A selective colorimetric chemosensor for detection of Cu(II) ions in aqueous samples

In this work we report a selective colorimetric chemosensor (S) for Cu2+ ion detection in aqueous media. It has been synthesized by a condensation reaction between 1-hydrazinophtalazine and 6,6′-dihydroxy-5,5′dimethoxy-(1,1′-biphenyl)-3,3′-dicarbaldehyde. The interaction between S and Cu2+ was studied and the results showed a bathochromic shift at 420 nm in UV–Vis spectra. The absorbance enhancement of S was attributed to complex formation with Cu2+ by 2:1 stoichiometry. The proposed analytical method can be applied to Cu2+ quantification with a linear range covering from 5.0 to 315.8 μM, and can be used to routinely analyze Cu2+ in environmental aqueous samples with satisfactory results.


Introduction
In the past few decades, the rapid increase in industry has resulted in considerable rise in the standard of people's living conditions.However, this vertiginous industrial development has also caused a negative environmental impact.Large volumes of wastewater containing different pollutants are generated daily by industrial processing activities, this being an anthropogenic source of contamination of water resources [1].
Heavy metals are a group of metals and metalloids that have relatively high density and are toxic even at low concentrations.Unlike organic pollutants, heavy metals tend to accumulate in organisms [2][3][4][5].
One of the polluting heavy metal ions is copper.The chemical pollution of copper begins when this metal enters the soil and groundwater through surface runoff, and then it relatively easily enters the living beings through feeding and drinking water [6].
Cupric ion (Cu 2+ ) is the third most essential transition metal ion for biological processes in the human body.It plays a vital role in electron transfer process of many biological reactions and in the development of many organs [7].The adverse effects arise when an imbalance in concentration takes place [8].The deficiency of copper in human body will affect enzyme activity and inhibit cell metabolism, leading to arterial diseases, high cholesterol and coronary heart diseases [9].Instead, the excessive intake of copper will burden the liver, resulting in liver ascites, cirrhosis, Wilson disease, Alzheimer disease, prioninduced diseases, kidney failure, necrotic hepatitis and hemolytic anemia [10][11][12].
As a result, the World Health Organization and the Argentine Food Code (CAA after its acronym in Spanish) have set the maximum concentrations of copper in drinking water at 2 mg/L and 1 mg/L respectively [13,14].
As evidenced, copper plays an important role in biology as well as in the environment, and therefore, developing selective detecting methods is an important research topic.The determination of metals such as copper in aqueous matrices is done with expensive technologies requiring low detection levels and accurate analysis with high sensitivity.The conventional techniques, for instance, atomic absorption spectrometry, atomic fluorescence spectroscopy, inductively coupled plasma atomic emission/mass spectrometry, electrochemical methods, enzymatic analysis and colorimetric assays are widely used in the determination of this analyte [15].
In the past years, sensitive and selective optical chemosensors have been developed as tools for analyte recognition [16].Their optical detection is usually performed via color modification or luminescence spectral changes.Detection using spectrophotometric methods has been in great demand due to its easy operation, low cost and simplicity.The naked eye detection of metal ion (color change) at very low concentrations using this method is attractive from a practical point of view.
Melting points were measured using an Electrothermal 9100 instrument and elemental analysis was performed on a CHONS LECO equipment.Mass spectra were measured on Waters Q-Tof micro ESI-MS. 1 H and 13 C NMR spectra were recorded using a Bruker DPX-300 (300 MHz) spectrometer.Chemical shifts were evaluated using TMS as internal standard.
The FTIR spectra were obtained on a Shimadzu FTIR Prestige-21 spectrophotometer in the region from 4000 to 1000 cm −1 .The UV absorption measurements were carried out with a Perkin Elmer Lambda 20 spectrophotometer.All instrumental measurements were performed at 25 °C.

Sensor synthesis
The general synthetic procedure of the sensor S is depicted in Scheme 1. Compound 1, necessary for the synthesis of S, was prepared by oxidative coupling according to the methods found in the literature [17], FeSO 4 (0.14 g, 0.89 mmol) and K 2 S 2 O 8 (3.38 g, 12.51 mmol) were added under stirring to an aqueous solution (20 mL) of vanillin (3.55 g, 23.33 mmol).
The mixture was heated at 50 °C for 120 h until the reaction was complete as indicated by TLC assay.After cooling at room temperature, the crude product was filtered and washed twice with cold ethanol.Product 1, which had a dark brown color, was obtained after a crystallization process with methanol and dried under vacuum.
For the synthesis of compound S, a suspension of HPT (2) (0.26 g, 1.32 mmol) and sodium acetate (1.85 g, 22.55 mmol) in water (10 mL) was added to a solution of compound 1 (0.20 g, 0.66 mmol) in ethanol (10 mL).The mixture was heated to reflux for four days, and the reaction was monitored by TLC.After cooling to room temperature, the solid product was collected by filtration and washed with water.The final product was obtained after crystallization with 2-propanol and dried under vacuum.

Evaluation of sensor selectivity toward cations by UV-Vis spectroscopy
The evaluation of the sensor with different cations were carried out by adding 2.0 mL of sensor solution (10 µM) to 2.0 mL of the cation solutions already described (10 µM).After mixing the solutions for 30 s, the absorbance spectra were recorded from 200 to 600 nm at a scanning speed of 240 nm/min against a blank of distilled water.

Evaluation of complex sensor-Cu 2+ stoichiometry
Aqueous solutions of sensor S (1.0 mM) and Cu 2+ (1.0 mM) were prepared.Then, both were mixed to generate a series of solutions with Cu 2+ mole fractions ranging from 0 to 1, keeping constant the total concentration system.According to this, volumes of 2.0, 1.8, 1.6, 1.4, 1.2, 1.0, 0.8, 0.6, 0.4, 0.2 and 0 mL of the sensing solution were taken and mixed with volumes of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mL of Cu 2+ solution.The total volume was always 2.0 mL.After 30 s of mixing, the absorbance at 420 nm of mixtures were recorded.Absorbances were normalized against solutions prepared by replacing the volume of the cation solution with distilled water.2+ with S Titration assays of Cu 2+ with S were done by adding 1.0 mL of the sensor solution (100 µM) at 2.0 mL of aqueous solutions of Cu 2+ with different concentrations.In these solutions, the final concentration of S was 33 µM, and the concentration range of Cu 2+ was from 0 to 133 µM.The absorption spectra of each solution were acquired after 30 s of mixing.

FTIR experiments
A mixture of 1.0 mL of stock solution (10 mM) of Cu 2+ in distilled water together with 1.0 mL of stock solution (10 mM) of sensor S in DMSO/ethanol (v/v = 2/8) was prepared.A mixture control of S was prepared using 1.0 mL of distilled water and the same volume of the stock solution of the sensor.Mixtures were heated at 100 °C until complete solvent evaporation.Solid products were diluted with KBr (1 wt%) and pressed into tablets before spectrum acquisition.

Theoretical modeling
The ground state geometry optimization of sensor S and complex cation sensor with Cu 2+ cation were carried out at the DFT level of theory using the CAM-B3LYP correlation function with a 6-31G(d,p) basis set.Frequency calculations allowed us to verify that currently found geometries were minimal.Molecular orbital calculations were performed to characterize the molecular properties.All of these calculations were carried out using Gaussian 09 program package [18].

Determination of Cu 2+ in water samples
Real water samples from the Setubal Lagoon and Colastine River of Santa Fe and two waste water samples were analyzed for Cu 2+ determination, previously filtered to remove solid particles in suspension.For the assay, 1.0 mL of the sensor solution (100 µM) was added to 2.0 mL of samples.After mixing the solutions, UV-Vis spectra were obtained.

Synthetic procedure of sensor
Sensor S has been synthesized following a schematic route depicted in Scheme 1 and described in the experimental section.Characterization of precursor 1 and sensor S are summarized below:

Photochromic properties of sensor
Selectivity is the first parameter that must be evaluated in the development of a new chemosensor [19].This defines the sensing performance, and in this context describes its ability to identify certain compounds in an unknown mixture [20].
The selectivity of sensor S toward different cations was studied using UV-Vis spectroscopy by analyzing absorption spectra changes of the solution sensor S (10.0 µM) when cation solutions of the same concentration were added separately.In these solutions, the final concentration of S and cations were 5 µM.Spectral changes of S in the presence of different cations are shown in Figure 1A.As can be seen in this figure, the UV-Vis spectrum of sensor S shows a characteristic absorption band at 378 nm.With the addition of cations into the sensing solution, the spectra observed were different.Upon addition of Li + , Na + , Mg 2+ and Fe 2+ cations in the solution of S, the band at 378 nm increased its intensity, whereas with the addition of K + , Pb 2+ , Zn 2+ , Ca 2+ , Ba 2+ , Sn 2+ , Cd 2+ , and Ni 2+ cations into S solution, band intensity at 378 nm decreased.On the other hand, the addition of other cations on sensor solution produced a band shift toward a lower wavelength (hypsochromic shift) together with the increase in the intensity of the band for Al 3+ cation and the decrease in the intensity of the band for Cu + , Fe 3+ , and Cr 3+ cations.Particularly, a significant change was observed when Cu 2+ C. R. Chimie -2021, 24, n 3, 385-396 cation was added; a new peak appeared at 420 nm for a bathochromic shift indicating that sensor S forms a chromogenic complex with Cu 2+ .
The absorbance at 420 nm of sensor S in presence of different cations are shown in Figure 1B.As can be seen in this figure, the intensity of sensor S solution at 420 nm increased about 50% after Cu 2+ addition, maintaining its maximum value for more than 1 h.These results demonstrated that S presents an excellent selectivity for Cu 2+ cation by UV-Vis spectroscopy.The pH of all samples was between 4.0 and 6.5, range in which our analysis method was verified.

Determination of binding mode between sensor S and Cu 2+
Continuing with the evaluation of sensor properties of S with Cu 2+ , the binding mode between S and Cu 2+ was determined according to continuousvariation method.To build Job's plot, the Cu 2+ molar fraction has been variated from 0 to 1 maintaining the total concentration of the solution constant (0.1 mM), and the absorbance value of these solutions has then been reported.The binding stoichiometry of the complex between Cu 2+ and S was determined from the Job's plot and corresponds to the value on the x-axis by the maximum value of absorbance.Figure 2 represents the absorbance variation value at 420 nm versus Cu 2+ mole fraction, and the maximum value of absorbance was obtained at a mole fraction of 0.33, indicating a 2:1 stoichiometry for the complex between sensor S and Cu 2+ .Continuing with the evaluation of binding mode between S and Cu 2+ , the UV-Vis titration of Cu 2+ Figure 3A shows the absorption spectra of S with increasing Cu 2+ concentration.Under experimental conditions, the UV-Vis spectrum of S exhibits an absorption band at 372 nm.Further addition of Cu 2+ solution into the S solution resulted in a gradual decrease in the absorbance band at 372 nm (Cu 2+ concentration from 0 to 14 µM) associated with the bathochromic shift of the band to higher wavelengths centered at 420 nm (Cu 2+ concentration from 20 to 133 µM).The molar extinction coefficient (ε) of S-Cu 2+ complex at 420 nm was 1.56 × 10 4 M −1 •cm −1 , two times larger than S coefficient.
Considering a ratio stoichiometry between S and Cu 2+ as 1:n, the equilibrium interaction is given by (1): In the equilibrium, the binding constant (K b ) will be expressed as: where [S] 0 , [Cu 2+ ] 0 are the initial concentration of sensor S and Cu 2+ , respectively, and [S(Cu 2+ ) n ] is the complex concentration at the equilibrium between S and Cu 2+ .Considering that [S(Cu 2+ ) n ] will be much lesser than [Cu 2+ ] 0 , the expression (2) can be simplified and reorganized as the expression (3): On the other hand, the absorbance intensity can be expressed for the ratio (4): where A 0 , A and A m are the absorbance intensity of sensor solution without Cu 2+ , the absorbance intensity of sensor solution presence of Cu 2+ , and the absorbance intensity of sensor solution at the maximum concentration of Cu 2+ , respectively.In the presence of an excess of [Cu 2+ ], [S(Cu 2+ ) n ] m is equal to [S] 0 , the expression (4) can be expressed as: Applying Ln to the expression (5) and considering n = 0.5, it is possible to obtain the following equation: The linear plot of expression ( 6) is shown in Figure 3B.
The binding constant was estimated with the intercept value of curve fit from Figure 3B; the value of Ln 9.755 corresponding to K b of 1.72 × 10 4 M −2 for the complex S 2 Cu 2+ .

Optimization of Cu 2+ chemosensing
To optimize the Cu 2+ chemosensing conditions of sensor S, the linear range of quantification was evaluated by absorption spectroscopy.It was assumed that the Beer's law is dependent on the analyte concentration and it is applicable to the quantitative detection of Cu 2+ .Plotting the absorbance ratio 420 nm/372 nm as a function of the Cu 2+ cation concentration from the data obtained at titration experiments, Figure 4A, the linear range curve was constructed.Two UV response regions were identified in Figure 4A, a 4A, a new calibration curve was plotted between absorbance at 420 nm versus concentration of Cu 2+ at 11 concentration levels (Figure 4B).Absorbances at 420 nm of standard solutions were normalized against the absorbance of a sensor solution at the same wavelength.The regression data together with the statistical parameters obtained by least-squares fitting of the calibration curve for Cu 2+ are summarized in Table 1.High correlation coefficient value (R > 0.99) indicates excellent linearity.The data used in the calibration was analyzed using lack of fit test to determine whether the linear model is suitable to describe the observed data, verified that the experimental data fit with the linear law.
The data obtained from the linearity study was used to determine the detection limit (DL) and quantification limit (QL) using the expression according to recommendation in literature [21], employing (7) and (8), respectively: where σ 0 is the standard error predicted for the blank sample according to Olivieri [22].Both DL and QL values are presented in Table 1 and they were analytically confirmed by proper dilution of the standard solution.The preferential selectivity of sensor S toward Cu 2+ in the presence of other cations was also analyzed.
The possible interference of other cations was evaluated by adding 33 µM of S to cations solutions (66 µM) in the presence and absence of 33 µM of Cu 2+ .After mixing, the absorbance of each solution was recorded at 420 nm and normalized with a blank sample.The results are shown in Figure 5, and they confirmed that the selective quantification and detection of Cu 2+ was not influenced by the presence of common cations.No significant absorbance variation was observed during sample comparison between Cu 2+ and other cations.
The accuracy of the analytical method was evaluated by analyzing the recovery (R) of five samples containing known Cu 2+ amounts.Recovery values in the range of 90-107% are considered as the criterion for a suitable recovery [19].Table 2 shows the results and, as it can be noticed, recovery percentages near 100% for all the samples were obtained.Although the concentration of sample 1 is less than QL, the concentration found and the recovery were adequate, indicating the excellent performance of the analytical method.These recovery values were tested statistically by the averaged recovery method, demonstrating that the method is accurate.
In order to further study the precision of the analytical method, three samples were analyzed five times.Data of standard deviations and relative standard deviations (RSD) for Cu 2+ determination in Table 2 showed a good repeatability of the results.The RSD values were in all cases lower than 1%.
According to these results it is possible to conclude that the proposed analytical method is selective, linear, precise and accurate for the qualitative and quantitative determination of Cu 2+ by UV-Vis spectroscopy.

Molecular modeling of the complex sensor-Cu 2+
Theoretical calculations based on DFT were performed to understand the binding mode of Cu 2+ cation with the sensor S.
C. R. Chimie -2021, 24, n 3, 385-396 The optimized structures of sensor S and sensor-Cu 2+ complex are shown in Figure 6.From these structures, it can be seen that nitrogen atoms of hydralazine moieties are participating in the complex formation.The N-Cu 2+ bonds range from 1.91 to 2.12 Å.
Considering a ratio stoichiometry between S and Cu 2+ as 2:1, the binding energy (BE) was estimated by the (9): where E (S 2 Cu 2+ ) , E (S) and E (Cu 2+ ) are the optimization energy of S 2 Cu 2+ complex, sensor S and the Cu 2+ cation, respectively.The BE obtained was −286.53Kcal/mol.With this definition, a negative binding energy indicated that the binding interaction was favored.
The complex formation between Cu 2+ and S involves charge transfer, which occurs between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO).The distribution of HOMO-LUMO orbitals of S and S 2 -Cu 2+ complex and the energy difference (gap energy) between those orbitals are shown in Figure 7.
In the presence of Cu 2+ ion, upon Cu 2+ binding with nitrogen atoms of phthalazine units, the energy levels of both HOMO and LUMO are lower than those of free S. Also, the decrease of energy in the LUMO is more significant than for the HOMO, indicating that the LUMO is more stabilized.
The decrease in energy gap between S and S 2 Cu 2+ is in agreement with the experimental bathochromic changes in spectra of S, upon complexing with Cu 2+ .

Theoretical-experimental evaluation of the complex sensor Cu 2+
In order to evaluate the functional groups involved in S 2 -Cu 2+ complex formation, FTIR spectra of S and S 2 -Cu 2+ were recorded, and are shown in Figure 8.
In the region from 1600-1000 cm −1 the main identification bands are observed.The vibrations at 1580 cm −1 and 1530 cm −1 are assigned to C=N and C-N stretching, respectively [24][25][26].The bands at 1630 cm −1 and 1427 cm −1 correspond to C=C and C-C stretching vibrations, respectively, and the band at 1228 cm −1 is assigned to C=C stretching vibrations present in aromatic systems [24][25][26][27].The bands observed at 1364, 1330 and 1263 cm −1 are assigned to N-N, N-N-H and C-N stretching vibrations respectively; while the bands at 1142, 764 and 640 cm −1 correspond to C-C=C, C-N-N and C-C=N bending modes [26].When the FTIR spectrum of S 2 -Cu 2+ was analyzed, (Figure 8A, blue line spectrum) the main assignations observed in S are identified, with slight differences in intensity and positions.It is observed that the stretching band between 3600-3200 cm −1 due to O-H and N-H groups has increased its intensity, which is attributed to S 2 -Cu 2+ complex association to water molecules through hydrogen bonds [27].In the region between 1600-1000 cm −1 , an intensity increase is observed followed by a shift to lower frequencies for C-C=N bending, and N-N, C-N stretching, at 623, 1359 and 1258 cm −1 respectively.Also, an intensity decrease is observed at 1506, 1464, 1452 and 1222 cm −1 assigned to C-C and C=C stretching vibrations.The band at 1115 cm −1 is attributed to SO 2− 4 counter ion present in the sample [24][25][26][27], while the band at 539 cm −1 corresponds to N-Cu stretching mode in the complex [28,29].These results suggest that phthalazine nitrogen atoms present in S ligands would participate in Cu 2+ complex formation.
In order to validate these experimental results, theoretical spectra for S and S 2 -Cu 2+ were calculated.
Mainly, when S and S 2 -Cu 2+ spectra are compared in the region between 3600-2900 cm −1 , the O-H, N-H and C-H stretching vibrations are observed (region not shown in Figure 8B) with a slight increase in intensity of these bands in complex S 2 -Cu 2+ spectrum.Significant changes are detected in the region between 2000-500 cm −1 (Figure 8B).
Just as observed when experimental FTIR spectra were compared, the main band changes correspond to N-N, C-N stretching and C-C=N bending.
An increase in intensity and a shift to lower frequencies is observed on comparing S 2 -Cu 2+ spectrum against free S. The band at 706 cm −1 in free S FTIR spectrum, assigned to C-C=N bending, is present in S 2 -Cu 2+ spectrum at 684 cm −1 ; while the bands at 1385 and 1267 cm −1 in S spectrum, assigned to N-N and C-N stretching, are identified in S 2 -Cu 2+ spectrum at 1360 and 1260 cm −1 respectively.These spectra changes are accompanied by a shift to lower frequencies for C-C=C, N-N-H and C-N-N bending signals in S at 1178 cm −1 , 1354 cm −1 and 786 cm −1 , respectively, in the complex at 1145 cm −1 , 1350 cm −1 and 778 cm −1 , respectively.Also, the interaction of Cu 2+ and ligand molecules is done through phthalazine nitrogens, which is concluded with the band at 553 cm −1 assigned to N-Cu stretching in the S 2 -Cu 2+ FTIR spectrum.In addition to this, in free S spectrum an increase intensity is observed for C=N and C-N stretching, positioned at 1538 cm −1 and 1455 cm −1 , respectively; along with intensity decrease for C=C and C-H stretching, present in the region between 1515-1490 cm −1 [28,29].This allows us to validate the observed FTIR results for S and complex, verifying the interaction of Cu 2+ in the S 2 -Cu 2+ complex.

Determination of Cu 2+ in real water samples
To evaluate the practical application of the validated method, different real water samples were analyzed.These samples were centrifuged, filtered and analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) and by our developed method (L).
The pH of all samples was between 4.0 and 6.5, which corresponds to the pH range in which our analysis method was verified.Results are presented in Table 3 (left) showing a good correlation between both techniques, indicating that our method would be suitable to routinely analyze the Cu 2+ content in real samples.
Furthermore, in order to analyze the method performance in real samples, two real water samples whose content was previously determined by ICP-MS, were enriched by adding known amounts of Cu 2+ .Subsequently, the samples were analyzed by our method and, based on the theoretical and experimental concentrations obtained, the Cu 2+ recovery was evaluated.The results (Table 3, right) showed a good correlation between found and theoretical concentrations, showing an adequate method performance to determine Cu 2+ cation concentrations in real water samples.

Conclusions
In summary, a new selective colorimetric chemosensor was synthesized and applied for Cu 2+ detection in aqueous samples.The sensor S was obtained through a simple synthetic pathway and it was obtained with good yield.The sensor has shown selectivity and sensitivity toward Cu 2+ , the limit of detection for this cation being 1.7 µM.The proposed analytical method is precise and accurate for Cu 2+ determination and it was successfully applied to real water samples, the results being in agreement to those obtained by more sophisticated techniques.

CScheme 1 .
Scheme 1. Schematic route used in the synthesis of sensor S (Compound 3).

Figure 1 .
Figure 1.Evaluation of sensor S selectivity with different cations by UV-Vis spectroscopy: (A) UV-Vis absorption spectra of sensor with cations; (B) absorbance at 420 nm of S solutions with different cations.

Figure 4 .
Figure 4. Linearity study of Cu 2+ : (A) Evaluation of the linear range of quantification of Cu 2+ ; (B) plot of normalized absorbance at 420 nm as a function of Cu 2+ concentration in the linear range.

Figure 5 .
Figure 5. Cation selectivity profiles of sensor S toward Cu 2+ when other cations are present.

Figure 6 .
Figure 6.Optimized structures of Sensor S and Sensor-Cu 2+ complex.

Table 1 .
Parameters obtained from calibration assay of Cu 2+

Table 2 .
Results from accuracy and precision assays

Table 3 .
Results obtained from Cu 2+ determination in real water samples