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Determination of zinc using single-drop microextraction based upon a natural deep eutectic solvent synthesized from oleic acid isolated from olive oil: Synthesis, design of semi-automated system, and analytical applications.
* Corresponding author E-mail address: hmsaidi@uqu.edu.sa (H.M. Al-Saidi)
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Received: ,
Accepted: ,
Abstract
In the present work, a new analytical methodology based on partial automation of direct immersion single-drop microextraction (DI–SDME) and electrothermal atomic absorption spectroscopy (ETAAS), abbreviated as DI-SDME-ETAAS, was developed for the preconcentration and determination of zinc in water samples. Zinc (II) complex with 1-[4-[(2-hydroxynaphthalen-1-yl) methylideneamino] phenyl] ethanone (HNE) was extracted by DI–SDME using a new natural deep eutectic solvent (NADES) as an extractor. Oleic acid (OLE) isolated from olive oil was used, for the first time, to synthesize a wide variety of ternary and binary NADES. All NADES were tested as extraction solvents. The NADES containing two hydrophobic components (OLE and thymol) and one hydrophilic component (Choline chloride) provided higher extraction efficiency than other ternary and binary NADES. The enhancement factor, the detection limit, and the limit of quantification were 45, 0.55, and 1.83 ngL–1 (ppt), respectively, when the sample volume was 1.00 mL. Certified reference water sample (LM24-OP-4000088) and other real samples were used for the evaluation of our methodology, and the results were compared using inductively-couple plasma-mass spectrometry (ICP–MS). On the other hand, the [Zn(C19H14O2N)2] compound was prepared and characterized by several techniques.
Keywords
Characterization
Determination
Partial automation
Single-drop microextraction
Water samples
Zinc
1. Introduction
Zinc plays an essential role in many biological processes, such as the division and growth of cells, protein synthesis, gene transcription, and the breakdown of carbohydrates (Zvěřina, et al., 2023, Rus, et al., 2022). The ability of zinc to form coordination bonds with certain amino acids, e.g., histidine and cysteine, is the driving force in stabilizing protein structures. This bonding can hold certain parts of the protein together, reinforcing its structural integrity. There is another method that enhances the stabilization of protein structures called zinc fingers. In some proteins, zinc binds to specific histidine and cysteine residues to form zinc fingers. Hence, the deficiency of zinc may lead to serious effects on human health, such as impaired cognition and immune dysfunction (Stevens, et al., 2017, Das, et al., 2019). On other hand, the accumulation of Zn in the tissues of organisms may cause some diseases like mental retardation, and Wilson’s disorders (Rezaee and Tajer-Mohammad-Ghazvini, 2022, Haq, et al., 2021). The main sources providing humans with the necessary amounts of zinc are food and water. Therefore, it is necessary to monitor zinc levels in these samples.
The concentration of zinc in the different samples has been monitored using several techniques (Mohammadi, et al., 2010, Escudero, et al., 2010, Kara, et al., 2005, Zhao, et al., 2012, Zhang, et al., 1994, Teixeira, et al., 2012). However, some of these methodologies suffer from several limitations, such as insufficient sensitivity, low selectivity, high cost, and different interferences. Electrothermal atomic absorption spectroscopy (ETAAS) is a sensitive detection technique. ETAAS uses very small amounts of samples. Therefore, this technique is used for the analysis of rare samples and monitoring ultra-trace levels of the analyte. However, the loss of zinc ions during pyrolysis is a common problem in ETAAS. This loss is a result of the presence of metal chlorides in the sample matrix, where the chloride ion reacts with zinc (II) to form volatile zinc chlorides. Thus, an extraction process is required to eliminate such interference (Kilic, et al., 2002).
Among the microextraction techniques, single drop microextraction (SDME) is one of the most widespread techniques in the extraction process. SDME provides diverse features such as simplicity and ease of combination with different detection techniques. The use of minimal solvent makes this technique eco-friendly (Kailasa, et al., 2021, Qi, et al., 2020). SDME is carried out by either headspace–single drop microextraction (HS–SDME) or direct immersing in sample solution (DI–SDME). HS–SDME is specifically used to extract volatile analytes; therefore, its applications are limited. In DI–SDME, a suspended drop of an organic solvent is directly immersed in the aqueous sample solution using a microsyringe (Tang, et al., 2018, Naeemullah, et al., 2017). Then, the micro-drop containing analyte is returned into the microsyringe and injected into the analyzer for further analysis. Although enrichment factors obtained using DI–SDME are high, the limited surface area and instability of the droplet are the main drawbacks of this method (Naeemullah, et al., 2017). Some of these problems result from the use of low-viscosity organic solvents (Mulia, et al., 2015).
Deep eutectic solvents (DESs) have been developed as appropriate alternatives to toxic ionic liquid solvents (ILs). DESs are defined as mixtures that exhibit melting temperatures less than those of their pure components by more than 100°C. DESs are formed by hydrogen bonding between hydrogen bond acceptor (HBA) and hydrogen bond donor (HBD). The physicochemical properties of DESs can be controlled by changing type and ratios of components (Mulia, et al., 2015, Sereshti, et al., 2021, Ferrone, et al., 2018). Natural deep eutectic solvents (NADES) are a new class of DES where the components of the eutectic mixture include an organic salt and primary metabolites (Dai, et al., 2013). A wide variety of metabolites including organic acids and bases, amino acids, phenols, sugars, and terpenes have been investigated to synthesize NADES with different physicochemical properties (Sereshti, et al., 2021). According to our knowledge, the use of metabolites isolated from their natural sources to synthesize NADES is limited util now.
Automation of SDME is still a challenge due to the instability of single droplet. Analytical methodologies based upon SDME consist of two main stages: the extraction and detection. The extraction is the critical step in the analysis process, because the possibility of contamination or loss of the sample is possible. Thus, the automation of this stage is very useful to avoid loss or contamination of samples. However, the full automation is sometimes highly cost. Therefore, the present study aims to develop a semi-automated methodology for determination of zinc by NADES-based DI-SDME. The Oleic acid (OLE) extracted from olive oil will be tested to synthesize NADES. Moreover, the complex formed between Zn (II) and HNE molecule will be prepared and characterized.
2. Materials and Methods
2.1. Reagents and chemicals
All chemicals and reagents were of a high degree of purity and were employed as received from the source. ZnCl2 obtained from Millipore Sigma (Saint Louis, USA) was used for preparing the zinc complex with HNE reagent and obtaining the aqueous standard solutions of Zn (II) ions. The chromogenic reagent (HNE) was prepared as previously mentioned in our work (Al-Saidi and Alharthi, 2021). Thymol (99.5%) was obtained from Sigma-Aldrich (Saint Louis, Missouri, USA). All organic solvents mentioned in this work were purchased from Thermo Fisher Scientific (Hampton, New Hampshire, USA). The standard solution of chromogenic reagent was prepared by dissolving 0.6 g in 25 mL of dimethylformamide (DMF). The systems of H3PO3-NaHPO4.2H2O, CH3COOH-CH3COONa, CH3COOH-CH3COONH4, NH4OH-NH4Cl and NH4Cl-NH3 were used to adjust the aqueous phase pH at 2, 4, 6, 8, and 10, respectively. Certified reference water sample (LM24-OP-4000088) containing 79 µg L–1 zinc was taken from Labmix 24 (Hamminkeln, Germany). The substrates of certified reference water sample are provided in the website of company.
2.2. Instrumentation
An UV-1280 spectrophotometer from Shimadzu (Kyoto, Japan) was employed in the spectral range of 190 to 1100 nm for recording electronic spectra of investigated compounds. Perkin Elmer elemental analyzer model 2400 (Waltham, Massachusetts, USA) was employed for studying the elemental compositions of [Zn(C19H14O2N)2] complex. The zinc concentration in the complex was determined by inductively-coupled plasma-optical electron spectroscopy (ICP-OES_ using an Ultima Expert spectrometer from HORIBA Scientific (Kyoto, Japan). Attenuated total-reflectance-Fourier transform infrared (ATR–FTIR) spectra of DESs were recorded using a JASCO FT–IR spectrometer model 4600 (Tokyo. Japan). The unit of attenuated total reflectance (ATR PRO ONE) was used to introduce samples. X-ray diffraction (XRD) data of [Zn(C19H14O2N)2] complex was collected using an X–ray diffractometer from Bruker model D8 ADVANCE (Massachusetts, USA). The morphology and the elemental distributions of the Zn (II) complex with HNE ligand were studied using the scanning electron microscope (SEM) model JEOL JEM-6390 (Massachusetts, USA). The determination of zinc after per concentration was performed by an atomic absorption spectrometer (AAS) model AA6650 with a Shimadzu hollow-cathode lamp model L233-30NQ, an ASC-6100 auto-sampler and a graphite furnace atomizer model GFA-EX7 (Kyoto, Japan). Table 1 shows the optimized temperature program of the atomizer.
| Stage | Temperature of furnace, (°C) | Time (s) | The flow rate of Ar gas, (mL min–1) |
|---|---|---|---|
| Drying | 260 | 20 | 25 |
| Drying | 400 | 40 | 35 |
| Pyrolysis | 1000 | 5 | 5 |
| Pyrolysis | 1000 | 3 | 0 |
| Atomization | 2500 | 5 | 0 |
| Cleaning | 2700 | 15 | 1000 |
2.3. The semi-automated DI–SDME system
The schematic representation of extraction system designed in this study has been given in Fig. 1. A 3 mL vial was employed to carry out extraction. The extraction vial contains a stopcock at the bottom for waste and cleaning. All required solutions were transferred into the extraction vial using Ismatec peristaltic pump ISM4212 (Landsberg, Germany) through Tygon tubes. Ismatec peristaltic pump uses 3-stop color-coded tubing and was controlled by built-in software. Therefore, this peristaltic pump is suitable for the present work, where three solutions, e.g., buffer, sample, and reagent, can be passed simultaneously or sequentially. Socorex tube feeding syringe model 187 (Ecublens, Switzerland) was used to form a single drop and its inflating. As shown in Fig. 1, this syringe has a feeding tube on the side, which can be used to inject air to inflate the solvent drop. A short hemisphere–shaped tube with 1.00 mm i.d was connected to the syringe inlet to increase the stabilization of the drop. The inflation of the solvent drop was carried out by creating a small air bubble inside the drop, using a feeding tube connected to an automatic syringe pump ISPLAB01(Athens, Greece). This pump was operated in withdrawal mode before injecting required solutions to remove the air present in the extraction vial. This procedure increases the stability of a single drop and speeds up transferring the solutions to the extraction vial.
- The semi-automated DI-SDME system designed for zinc determination.
2.3. Preparation of [Zn(C19H14O2N)2].
Ethanolic solutions of HNE (2 mmol, 10 mL) and ZnCl2.6H2O (1 mmol, 10 mL) were mixed and then refluxed at 80°C for 2 h. After evaporation of the solvent using a rotary evaporator, Zn complex with HNE in its solid state was obtained. The complex in its pure form was obtained by recrystallization using methanol. Color: Light brown, M.p.180°C, Λm: 16.31 Ω-1 cm2 mol-1, Elemental analysis of [Ni(C19H14O2N)2] (MW - 642.032 g/mol): Calculated (C %, 71.09; H %, 4.39; O %, 9.97; N%, 4.36; Zn%, 10.18). Found (C %, 71.12; H %, 4.35; O %, 9.88; N%, 4.28; Zn%, 10.22).
2.4. Extraction of oleic acid from olive oil
OLE was isolated from olive oil using the method previously described in (Aldaw, et al., 2018). The procedure includes three stages. Firstly, the conversion of triglycerides into fatty acids. 100 g of olive oil was treated using 48 g of NaOH and 0.5 g of EDTA solution prepared in a mixture of water and ethanol (1:1 v/v). Then, 700 mL of hexane and 80 mL of deionized water were added to the mixture with stirring for 1.5 h. The unconfiscated material present in the upper layer was removed. Concentrated HCl was added to the mixture with shaking to adjust the pH of the aqueous phase (lower layer) to 1. After phases separation, the hexane layer rich in fatty acids was recovered and the solvent was evaporated by the rotary evaporator. The second stage involves extraction of the OLE from the mixture of fatty acids obtained in the first stage. Then, 100 g of the fatty acid mixture was gradually added to a urea solution prepared by mixing 150 g of urea with 400 mL of methanol. The resulting mixture was filtered under reduced pressure, and the filtrate was evaporated by a rotary evaporator for obtaining a solid product. This product was purified by a mixture of 400 mL water and a small volume of concentrated HCl with stirring to remove unreacted urea. The stirring was continuous until the unsaturated fatty acids were separated as a sufficient layer on the surface of the mixture. Then, the layer of the unsaturated fatty acids was recovered and treated with a hot urea solution. The urea solution was added gradually with cooling to room temperature. The cooling rate was 0.3 0C/min. The reaction mixture was filtrated to obtain solid particles. The particles were then treated by a mixture of water and hexane (1:1 v/v) and few drops of concentrated HCl. The hexane layer rich in concentrated OLE were separated and the solvent was evaporated by a vacuum rotary evaporator. Finally, the OLE was purified by re-dissolving in hexane with cooling in the range of -5 to -10°C. The obtained crystals were filtrated and the solvent was evaporated to obtain the OLE in pure form. The purity of OLE was tested by measurement of physical properties and the analysis of gas chromatography (GC–FID) to be 99%.
2.5. Preparation of NADES
Novel ternary NADES were synthesized using choline chloride as a hydrogen bond acceptor (HBA), while OLE obtained from olive oil and thymol were used as hydrogen bond donors (HBD). NADES components were mixed in different molar ratios in a suitable glass beaker with heating in an oil bath at the temperatures shown in Table 2. The mixtures of NADES were mixed well at 1000 rpm using magnetic stirring until they became transparent liquids. The formed NADES were then cooled to room temperature. NADES were kept in a desiccator before use. All synthesis conditions have been demonstrated in Table 2. For comparison, binary NADES composed of choline chloride as HBA with either OLE or thymol as HBDs were prepared according to the conditions shown in Table 2.
| No. | The composition of NADES | Molar ratio | Temp. (°C) | Synthesis time (min) |
|---|---|---|---|---|
| NADES.1 | ChCl:TH: OLE | 1:1:1 | 150 | 20 |
| NADES.2 | ChCl:TH: OLE | 1:1:2 | 150 | 20 |
| NADES.3 | ChCl:TH: OLE | 1:2:2 | 150 | 20 |
| NADES.4 | ChCl:OLE | 1:1 | 150 | 20 |
| NADES.5 | ChCl:TH | 1:1 | 150 | 20 |
2.6. DI-SDME procedure for zinc determination
The cleaning process was performed by injecting the buffer solution into the extraction vial with continuous stirring. Then, the stopcock was opened to waste the washing solution. After that, the stopcock was closed and the air was removed from the extraction vial by the automatic syringe pump ISPLAB01. Then, 1.50 mL of either samples solutions or zinc standard solutions (2–150 ngL–1) were injected into the vial at 0.6 mL min–1 flow rate, whereas, 0.8 mL of acetate buffer (pH 6) was transferred into the extraction vial at 0.3 mL min–1 flow rate with constant stirring to mix the solutions well. 100 µL of HNE (1.00×10–5 mol L–1) was then introduced into the mixture at a flow rate of 0.1 mL min–1. Thereafter, the feeding syringe was pushed slowly to form a drop of hydrophobic NADES.1. A small volume of air was injected using the ISPLAB01syringe pump for inflating the solvent drop to increase the interfacial area available for extraction. The extraction time was 5 min at a stirring rate of 800 rpm. The stirring was stopped, and the DES microdrop was slowly drawn into the feeding syringe. Then, the DES drop containing the analyte was injected into the GFAAS spectrometer for analysis.
2.7. Analysis of zinc in sea-water
The seawater samples were collected from the Red Sea, Jeddah, KSA, and filtered through a 45 µm Millipore cellulose membrane to clean polyethylene bottles. The samples were acidified to pH 3 with HCl. Before the application of the recommended procedure, the seawater samples were neutralized and then buffered to the desired pH.
3. Results and Discussion
3.1. Characterization of [Zn(C19H14O2N)2] complex
ATR–FTIR spectra of the ligand and [Zn(C19H14O2N)2] complex shown in Fig. 2 revealed that Zi (II) ion is coordinated to N and O atoms in the HNE molecule. The disappearance of the ν(OH) signal at 3400 cm−1 and the appearance of a new band at 574 cm−1 for ν(M–O) in the ATR–FTIR spectrum of [Zn(C19H14O2N)2] confirm the Zn (II) ion coordination with the oxygen atom in the HNE ligand. On the other hand, the stretching frequency at 1594 cm–1 for C=N in the HNE molecule was observed at 1489 cm–1 after complex formation. This shift and the appearance of a new band in the spectrum of [Zn(C19H14O2N) Cl2] complex at 546 cm−1 for ν(M–N) confirms the coordination of Zn (II) ions with the HNE through the nitrogen atom (Prabhu, 2018).
![ATR–FTIR spectra of chromogenic reagent HNE (Red) and [Zn(C19H14O2N)2] (Black).](/content/185/2025/37/4/img/JKSUS-37-5612025-g2.png)
- ATR–FTIR spectra of chromogenic reagent HNE (Red) and [Zn(C19H14O2N)2] (Black).
The 1HNMR spectrum of [Zn(C19H14O2N)2] complex recorded in CDCl3 and demonstrated in Fig. S1A provides good information about the coordination mechanism of HNE ligand with Zn (II) ions. The signal of imine proton (CH=N) of HNE at 9.623 ppm shifted to downfield (9.311 ppm), and the singlet at 12.23 ppm corresponding to the proton of the OH group in the naphthalene moiety disappeared as shown in Fig. S1A. These changes provide additional indicators for the coordination of Zn (II) with HNE through the N-atom of the imine group and the O-atom in the naphthalene moiety. The 1HNMR spectrum of HNE reagent has been shown in Fig. S1B for comparison.
The electronic spectrum of the [Zn(C19H14O2N)2] complex shown in Fig. 3 reveals that the absorption peak at 228 nm corresponding to π–π* transitions in naphthalene rings of free ligand shifted to 240 nm after complex formation, while, the peak corresponding to π–π* transitions in azomethine group was observed at the same wavelength (324 nm) with a significant increase of absorbance. The coordination of Zn (II) ion with HNE ligand through nitrogen and oxygen atoms was confirmed by the shift of the absorption peak corresponding to the n–π* transition from 445 to 462 nm with a slight decrease in absorbance. Characterization of the newly synthesized complex using ATR–FTIR spectra and UV-visible measurements suggests the chemical formula of [Zn(C19H14O2N)2], and the chemical structure has been shown in Fig. 4.
![The absorption spectra of (Black) HNE ligand, and (Red) [Zn(C19H14O2N)2] complex recorded using methanol as a solvent. [HNE] = 4×10-2 M and [Zn2+] = 1×10-4 M.](/content/185/2025/37/4/img/JKSUS-37-5612025-g3.png)
- The absorption spectra of (Black) HNE ligand, and (Red) [Zn(C19H14O2N)2] complex recorded using methanol as a solvent. [HNE] = 4×10-2 M and [Zn2+] = 1×10-4 M.
![Proposed chemical structure of the [Zn(C38H28O2N)2] complex.](/content/185/2025/37/4/img/JKSUS-37-5612025-g4.png)
- Proposed chemical structure of the [Zn(C38H28O2N)2] complex.
3.2. Synthesis and characterization of NADESs
Although there are various methods for the synthesis of DESs, e.g., heating and stirring (thermal mixing), ultrasonication, freeze drying, and grinding, thermal mixing is one of the widely preferred methods due to its simplicity and effectiveness. Therefore, all ternary and binary NADESs composed of choline chloride, OLE, and thymol were synthesized as homogeneous liquids using thermal mixing at the experimental conditions mentioned in Table 1. Extraction solvents with high viscosity are not preferred in analytical applications due to the difficulty of transportation and stirring. It is well known that the viscosity of NADES is inversely proportional to the temperature used during the preparation of NADES. Thus, a high temperature (150 0C) was employed to obtain NADESs with low viscosity. On the other hand, one of the factors reducing the viscosity is increasing the mole fraction of HBD (Negi, et al., 2024). However, the increase in alkyl chain length of HBD enhances the viscosity of NADES. Therefore, NADES.1 with a molar ratio of 1:1:1 was selected as an extractor due to its suitable viscosity. The high molar ratio of OLE made NADES.2 and NADES.3 has high viscosity. Therefore, they were excluded from subsequent experiments because of their lack of usefulness in analytical applications. The NADESs formation was confirmed using the ATR-FTIR technique and comparing the physical properties of NADESs with those of their components. The ATR-FTIR spectra of OLE, TH, ChCl, and NADES.1 have been demonstrated in Fig. 5, respectively. The characteristic bands in the ATR–FTIR spectrum of TH are stretching vibrations of phenolic O-H at 3250 cm−1, stretching frequencies of aromatic C=C at 1625 cm−1, and bending vibrations of O-H at 1351 cm−1. The spectrum of pure OLE shows a sharp band at 3050 cm−1 for the acidic O-H group. The spectrum also contains a sharp band at 2925 and a weak one at 2857 cm−1 corresponding to the symmetric and asymmetric stretching of the -CH2 group, respectively (Zhang, et al., 2006). The stretching frequency of C=O and O-H bending is observed at 1708 cm−1and 1340 cm−1, respectively. The band corresponding to O-H stretching vibrations in NADES.1 spectrum is broader compared with those observed in TH and OLE spectra, and its center has shifted to lower wavenumber (3000 cm−1). The bending vibrations of the OH group in NADES.1 spectrum were observed as a sharp band at 1280 cm−1. The previous changes confirm the formation of hydrogen bonding in DES.1, as shown in Fig. 6.
- The ATR–FTIR spectra of (red) OLE, (green) TH, (mauve) ChCl, and (blue) NADES.1.
- The formation of hydrogen bonding in NADES.1
3.3. Design of semi-automated system based upon SDME for zinc determination
The analytical methodology developed in the present work is based upon: (i) the use of NADESs synthesized from natural components as an extraction solvent. (ii) designing simple flow system in which DI–SDME is carried out. (iii) determining zinc in sing drop by ETAAS.
3.3.1. Optimization of ETAAS
One of the main drawbacks of ETAAS in the determination of some metal ions is the loss of analytes during pyrolysis because of the formation of volatile metal chlorides (Haese,, et al., 1992). However, the effect of the sample matrix is neglected in our method since the zinc (II) ions are extracted in the form of [Zn(C19H14O2N)2] complex before determination by ETAAS. Therefore, high temperatures can be used in pyrolysis and automation stages to increase the sensitivity of the method without loss of analyte. The furnace temperature was studied and optimized (Tabe 1). Hence, the shape of absorbance peaks was normal and the background signal was minimum. Generally, DESs dissociate into their components (HBDs and HBAs) at high temperatures by weakening of the hydrogen bonding. Wenjun et al mentioned that most of DESs containing ChCl as HBA start to decompose at around 250°C (Wenjun, et al., 2018). Therefore, the drying stage in our methodology was carried out in two steps. Firstly, the drying temperature of 260°C was used for 20 s to ensure the decomposition of NADES.1 to its components. Then, the samples were dried at 400°C for 40 s to evaporate the components of the extraction solvent completely, where OLE is the ingredient that has the highest boiling point (360°C). On the other hand, a series of pyrolysis and atomization temperatures were examined. The results showed that the optimized absorbances were obtained at the temperatures of 1000 and 2500°C for pyrolysis and atomization, respectively, as demonstrated in Table 1.
3.3.2. Optimization of DI–SDME
3.3.2.1. Selection of extractant (DESs)
The solvents used in DI–SDME should be water-insoluble, good extractors of the analyte, and sufficiently low viscous for easy transfer to the detection tool. The physicochemical properties of NADESs are dramatically changed by controlling their components type and ratio. Thus, a series of ternary and binary NADESs was synthesized as shown in Table 1. Preliminary examinations for choosing a suitable extractant were manually performed by mixing 5 mL of a zinc standard solution (150 ngL–1) and 1 mL of HNE (1.00×10–5 molL–1) at pH 5 with 800 μL of NADES, then the previous mixture was shaken for 1 min. Then, 10 μL of NADES phase was directly injected into the GFAAS spectrometer. NADES.1 displayed higher absorbance compared to binary NADESs (NADES.4 and NADES.5) as shown in Fig. 7. NADES.1 is composed of two hydrophobic components (TH and OLE), while the third component is hydrophilic (ChCl). Thus, the hydrophobicity of NADES.1 is high (Sereshti, et al., 2021). The hydrophobic complexes like [Zn(C19H14O2N)2] tend to be extracted in hydrophobic solvents. This means that the extraction efficiency of [Zn(C19H14O2N)2] complex is high using NADES.1 as an extractor. Therefore, NADES.1 was used as an extractor in further work.
- The influence of NADES type on the extraction efficiency. Extraction conditions: standard zinc solution volume, 5 mL; Zn (II) ions concentration, 150 ngL–1; 1 mL of HNE (1.00×10–5 M); pH 5.
3.3.2.2. pH optimization
The influence of pH on [Zn(C19H14O2N)2] formation was investigated at pH values in the range of 2.0–10.0 using different buffer systems, as shown in the experimental part. Fig. 8 shows that the maximum signal was observed at pH 5–7. The pKa of the N atom is 4.98 (Al-Saidi and Alharthi, 2021). Therefore, the pH appropriate for the formation of [Zn(C19H14O2N)2] complex should be higher than 5 since the deprotonated nitrogen atoms in the HNE molecule are involved in the formation of the extracted complex. At pH less than 4 or more than 7, the absorbance values were very small, as shown in Fig. 8. On the other hand, the minimum value of relative standard deviation (RSD) was obtained when using the buffer system of CH3COOH-CH3COONH4. Therefore, the aqueous phase was adjusted at pH 6 using a CH3COOH-CH3COONH4 system in further experiments.
![Impact of aqueous phase pH on the efficiency of preconcentration of Zn2+ by DI–SDME. conditions: standard zinc solution volume, 5 mL; Zn2+concentration, 150 ngL–1; [HNE] = 1 mL of 1.00×10–5 M; Extraction solvent, NADES.1.](/content/185/2025/37/4/img/JKSUS-37-5612025-g8.png)
- Impact of aqueous phase pH on the efficiency of preconcentration of Zn2+ by DI–SDME. conditions: standard zinc solution volume, 5 mL; Zn2+concentration, 150 ngL–1; [HNE] = 1 mL of 1.00×10–5 M; Extraction solvent, NADES.1.
3.3.2.3. The impact of HNE concentration
The influence of complexing agent concentration on the extraction efficiency of 150 ngL–1 Zn (II) ions by the developed method was studied using 0.3×10–6-1.9×10–6 mol L–1 of HNE at optimized conditions. The absorbance signal increased with increasing HNE concentration; however, the increase of signal becomes constant at concentrations higher than 1.0 × 10–6 mol L–1. Thus, the concentration of 1.0 × 10–6 mol L–1 was employed in the recommended procedure.
3.3.2.4. The effect of DES drop volume
The preconcentration factor is increased by increasing the drop volume. However, the increase in drop volume usually led to the release of microdrops. The stability of the microdrop is controlled by three forces: the downward force of gravity, the upward floating force, and the adhesion force (Abolhasani, et al., 2013). In our methodology, the balance between adhesion force and gravity determines the microdrop stability at the tip of the needle, since the density of NADES.1 is greater than that of aqueous solution. To enhance the adhesion forces, a plastic hemisphere–shaped tube with a rough inner surface was fixed at the tip of syringe needle. Therefore, larger volumes of NADES.1 and high stirring rates may be used without the fear of drop release. The influence of NADES.1 drop volume on the absorbance signal of zinc at 213.86 nm was investigated at 1–15 μL. The absorbance dramatically enhanced with increasing the microdrop volume until 15 μL, as demonstrated in Fig. 9. After this value, we could not maintain the drop, and therefore the values after 15 μL were not studied.
![Influence of NADES drop volume on the efficiency of preconcentration of Zn (II) ions by DI–SDME. conditions: standard zinc solution volume, 5 mL; Zn2+concentration, 150 ngL–1; [HNE] = 1 mL of 1.00×10–5 M; pH, 6; Extraction solvent, NADES.1.](/content/185/2025/37/4/img/JKSUS-37-5612025-g9.png)
- Influence of NADES drop volume on the efficiency of preconcentration of Zn (II) ions by DI–SDME. conditions: standard zinc solution volume, 5 mL; Zn2+concentration, 150 ngL–1; [HNE] = 1 mL of 1.00×10–5 M; pH, 6; Extraction solvent, NADES.1.
3.3.2.5. The effect of bubble-in-drop
In SDME, the increase of interfacial area can be enhanced the extraction efficiency. Creating a small air bubble inside of drop increases the interfacial area, thus, the extraction efficiency improves. 8 μL was the largest volume of air required for the formation of a stable drop with a volume of 15 μL at the syringe inlet. Drop detachment or partial dispersion was observed when using air volumes larger than 8 μL.
3.3.2.6. Extraction time and stirring rate
One of the main drawbacks of SDME compared to some microextraction techniques is the relatively long extraction time. This is most likely attributed to the small surface area of the acceptor (Rodríguez Cabal, et al., 2019). However, the stirring of the sample solution dramatically reduces the extraction time. Unlike some automated systems previously used with SDME, the semi-automatic system developed in the current study enabled us to use the stirring rate of 800 rpm; therefore, the extraction time was reduced to 5 min.
3.3.2.7. The effect of flow rate
Samples, solutions, or zinc standard solutions (2–150 ngL–1) and acetate buffer were passed simultaneously into the extraction vial with continuous stirring. This procedure ensures complete mixing between the two solutions before introducing the reagent solution (HNE) into the previous mixture. The sample volume transferred to the extraction vial was nearly twice as large as that of the buffer. Therefore, the flow rate of sample solutions or zinc standard solutions should be higher than that of the buffer solution for introducing these solutions into the extraction vial simultaneously. Thus, the flow rates of 0.60 and 0.30 mL min–1 were selected to transfer 1.50 and 0.8 mL of sample solution and buffer, respectively. The extraction efficiency was slightly affected by the flow rate of the complexing agent (HNE) where the absorbance decreased with the increase of the flow rate. This behavior is most likely attributed to the short reaction time between HNE and zinc ions.
3.3.3. Proposed method selectivity
One of the main problems when determining zinc by ETAAS is iron interference at the spectral line of 213.856 nm (McKay and Latham, 1973). Therefore, the influence of iron presence on the determination of zinc by our method was studied at a concentration level of 10 ng L–1 Zn2+. The zinc concentration in binary solutions (Zn2+ & Fe2+) was monitored by the developed method at a tolerable level of 1500 without significant interference. On the other hand, the transition elements ions, which can form complexes with Schiff bases such as Mn2+, Co2+, Cu2+, and Ni2+, didn’t interfere at the tolerable level of 2000. In fact, the use of ETAAS for detection in the proposed methodology provided a good solution to avoid the interferences of colored complexes. The major source of interferences when using ETAAS as a detection technique is the chloride ion due to the formation of highly volatile ZnCl2. However, in our present method, the addition of Cl− ions to the aqueous standards at the tolerable level of 1500 did not affect the signal (absorbance). Moreover, the interferences of other ions were studied at a concentration of 10 ng L–1 Zn2+, and the results have been revealed in Table 3.
| Ions | Tolerance limits |
|---|---|
| Mn2+, Co2+, Cu2+, Pb2, Mg2+, Ag+, Hg2+, Pb2+, Cr3+, Zn2+, Cd2+ Na+, Li+, K+, Sr+, Ca2+, | 2000: 1 |
| Fe3+, Fe2+, NO3−, Cl−, Br−, NO2−, SO42 -, CO32 -, I− | 1500: 1 |
3.3.4. The performance of the proposed analytical methodology
The analytical methodology, consisting of semi-automated DI–SDME and ETAAS, provided a good performance. The calibration curve was linear in the range of 1−150 ngL−1, and the regression equation is:
The detection limit (LOD) and limit of quantification (LOQ) calculated using a sample volume of 1.00 mL were 0.55 and 1.83 ngL−1, respectively. LOD and LOQ are calculated based upon a signal-to-noise ratio of 3 and 10, respectively, and using the equations previously reported in (Al-Saidi and Alharthi, 2021). The enhancement factor (EF) calculated using Eq. (2) was 45.
where, is the calibration curve slope of the determination of zinc using the designed microextraction system, while is the calibration curve slope of direct determination (without preconcentration). The relative standard deviation (RSD) for ten replicate measurements at a concentration of 100 ngL−1 Zn2+ was 2.8%. On the other hand, the analytical methodology proposed in our present study ensures no contamination or loss of samples during the entire extraction stage.
3.3.5. The application of the developed method for zinc determination in some samples
The accuracy of the DI-SDME system combined with ETAAS was established by determining zinc in a certified reference water sample (LM24-OP-4000088). The zinc concentration in CRM determined by the proposed method was 80 ±1.01 µg L–1 with the relative error of 1.27%. The certified value is 79 µgL–1. The statistical analysis (Student’s t-test) showed that the difference between the certified value and the determined amount is the result of random errors because the calculated value of the t-test (1.55) is less than the critical t value (1.812) at t0.95 and freedom degrees n = 10. Thus, our developed method is free from systematic errors. We tested the developed method for the determination of zinc in some real samples rich in high levels of chloride and nitrate ions, such as sea water. The spiked sea-water samples were analyzed by the developed method (A) and ICP–MS (B). The results in Table 4 show that the method was applicable to determine zinc even in the presence of these ions in high concentrations. The relative recovery values ranged from 97.3 to 102.2%. On the other hand, the statistical evaluation of the results using F-test showed that no statistical difference between the developed method and the standard one, where tabulated F value (3.179) for ten replicate measurements is larger than the calculated value of F (1.79). Hence, the precision of both methods at the 95% confidence level is statistically acceptable.
| Sample | Spiked (μg L-1) | Found (μg L–1) | Recovery (%) | F–value a | ||
|---|---|---|---|---|---|---|
| A | B | A | B | |||
| Red sea water | ___ | 187 ± 0.3 | 189 ± 0.2 | ___ | ___ | 1.79 |
| 100 | 286 ± 0.5 | 290 ± 0.7 | 99.2 | 102.2 | ||
| 200 | 382 ± 1.5 | 390 ± 0.7 | 97.3 | 102.2 | ||
3.3.6. Comparison with other methods
The developed analytical method was compared with previously published methods as shown in Table 5. Our method provides better analytical performance in terms of linear range, RSD, LOD, EF, and LOQ. The use of DESs reduces the harmful effects on the environment because DESs are less toxic than normal organic solvents and ILs. The semi–automated DI–SDME system developed in this study is simple and convenient to work with many detection techniques.
| Detection System a | Analyzed sample | LOD b (µg L-1) | LOQ c(µg L-1) | D.LR d (µg L-1) | Sample volume (mL) | EF e | Optimum pH | RSD f (%) | R2 g | Ref. |
|---|---|---|---|---|---|---|---|---|---|---|
| HLLE-FAAS | mineral water | 5 | N.D. h | 5-1000 | 100 | 100 | 9.0 | 2.3 | N.D.h | (Abkenar, et al., 2011) |
| DLLME-UV-VIS | waste water | 11.2 | N.D. | 30-220 | 80 | 130 | 9.4 | 1.4 | N.D. | (Niazi, et al., 2012) |
| UV-VIS | tap water | 200 | N.D. | 200-2000 | 30 | 45.0 | 7.0 | 2.5 | 0.9994 | (Elsherif, et al., 2022) |
| UV-VIS | pharmaceutical sample | 0.381 | 1.156 | 0.001-0.15 | 5.5 | N.D. | N.D. | 1.1 | 0.996 | (Najim, et al., 2019) |
| UV-VIS | soil samples | 6.0×10-5 | N.D. | 2.6×10-4-2.62×10-3 | N.D. | N.D. | 6.0 | 0.52 | 0.999 | (Admasu, et al., 2016) |
|
Coprecipitation FAAS |
natural water | 0.28 | N.D. | N.D. | 50 | 25 | 9.0 | <5.0 | N.D. | (Mendil, et al., 2015) |
| FI-FAAS | tap water | 2.2 | N.D. | 5-50 | 12.6 | 40 | 5.0 | 1.2 | 0.998 | (Yılmaz, et al., 2013) |
| PSHF-DPASV | river water | 0.015 | N.D. | 500-0.05 | 5 | 5140 | 5.0 | 3.6 | 0.998 | (Es’haghi, et al., 2017) |
| DI-SDM-ETAAS | red sea water | 0.55 | 1.83 | 1-150 | 1 | 45 | 6.0 | 2.8 | 0.9995 | This study |
DLPME: Dispersive liquid phase microextraction; SFODME: Solidified floating organic drop microextraction; SFO: Solidified floating organic; FAAS: Furnace atomic absorption spectrometry; MME: Micelle-mediated extraction; PVG: Photochemical vapor generation; BT: Batch type; UAGLS: Ultrasonication assisted gas liquid separator; AAS: Atomic absorption spectrometry; FPSE: Fabric phase sorptive extraction; DAD: diode array detection; PV: Pressure variation; IS: In-syringe; SQT: Slotted quartz tube; UAE: Ultrasound assisted emulsification; DI-SDME: direct immersion-single drop microextraction; PSHF: Pseudo stirbar hollow fiber; DPASV: Differential pulse anodic stripping voltammetry.
4. Conclusion
In this study, we developed an analytical methodology for the microextraction and estimation of zinc in water samples using a semi-automated DI-SDME system combined with ETAAS. The efficiency of the analytical methodology developed in this study was improved by designing a semi-automated DI-SDME system to introduce samples and reagent solutions automatically into the extraction vial and conduct DI-SDME. The new methodology is sensitive and selective. On the other hand, our method is eco-friendly, where OLE isolated from olive oil was used to synthesize NADES. The use of the bubble-in-drop approach enhanced the stabilizing of the submersed drop and increased the interfacial area. Therefore, a high stirring rate (800 rpm) was used, and the extraction time was reduced to 5 min. The synthesized NADESs were characterized using the ATR-FTIR technique and compared with the physical properties of NADESs with those of their components.
CRediT authorship contribution statement
Hamed M. Al-saidi: Writing – original draft, Salman S. Alharthi: Writing – original draft, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Declaration of Generative AI and AI-assisted technologies in the writing process
The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.
Supplementary data
Supplementary material to this article can be found online at https://dx.doi.org/10.25259/JKSUS_561_2025.
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