgulu
aogorgulu@firat.edu.tr
Gonul Akin Geyik
gnlakin@gmail.com
1
Department of Chemistry, Kamil Ozdag Science Faculty,
Karamanoglu Mehmetbey University, 70100 Karaman, Turkey
Kenan Koran
kenan.koran@gmail.com
2
Van’t Hoff Institute for Molecular Sciences, Analytical–Chemistry
Group, University of Amsterdam, Amsterdam, Netherlands
3
Furkan Ozen
furkanozen@akdeniz.edu.tr
Department of Chemistry, Firat University, Science Faculty,
23119 Elazıg, Turkey
4
Duygu Aydin
Department of Mathematics and Science, Akdeniz University,
duyguaydin@kmu.edu.tr
Faculty of Education, 07058 Antalya, Turkey

J Fluoresc
human body may intake copper ions through food, drinking
water, environmental and biological sources, as well as through
skin contact to copper containing compounds. However, main-
taining the copper level within a particular range below toxic
concentrations is a challenge [4–6]. It is well known that high
level of copper ions in human body causes the disturbance of
cellular homeostasis leading to oxidative stress and disorders
such as brain and neurodegenerative diseases such as
Parkinson’s and Alzheimer’s diseases, and also leads to liver
or kidney damages [1–10]. For drinking water samples, the
maximum admissible level of copper ions is 2 mg.L−1 accord-
ing to the WHO and EU–WFD [4, 6–10]. Since copper ions
can accumulate into the human body through drinking water,
the detection of its levels in mineral or drinking water samples
is vital to control its impact on the human health.
chemosensors based on coumarin derivatives for copper (II)
ion sensing with high accuracy and recovery values is notice-
ably urgent.
In an effort to contribute towards the above issues, a
new 6,7–dihydroxy–3–(4–(trifluoro)methylphenyl)coumarin
(HMAC) sensor based molecular system is fabricated for
the sensitive and selective sensing of copper (II) ions.
Coumarin has been employed to be a fluorophore due to
its high fluorescence quantum yield, high chemical stabil-
ity, large Stoke’s shift and moderately high water solubil-
ity. Besides, coumarin derivatives are less toxic to envi-
ronment and the livings [31–33]. Herein, we reported the
preparation of a fluorescent “turn on–off” chemosensor
based on coumarin derivative for copper (II) monitoring
in mineral and drinking waters with a quite simple oper-
ation. Firstly, HMAC fluorescent sensor was synthesized
and then successfully characterized by 13C–NMR, 1H–
NMR and FT–IR techniques. Afterwards, it was evaluated
for the selective determination of copper (II) ions in
CH3CN/HEPES media (95/5, v/v) at pH = 7.0. The theo-
retical calculations, DFT, were also carried out for the
geometric optimized structures. In the last part of study,
the sensor HMAC was applied for the various real min-
eral and drinking water samples. Fabricated sensor
HMAC presents low detection limits and shows a good
performance for a series of mineral and drinking water.
Although a wide range of traditional methodologies for
example inductively coupled mass atomic emission spectrom-
etry (ICM–AES) [11], plasmon resonance sensor [12], graph-
ite flame atomic absorption spectrometry (AAS) [4, 13], elec-
trochemical assays [14], Rayleigh scattering spectroscopy
(RSS, plasmon–resonance) [15], inductively coupled plasma
optical emission spectroscopy (ICP–OES) [16], and etc. have
been employed to monitor copper (II) ions, as a consequence,
fluorescence chemosensors based techniques remain attrac-
tive, because of their high efficiency, operational simplicity,
sensitive detection and non–destructiveness [1, 2, 4–7, 9, 10,
17]. Recently, thousands of investigations have been placed
on the development of novel, highly selective and sensitive
fluorescent chemosensors for the determination of copper ions
[18–27]. For this purpose, various organic molecules such as
rhodamine, BODIPY, coumarin, fluorescein and cyanine are
widely used to monitor copper (II) ions in different media
[20]. Among these organic molecules, fluorescent sensors
based on coumarin derivative in the literature offer the selec-
tive and sensitive monitoring of copper (II) ions as well as
high accuracy. However, the studies based on the fluorescent
chemosensor based on coumarin derivative for copper (II)
monitoring in real drinking and water samples are quite lim-
ited. For example, a fluorescent chemosensor based on cou-
marin derivative for the efficient monitoring of copper (II)
ions in wine samples was successfully developed by Wu and
coworkers [28], and the limit of detection was found as
62 nM. In another study, Yan and coworkers designed a novel
FRET ratiometric sensor for the monitoring of copper (II) ion
in tap and lake water samples with satisfactory recovery ratios,
and the limit of detection was found as 210 nM [29]. Duan and
coworkers presented a novel fluorescent chemosensor based
on coumarin derivative for sensing copper (II) ion present in
deionized water samples. The detection limit was measured to
be 52 nM [30]. However, detection limits in the mentioned
studies are still above the legal acceptance values reported by
WHO and EU–WFD guidelines for drinking water samples
(31.5 μM). Hence, the fabrication of novel fluorescent
Materials and Methods
Chemicals and Instruments
All chemicals were of analytical reagent grade and pur-
chased from VWR international (Poole, UK), Sigma–
Aldrich (Lyon, France) and Merck (Darmstadt,
Germany) chemical companies. Perchlorate salts of differ-
ent metal ions (Cu2+, Zn2+, Cd2+, Ca2+, Sr2+, Ba2+, Mg2+,
Mn2+, Fe2+, Fe3+, Co2+, Na+, Hg2+, Pb2+and Al3+) were
used. Ultrapure water was supplied by Millipore water
1
purification system (Millipore Corp., Billerica, MA). H
and 13C NMR spectra were obtained on a Bruker DPX
400 MHz spectrometer. A spectrum 100 FT–IR spectrom-
eter (Perkin Elmer Inc., Wellesley, MA) was employed to
measure the FT–IR spectra of compounds. Fluorescence
spectra were recorded on a Varian Cary Eclipse fluores-
cence spectrophotometer (Agilent Technologies Inc.,
Santa Clara, CA, USA). A mass spectrum was obtained
on a Bruker Mass Spectrometer (Daltonics Microflex)
equipped with an ESI source. An inductively coupled
plasma–optical emission spectroscopy (ICP–OES,
Agilent 720) system (Agilent Technologies, Wilmington,
USA) equipped with an axial plasma torch was used.

J Fluoresc
Preparation of the Chemosensor HMAC
Procedure of Copper (II) Ion Determination
by Fluorescence Spectroscopy
T h e c o m p o u n d s 6 , 7 – d i h y d r o x y – 3 – ( 3 –
methylphenyl)coumarin (probe HMAC) and 2–(2,4,5–
trimethoxyphenyl)–1–(3–(methylphenyl))acrylonitrile
(HMAN) were fabricated and purified according to the liter-
ature [17, 34, 35]. The procedures are given in supporting
information file.
Following the centrifugation and filtration procedures, the
spectroscopic measurements were carried out by the following
procedure. Briefly, 5.0 μM of ligand (probe HMAC) solution
was freshly prepared in 95/5, v/v, CH3CN/HEPES buffer me-
dia (pH = 7.0) before analysis. For each experiment, 3 mL of
the ligand solution was added to fluorescence cuvette and
spectra were recorded. Then, 15 μL of real sample was added
to this solution and it was mixed for a few seconds.
Fluorescence spectra of the mixed solution (probe HMAC +
Fluorescence Studies of Probe HMAC
The stock solution of HMAC (10 mM) was prepared in
CH3CN and diluted to appropriate concentration (5.0 μM).
The emission intensities of the probe HMAC system
(5.0 μM) were performed in CH3CN/HEPES (95/5, v/v) at
pH = 7.0 buffer media, and the excitation wavelength was
364 nm. 1.10−3 M of perchlorate salts of the metal ions was
prepared in ultrapure water. The excitation and emission
wavelengths were adjusted as 364 nm and 458 nm (slit width
5 nm), respectively.
real sample) were taken again at room temperature (λexc
=
364 nm, λemm = 458 nm with 5 nm of slit size). Also, to eval-
uate the method accuracy, standard addition method was per-
formed by adding 15 μL of copper (II) solutions (0.1 μM and
0.2 μM) into the mixed solution.
Procedure of Copper (II) Ion Determination
by ICP–OES
The fluorescence titration experiments were carried out
triplicate at 458 nm, and the LOD value of the chemosensor
HMAC was calculated using the 3σ/k equation. (σ = standard
deviation of blank solution, k = the slope of the calibration
curve between the amount of copper (II) and fluorescence
intensity of chemosensor HMAC). The interference behav-
iours were tested by excitation at 364 nm, in the presence of
various metal ions [10–fold excess solutions,
M(ClO4)nxH2O)] and copper (II) ion.
Before the ICP–OES analyses, the pre–treatment procedure
for real samples was done according to the method proposed
by Mendil et al. [41]. Briefly, the mineral and drinking water
samples were acidified with 1% of nitric acid and the solutions
were adjusted to pH = 9 by NH3/NH4Cl buffer solution. They
were kept approximately 10 min and then the solutions were
centrifuged at 8.000 rpm for 15 min. The upper layers were
suspended and the precipitates were dissolved with 0.5 mL of
concentrated nicric acid. Finally, the samples were diluted to
2.5 mL of HPLC grade water and analyzed by ICP–OES in
triplicate. The operating parameters of system were given in
Table S1. The measurement results were reported as mean
value SD.
Theoretical DFT Calculation
The calculations were performed using the Gaussian 09 soft-
ware (Gaussian, Inc., Wallingford CT) with B3LYP and 6–
31 g(d) basis sets [36–40]. The theoretical calculations were
carried out for the geometrically optimized structures, the en-
ergy levels (HOMO and LUMO) of the probe HMAC and
complex of the HMAC–Cu2+.
Results and Discussion
Fabrication and Characterization of HMAN and Probe
HMAC
Sensing of Copper (II) Ions in Real Samples
Synthesis and purification of HMAN were done according to
previous literatures [17, 34, 35]. After filtering the synthesized
phenylacrylonitrile compound, it was washed with ultrapure
water until the neutral pH. HMAC was synthesized in a non–
solvent silica gel condition under microwave irradiation at
high yield in the presence of HMAN with pyridinium hydro-
chloride, and column chromatography was chosen to purify
the crude product. The general procedure for synthesis is pre-
sented in Scheme 1.
To demonstrate the applicability of the probe HMAC in real
samples, various mineral and drinking water samples were
analyzed. Samples were obtained from local markets
(Karaman city, Turkey). As an initial step, the waters were
degassed to remove the carbonate present in the samples;
and then they were put on an ultrasonic bath and sonicated
for 15 min. Afterwards, tested samples were centrifuged at
8.000 rpm for 10 min, and then filtered using a 0.45.0 μM
pore–size membrane filter to get rid of suspended species or
contamination.
Silica gel was used as a solid support for the synthesis
step of HMAC. This support material reduced formation
of side products and has been effective for obtaining the

J Fluoresc
Scheme 1 Synthetic scheme of
HMAN and HMAC
main products with high yields. Pyridinium hydrochloride
was used to transform hydroxyl groups of methoxy
groups in the compound HMAC. Therefore, the lactone
ring was formed and resulted in a changing from the hy-
spectrum of the compound HMAC. Also, the disappear-
ance of methoxy and formation of –OH stretching vibra-
tions are clearly monitored at 3190 and 3412 cm−1 in the
HMAC spectrum. The lactone ring of the coumarin com-
pound has specific –C=O stretching vibrations monitored
at 1661 cm−1. The disappearance of methoxy protons and
formation of –OH proton peaks prove that there is a clear
product formation. All of the characterization data is giv-
en in Fig. S1 to S7.
1
droxyl groups to nitrile carbon. FT–IR, H–, 13C– and
APT–NMR techniques were used to confirm the structure
of synthesized compounds. When comparing the NMR
and FT–IR spectra of HMAN and HMAC, it is easily
seen that the –C ≡ N peaks of HMAN do not exist at
Fig. 1 (a) Fluorescence intensity changes of probe HMAC (5.0 μM) in
the presence of various cations (50 μM) in ACN/HEPES buffer solution
(95/5, v/v, pH = 7.0), (b) Changes in the fluorescence intensity ratio (Io/I)
of probe HMAC in the presence of various metal ions in ACN/HEPES
buffer solution (95/5, v/v, pH = 7.0) (λemm = 458 nm, λexc = 364 nm)

J Fluoresc
Fig. 2 (a) Selectivity of the HMAC–Cu2+ sensor over the different metal
ions in ACN/HEPES buffer solution (95/5, v/v, 5.0 μM, pH = 7.0) (b) the
fluorescence spectra of HMAC with the increasing concentrations of
copper (II) in ACN/HEPES buffer solution (95/5, v/v, 5.0 μM, pH =
7.0) (λemm = 458 nm, λexc = 364 nm)
Fluorescence Studies
obtained results were presented in Fig. S8. In the presence of
copper, an increase in the absorbance intensity of the sensor
HMAC at 230 nm was observed, while the peak at 370 nm
was shifted to 300 nm, in other words towards the blue wave-
length. Thus, chemosensor HMAC demonstrated a high se-
lectivity for copper (II) ions over tested metal ions.
To study the impact of the other metal cations on the rec-
ognition of probe HMAC system to copper (II) (10.0 eq.),
each of the other metal cations (10.0 eq.) was added into the
probe HMAC–copper (II) solution, respectively (Fig. 2a).
The selectivity studies of probe HMAC (5.0 μM) were per-
formed at 458 nm in CH3CN/HEPES buffer media (95/5, v/v)
at pH = 7.0. As depicted in Fig. 2a, the fluorescence emission
Selectivity is one of the most important performance parame-
ters for fluorescent sensors. In this study, the ability of the
chemosensor HMAC for the detection of copper (II) in the
existence of 10–fold excess of various cations (Mg2+, Mn2+,
Ca2+, Sr2+, Co2+, Ba2+, Fe2+, Hg2+, Cu2+, Zn2+, Fe2+, Pb2+,
Cd2+, Al3+ and Na+) was evaluated. As shown in Fig. 1, when
these ions were added into the HMAC solution, only copper
(II) ions result in a significant fluorescence quenching at
458 nm. None of the other metal cations generated obvious
impact on fluorescence intensities. In addition to fluorescence
emission study, UV–Visible experiments were performed, and
Fig. 3 (a) The plot of emission intensities of probe HMAC versus various Cu2+ concentrations, (b) Benesi–Hildebrand plot of 1/(I–I0) vs 1/[Cu2+] based
on 1:1 association stoichiometry between probe HMAC–Cu2+ complex (λemm = 458 nm, λexc = 364 nm)

J Fluoresc
Fig. 4 ESI–MS spectrum of
probe HMAC–Cu2+ complex
intensity of probe HMAC–copper (II) solution changed only
a little when adding the tested cations, which proposed that the
recognition process of copper (II) was scarcely influenced by
tested cations. Consequently, probe HMAC system had high
selectivity on monitoring copper (II) ion. To obtain quantita-
tive relation of probe HMAC (5.0 μM) solution on sensing
copper (II) ion, the fluorescence titration experiments in
CH3CN/HEPES buffer media (95/5, v/v) at pH = 7.0 were
performed (Fig. 2b). As can be seen from Fig. 2b, the fluores-
cence emission intensity at 458 nm gradually decreased with
the copper (II) concentration (0 to 50 μM) increased. After the
addition of 2.0 equivalent of copper (II) ion, quenching of the
fluorescence intensity reached a constant value. This
quenching which resulted from the formation of the
HMAC–Cu2+ complex, may be attributed to the paramagnetic
effect and heavy atom effect of transition metals [42]. Hence,
the probe HMAC could be employed as a “turn–on–off” fluo-
rescent chemosensor for sensing copper (II) ions.
to be 24.5 nM on the basis of 3σ/k and the linearity range was
so extensive. The LOD value of probe HMAC enables the
copper (II) ion monitoring in nanomolar levels, and therefore
it is lower than the acceptable limit of the drinking water
guidelines of WHO and EU–WFD (31.5 μM) [4, 6–10].
Furthermore, the correlation constant value was calculated to
be 9.00 × 104 M−1 using the Benesi–Hildebrand equation
based on the data of fluorescence titration (Fig. 3b). Thus,
these results showed that the probe HMAC system had good
sensitivity on copper (II), and the standard curve with good
linearity (R2 = 0.9916) could be used to quantify.
During detecting copper (II) ion with probe HMAC system
as fluorescent probe, fluorescence quenching occurred, which
meant copper (II) ion interacted with probe HMAC system. To
investigate the structure of HMAC–Cu2+ complex, ESI–MS
spectra were obtained (Fig. 4). The formation of a complex
between HMAC–Cu2+ (Scheme 2) was verified by the ESI–
MS technique. As can be seen from MS spectrum, the peak at
m/z = 405.8 corresponds to the [HMAC+ Cu + Na+] was in-
dicated 1:1 ratio in Fig. 4.
Based on the fluorescence titration experiments, the cali-
bration curve of the quantitative correlation between the emis-
sion value and metal concentration was y = −3.56 × 107x +
366.38 (R2 = 0.9916) (Fig. 3a). The detection limit of the
chemosensor HMAC for copper (II) ion sensing was found
Also, to detect the stoichiometric ratio of copper (II) ion
and probe HMAC in HMAC–Cu2+ complex, Job’s graph was
plotted based on fluorescence titration data (Fig. 5). As shown
Scheme 2 The proposed binding
mechanism of probe HMAC and
copper (II) complex in ACN/
HEPES buffer solution (95/5, v/v,
5.0 μM, pH = 7.0)

J Fluoresc
Fig. 5 Job plots of HMAC–Cu2+
complex in ACN/HEPES buffer
solution (95/5, v/v, 5.0 μM, pH =
7.0) (λemm = 458 nm, λexc
=
364 nm)
in Fig. 5, the stoichiometry of the HMAC–Cu2+ complex was
found to be 1:1 ratio, due to the maximum of relative intensity
was about 0.5 value.
with EDTA, and the obtained results were presented in
Fig. S10.
The response time of the probe HMAC were also per-
formed for real–time monitoring of copper (II) ion, and
presented in Fig. S9. The emission intensity of HMAC–
Cu2+ complex was balanced within only 1 min, and it was
proved that the extraordinarily rapid reaction between
chemosensor HMAC and copper (II) ion was occurred.
Moreover, the reversibility experiments were performed
Computational DFT Studies
To support the experimental results, DFT calculations were
performed on probe HMAC and HMAC–Cu2+. Molecular
orbital (MO) energies and figures of HOMO and LUMO
levels are given in Fig. 6. The HOMO and LUMO of probe
HMAC were spread over the molecules, but the electronic
Fig. 6 Frontier molecular orbital
profiles of probe HMAC and
HMAC–Cu2+complex

J Fluoresc
Table 1 The analysis of Cu2+ in
various mineral and drinking
water samples by probe HMAC
and ICP–OES
Cu (II) found (μmol L−1
)
difference of
the means
t test statistics (df = 2)
HMAC
ICP–OES
t
probability > |t|
Statistic
mineral water samples
mineral
water–A
mineral
water–B
mineral
water–C
mineral
water–D
mineral
water–E
drinking water samples
drinking 0.25 0.003 0.25 0.003 0.0048
water–A
drinking
water–B
drinking
water–C
drinking
water–D
drinking
water–E
drinking
water–F
1.18 0.003 1.18 0.001 0.0056
0.29 0.006 0.30 0.002 −0.0090
1.23 0.002 1.23 0.001 −0.0016
0.24 0.003 0.25 0.001 −0.0053
0.55 0.003 0.56 0.004 −0.0059
3.2095
0.0849
t calculated < t
tabulated
−2.0718 0.1740
−0.9351 0.4484
−2.0276 0.1797
−1.4388 0.2868
t calculated < t
tabulated
t calculated < t
tabulated
t calculated < t
tabulated
t calculated < t
tabulated
2.6381
2.9099
1.3326
0.1186
0.1005
0.3141
t calculated < t
tabulated
0.50 0.002 0.49 0.001 0.0056
0.31 0.007 0.31 0.002 0.0045
0.20 0.002 0.20 0.001 −0.0027
0.21 0.004 0.21 0.004 −0.0002
0.10 0.003 0.10 0.001 0.0044
t calculated < t
tabulated
t calculated < t
tabulated
−1.3361 0.3132
−0.0841 0.9406
t calculated < t
tabulated
t calculated < t
tabulated
3.0072
0.0950
t calculated < t
tabulated
† Null Hypothesis: mean1–mean2 = 0
†† Alternative Hypothesis: mean1–mean2 <> 0
††† At the 0.05 level, the difference of the population means is NOTsignificantly different with the test difference
(0)
distribution weren’t the same for the HMAC–Cu2+ complex.
Although the LUMO of HMAC–Cu2+ complex was localized
over the copper (II) ion and the coumarin moiety, HOMO of
HMAC–Cu2+ complex was distributed around the molecules
and copper (II) ion. The energy gap between HOMO to
LUMO for probe HMAC and HMAC–Cu2+ is found as
3.87 and 1.17 eV. Probe HMAC provides a suitable molecular
structure to coordinate with the copper (II) ion. After complex-
2+
)
ation, the interaction energy (Eint = Ecomplex – EHMAC – ECu
is lowered by −11.94 eV (−275.348 kcal/mol), which proves
Table 2 Comparison of the performance of coumarin–based fluorescent probes reported in literature and probe HMAC
fluorescent probe
method performance
application
reference
linear range
LOD
λex/ λem
coumarin 7–yl picolinate based probe
0.1–0.9 μM
1.0–3.2 μM
0.0–15.0 μM
35 nM
256 nM
52 nM
325 nm/ 454 nm
350 nm/ 475 nm
365 nm/ 410 nm
tap water, deionized water
tap water, river water
deionized water
[29]
[30]
[45]
4,7–dihydroxy–3–aminocoumarin based probe
CQP, (E)–ethyl–7–hydroxy–8–[[2–[2–(quinolin–8
–yloxy)acetyl] hydrazono] methyl]–coumarin–3
–carboxylate based probe
7–diethylaminocoumarin–3–carbohydrazide
(HCM) based probe
6,7–Dihydroxy–3–(4–(trifluoro)methylphenyl)
coumarin based probe
0.0–10.0 μM
0.0–30.0 μM
210 nM
24.5 nM
340 nm/ 458 nm
365 nm/ 458 nm
tap water, lake water
[46]
drinking water, mineral water
this work
† LOD; limit of detection

J Fluoresc
the high affinity of probe HMAC towards copper (II) ions and
the formation of a stable complex between probe HMAC and
copper (II) ions. Thus, the obtained results indicate that the
stabilized complex between probe HMAC and copper (II) ion
has a lower HOMO–LUMO energy gap when compared to
probe HMAC. This emission quenching response of probe
HMAC was carried out due to the strong interaction between
copper (II) ions and –OH as a result of the charge transfer
process from the variation of electronic structure of probe
HMAC [43, 44].
24.5 nM and it was below the current literature [29, 30, 45,
46] fluorescent probe’s detection levels used for analysis of
copper (II) ion in drinking water samples (Table 2). Overall, it
is clear that the obtained results were accurate and had good
recovery values, which confirm the reliability of the proposed
assay, and could be applied to trace copper (II) ion sensing in
real beverage samples quantitatively.
Conclusion
ICP–OES Analysis of Mineral and Drinking Water
Samples
A fluorescent chemosensor based on coumarin derivative for
copper (II) ion sensing with signal “turn–on–off” has been
developed. The fabricated probe HMAC is a quite simple
operation and offers a realistic potential for the trace copper
(II) ion monitoring in the presence of other cations. The cali-
bration plot of 1/(I–I0) versus concentration of 1/Cu2+ was
linear with a correlation coefficient of 0.9875. The LOD was
found as 24.5 nM on the basis of 3σ/k. The stoichiometry of
the HMAC–Cu2+ complex was established by Job’s method
(1:1 ratio) and ESI–MS technique. In mineral and drinking
water samples, copper (II) ions can be detected with excellent
recovery values (95.48–109.20%). Furthermore, the ICP–
OES results confirmed the results of HMAC for copper (II)
ion sensing. Consequently, the presented approach is expected
to have a great potential application for safety copper (II)
monitoring in various drinking samples including water used
in agriculture and environmental applications as well as in
commercial distribution of beverages.
To prove the applicability of the probe HMAC, we performed
analyses for copper (II) ion detection in various mineral and
drinking water samples. The amount of copper (II) ion in
tested samples was directly analyzed with a simple operation
and quantitatively determined using the calibration curve.
Also, a standard addition method was carried out (final con-
centrations = 0.1 μM and 0.2 μM). The satisfactory recovery
and relative standard deviation (RSD) values were obtained
for all tested samples (Table S2). The recovery values of the
method were obtained within a range of 95.48% to 106.01%
for drinking waters and 96.18% to 109.20% for mineral wa-
ters, respectively. The results show the great potential and
feasibility of the developed probe HMAC for the sensing of
copper (II) ion in mineral and drinking water samples (Table
S2).
The ICP–OES analyses were also performed (Table S3).
Before ICP–OES measurements, the calibration curves were
constructed by standard copper (II) ion solutions prepared in
the concentrations from 10 to 1000 μg.L−1. Besides, two dif-
ferent concentrations (10 and 20 μg.L−1) of the standard so-
lutions were spiked to the tested samples. The good agree-
ments were obtained between the spiked and found amounts
of copper (II) ion. The recovery values were between
91.05%–109.57% and the relative standard deviation (RSD)
values were also obtained as lower (0.05%–2.78%) from ICP–
OES measurements. The accuracy values of the probe
HMAC and ICP–OES procedures were evaluated (Table 1).
Moreover, the results were statistically evaluated employing
Student’s t test (tcalculated values < ttabulated values, df = 2, con-
fidence level = 95%). As a result, the results of probe HMAC
and ICP–OES were in good agreement (Table 1). Therefore,
the results of ICP–OES method prove the results of
chemosensor HMAC for copper (II) ion sensing present in
various mineral and drinking water samples.
Acknowledgments The authors are grateful to the Research Fund of the
TUBITAK for their support (for the synthesis of compound) with the
project No–110 T652. The authors would also like to thank Scientific
Research Project Center of Karamanoglu Mehmetbey University with
the project 30–M–16 and for providing the financial support to use
Gaussian 09 (Gaussian, Inc., Wallingford CT) program.
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