2.1 Reagent and standards

Hydroxy-terminated polydimethylsiloxane (OH-PDMS), tetramethoxygermane (TMOG) and trifluoroacetic acid (TFA) 99% were purchased from Sigma-Aldrich. HPLC grade solvents like acetonitrile (ACN), methanol and dichloromethane were purchased from Fisher Scientific. The analyte of interest, 4-chloro-1-naphthol, was obtained from Sigma-Aldrich.

A 1.0 mg mL⁻¹ stock solution of the 4-CN was prepared in methanol. All the working solutions or the extraction solutions were prepared freshly each time by diluting the stock solution in de-ionized water. All the optimizations of the major parameters were conducted at 200 ng mL⁻¹ of 4-CN. All the standards and stock solutions were kept at 4 °C in the refrigerator.

2.2 Instrumentation

HPLC system (Agilent Technologies, USA) equipped with a quaternary pump (G1311B/C), a DAD (G4212B), an auto-sampler (G1329B), and Chemstation for LC software Rev.B.04.03[16] was used. The column used for separation was Agilent ZORBAX Eclipse XDB C-18 (5 µm, 4.6 mm id × 250 mm). For the preparation and homogenized mixing of PDMS-Ge sol, Thermofisher Scientific MaxiMix vortex mixer was used (model M16715). The precipitates of the sol were separated by using Sorvall™ Legend™ micro17 Microcentrifuge. The extraction from the sample was done using Fisher Scientific™ Isotemp™ basic stirring hotplate and desorption was done in Branson 3800 ultrasonic cleaner.

Thermo Scientific ESCALAB 250Xi (PHI 5000 Versa Probe II, ULVAC-PHI Inc.) X-ray photoelectron spectroscopy (XPS) was used to determine the bonding state and surface chemical composition. Prior to analysis, a representative sample from polymer sample was mounted on holder made of carbon tape, and vacuumed under high pressure to remove impurities and/or moisture adsorbed on the sample. Thermogravimetry analysis (TGA) was conducted from room temperature (RT) to 600 °C, at constant heating rate of 10 °C, under the flow of pure N₂ gas environment by using SDT Q600, V20.9 Build 20, thermal analyzer. The morphological information of the polymer was examined by field emission scanning electron microscope (FE-SEM) from TESCAN (LYRA 3 Czech Republic), using both secondary electron (SE) and back scattered electron (BSE) modes at accelerating voltage of 30 kV. The FE-SEM is equipped with energy dispersive X-ray spectrometer (EDS, Oxford Inc.) detector for elemental analysis.

This analysis is not contrary to any human and animal right.

2.3 Preparation of PDMS/Ge coated stir bars

2.4 SBSE procedure and optimizations

For the SBSE procedure, extraction of 4-CN was done by stirring, and liquid desorption was carried out by ultrasonication. Typically, the coated stir bar was introduced into a 15.0 mL of a 5% w/v NaCl aqueous solution of 4-CN at room temperature and stirred for 15 min at 500 rpm. Afterward, the stir bars were taken out, rinsed with de-ionized water, and dried using lint-free tissue paper. For the desorption step, the stir bar was placed in a microcentrifuge tube containing 100 µL methanol and kept in an ultrasonic bath for 10 min then followed by 20 µL injection into the HPLC system. After each extraction and desorption step, the stir bar was added to the 1.0 mL ACN and kept inside the ultrasonic bath for 10 min to ensure cleaning and removal of expected carry-over.

Furthermore, various important parameters of the extraction procedure were optimized, and these parameters include ionic strength, a solvent for the desorption, extraction time, and sample volume for extraction and desorption time. The extraction efficiency of germania-coated stir bar was also compared with PDMS-coated stir bar. All the experiments were done in triplicates.

2.5 Chromatographic condition

The chromatographic analysis was performed in reverse phase mode using an isocratic mobile phase of acetonitrile and water (v/v) 80:20 respectively with a flow rate of 1.00 mL min⁻¹. An Agilent Eclipse XDB-C18 4.6 mm ID × 250 mm (5 µm) was selected. The analysis was carried at 274 nm.

2.6 Sample analysis

The literature suggests that the chances of the hydroxyl group containing degradation products of naphthalene are mostly in water resources, blood, and urine. Therefore, three different types of samples were collected and tested for 4-CN. Firstly, the wastewater sample was collected from a drain leading to the ocean. Secondly, a sample was collected from a swimming pool. Lastly, to check the presence and the recovery of 4-CN, a urine sample was also collected.

For the extraction purpose, 5.0 mL of the sample was taken and diluted with 5.0 mL of deionized water. The germania-coated stir bar was added to the sample along with 35% NaCl and kept in it for 10 min at a 600 rpm stirring speed. After the germania-coated stir bar was taken out of the sample, it was rinsed with DI water for the removal of any sludge or impurity physically attached on the surface. Then the analyte was desorbed into a 100 µL ACN by ultrasonication for 20 min. Finally, from the 100 µL desorbed solvent, an aliquot of 20 µL was injected into HPLC system.

3.1 Characterization of PDMS/Ge coated stir bars

XPS analysis of polymer obtained from tetramethoxygermane and hydroxy-terminated polydimethylsiloxane (PDMS) is presented in Fig. SI-1 to identify the bonding state and oxidation state of the polymer’s constituent. As shown in the figure, germanium (Ge 3d), is present in (+2) oxidation state by the presence of peak at binding energy (BE) of 31.15 eV, which is mainly attributed to GeO in —Ge—O—Si— network (Murphy et al., 2014; Schmeisser et al., 1986). The Si 2p peak in the as-synthesized polymer is symmetrical and binding energy is centered at 100.07 eV, which is a characteristic of Si bounded with mainly oxygen. The BEs of O 1s as observed are found at 530.06 eV (22%) and 530.85 eV (78%), corresponding to two different components bounded with oxygen. These two components can be referenced to oxygen bounded elements in form of Si—O (530.06) and Ge—O (530.85) (Prabhakaran and Ogino, 1995). C 1s is observed in a single environment, which can be associated with the carbon from PDMS employed for the synthesis polymer coated on the of stir bar. The corresponding BE of this carbon is found at 282.48 eV, and is a characteristic of carbon bonded with silicon in PDMS. Table 1 shows the surface composition of the polymer with carbon and silicon being the main elements with total contribution of 47% and 27%, respectively. The surface contribution of oxygen is about 25%, while the germanium concentration found on the surface is less than 1%.

The PDMS/Ge polymer was subjected to thermal and structural stability test under pure N₂-gas environment by thermogravimetry analysis. As shown in Fig SI-2 there is no significant mass loss (approx. 4%) and phase transition up to temperature of 300 °C. The total mass loss due to adsorbed water and impurities associated with the polymer is approximately 8% up, with no phase change. At a temperature relatively above 450 °C, there is a drastic loss of weight until 600 °C, which accounted for 75% of total mass loss. In addition, a phase transition or change is observed and could be attributed to decomposition of organic framework of the polymer with the formation of carbon or soot (Graziella Trovati, Edgar Ap Sanches, Salvador Claro Neto, Yvonne P. Mascarenhas, 2010; Prime et al., 2009). Therefore, the potential application of this polymer in high temperature environment will be from room temperature (RT) to 400 °C, with minimal loss of material due to heating effect.

Since Stir bar sorptive extraction (SBSE) involves coating of polymeric materials onto the surface of stir-bar for extraction and pre-concentration of analytes, the distribution and uniformity of the coated PDMS/Ge polymer will be an important parameter for maximum and efficient extraction of analytes in the liquid phase. Fig. 1(a and b) presents the SEM micrographs of the PDMS/Ge coated on the stir bar at lower and higher magnifications, respectively. As shown in the figure, there is uniform distribution of polymeric materials on the stir bar without cracks, which confirms the successful coating operation for extraction of 4-CN at optimum conditions. In addition to X-ray photoelectron spectroscopy (XPS), the elemental analysis confirmed the presence of all constituents (Ge, Si, C and O) of the as-synthesized polymer coated on the bar, and there is a good agreement between the calculated (theoretical) and instrumental results, as obtained from energy dispersive X-ray (EDX) analysis shown in Fig. SI-3 (inset).

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Fig. 1 Scanning electron microscope (SEM) analysis of as-synthesized polymer (PDMS/Ge) coated on the stir bar, at lower and higher magnifications (a and b), respectively.

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3.2 Optimization of PDMS/Ge SBSE parameters

3.2.1

3.2.1 Sample volume

Sample volume is a simple but important factor for the extraction process. Considering the practical application of the analysis, it is important to determine the amount of sample that is sufficient for analysis. The sample volume was varied between 2.0 and 30.0 mL. The other parameters were 5% w/v NaCl salt, 15 min extraction time with stirring rate at 500 rpm, and 10 min desorption in methanol. The increment in the volume of sample leads to an increase in the response, but the larger volumes have no significant effect on the extraction process. The data presented in Fig. 2(a) shows the optimized sample volume to be at 10.0 mL.

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Fig. 2 Optimization of parameters; (a) Effect of the sample volume (2–30 mL), Conditions: concentration 200 ng mL⁻¹, salt addition 5% w/v, extraction time 15 min, stirring rate 500 rpm, desorption time 10 min, desorption solvent Methanol. (b) Effect of Extraction time, (2–30 min): Conditions: Sample volume 10 mL, concentration 200 ng mL⁻¹, salt addition 5% w/v, stirring rate 500 rpm, desorption time 10 min, desorption solvent Methanol. (c) Effect of Stirring rate, (300–1200 rpm): Conditions: Sample volume 10 mL, concentration 200 ng mL⁻¹, salt addition 5% w/v, extraction time 10 min, desorption time 10 min, desorption solvent Methanol. (d) Effect of the type of solvent for desorption: Methanol, Acetonitrile, Mobile phase (Acetonitrile: water 80:20). Conditions: Sample volume 10 mL, concentration 200 ng mL⁻¹, salt addition 5% w/v, extraction time 10 min, stirring rate 600 rpm, desorption time 10 min. (e) Effect of the desorption time, (5–40 min): Conditions: Sample volume 10 mL, concentration 200 ng mL⁻¹, salt addition 5% w/v, extraction time 10 min, stirring rate 600 rpm. Desorption solvent Acetonitrile. (f) Effect of the Salt addition, (5–50% w/v): Conditions: Sample volume 10 mL, concentration 200 ng mL⁻¹, extraction time 10 min, stirring rate 600 rpm. Desorption solvent Acetonitrile, Desorption time 20 min.

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3.3 Analytical performance

Under the optimum condition for extraction and desorption for the proposed PDMS-Ge SBSE HPLC-UV method, a calibration curve was obtained as shown in Fig. SI-4 (Overlaid Chromatograms in Fig. SI-7). The analyte (4-CN) showed linearity between the range of 0.4–800 ng mL⁻¹ with enrichment factor of 135. The chromatograms of the lowest and highest concentrations within the linear ranges are shown in Figs. SI-6 and SI-5, respectively. It also displayed very good linear equation y = 1.3419x + 47.458 and R² = 0.9992. The analysis proved remarkably good in terms of the limit of detection (LOD) and limit of quantification (LOQ), 0.034 (S/N = 3) and 0.400 ng mL⁻¹, respectively. The carryover of the stir bar after the complete SBSE process was also determined by desorbing the stir bar in the desorption solvent and later injecting into HPLC system, where no peak at the desired retention time was seen. Therefore, the PDMS/Ge coated stir bars proved to produce reliable results.

3.4 Preparation reproducibility of PDMS/Ge coated stir bars

In this work, preparation reproducibility of the PDMS/Ge stir bars were also analyzed. All the experimental conditions were kept similar, and two different sol-gel solutions were prepared. Two pretreated glass bars of identical size were kept inside the sol for 30 min separately as described in Section 2.3. The extraction efficiency of these two different bars was tested, and both produced similar results with 1.7% RSD for the same batch and 3.5% RSD for different batches from each other as shown in Fig. 3A.

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Fig. 3 (A) Preparation reproducibility of PDMS/Ge coated stir bar, in the same batch (Batch 1 bar 1 & 2) in different reaction batches (batch 1 bar1 and batch 2 bar 1), (B) Comparison between in-lab prepared PDMS coated bar vs PDMS/Ge coated stir bar for the extraction efficiency for 4-CN, Conditions: Sample volume 15 mL, salt addition 5% w/v, Extraction time 15 min, stirring speed 500 rpm, Desorption solvent volume 100 µL, desorption solvent methanol, Desorption time 10 min.

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3.5 Comparison of PDMS/Ge with laboratory prepared PDMS stir bar

Based on previous methods (Segro and Malik, 2010), we prepared a PDMS coated on glass stir bars, and compared its extraction efficiency with our newly developed PDMS/Ge coated stir bar. The extraction results in Fig. 3B (Chromatogram in Fig SI-8) presented that PDMS/Ge coated stir bar exhibited approximately four times more extraction as compared to PDMS coated stir bars. This increase can be explained by presenting the nature of germanium oxide. The oxides of germanium are amphoteric in nature. The addition of germanium oxide to silica-based material has provided a new network that can present both acidic and basic sites (according to Bronsted-Lowry theory) to the target analytes. Oxygens attached to germanium will have partial negative charge and germanium will get a positive charge. 4-CN also has positive and negative sites for example hydrogen of hydroxyl group will be partially positive and chlorine will have partial negative charge based on electronegativity difference. Hence, such interactions have resulted to increase the extraction efficiency of 4-CN from the sample matrix.

3.6 Wide-ranging applicability of method

In addition to various factors taken into consideration, method was tested for applicability and selectivity. For this purpose, 1-naphthol and 2-naphthol were taken as an interfering agent in the same extraction media because the chances of its existence in the same media are very high. The peaks of 1-naphthol and 2-naphthol were baseline separated from 4-CN using our developed method. Fig. 4 shows the chromatogram of 4-CN in the presence of 1-naphthol and 2-naphthol. This also proves that our SBSE-HPLC-UV method is widely applicable for various naphthols.

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Fig. 4 Chromatogram of 4-CN (t_R: 3.752, identified with *) in the presence of 2-Naphthol (t_R: 2.860), 1-Naphthol (t_R: 3.094).

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3.7 Sample analysis

The performance of the method was determined by the analysis of 4-CN in wastewater, swimming pool water, and urine. The extraction was done from the sample and from a spiked sample. However, 4-CN concentrations were found to be 0.38 ng mL⁻¹ in urine, 0.13 ng mL⁻¹ in wastewater, and 0.39 ng mL⁻¹ in pool water. Each sample was also spiked with 4-CN to have a concentration of 0.80, 150, and 500 ng mL⁻¹ (Fig. 5). Recoveries were estimated and showed in Table 2. All the extraction recoveries were ranged between 87.4 and 141.3% with acceptable % RSD between 4 and 11%.

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Fig. 5 Chromatogram showing the 4-CN (t_R: 3.752, identified with *) in pool water (A) urine (B) and waste water (C) unspiked (dotted lines) and 0.8 ng mL⁻¹ spiked (solid lines).

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