Determination of ultra-trace amounts of chlorophenols in rain, tap and river water by an electrochemically controlled in-tube solid phase microextraction method

Abstract

An electrochemically controlled in-tube solid phase microextraction method has been developed for the extraction of ultra-trace amounts of three groups of chlorophenols (CPs): mono-chlorophenols, di-chlorophenols and tri-chlorophenols in rain, river and tap water samples. Determination of the extracted CPs was performed by high performance liquid chromatography and UV detection. A new polypyrrole/Nafion composite coating was electrochemically synthesized on the inner surface of the stainless steel capillary as a working electrode (anode) for the electrochemical extraction of chlorophenols from water samples. By passing the sample solution over the inner surface of the coated electrode, the chlorophenols could be extracted by applying a positive potential under flow conditions. This method is an ideal sample preparation technique because it is easy to automate, solvent-less, and inexpensive. Some important experimental parameters that could affect the extraction and separation of the analytes, such as the extraction and desorption voltages, the pH of sample solution, the extraction and desorption times and the flow rates of the sample solution and eluent were investigated and optimized. Under optimal conditions, the limits of detection were in the range of 0.07–0.20 μg L⁻¹. This method showed good linearity for chlorophenols in the range of 0.20–500 μg L⁻¹, with coefficients of determination greater than 0.9984. The inter- and intra-assay precision (RSD%, n = 3) was in the range of 5.4–6.8% and 4.0–5.9% at three concentration levels of 2, 10 and 20 μg L⁻¹, respectively. The validated method was successfully applied for the analysis of 2-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, 2,3,6-trichloro-, and 2,4,6-trichloro phenols in water samples.

---

Introduction

Water pollution has become an urgent environmental challenge across the world, especially in developing countries with growing chemical industries. However, the complexity of water samples demands a powerful sample preparation technique, which is usually the most critical and time-consuming step for the analysis of contaminants in water matrices. Chlorophenols (CPs), as a group of hazardous pollutants, are usually released into the environment during the degradation of some pesticides,¹ chlorination of drinking water,² and industry processes such as manufacturing, refinery waste or the production of petrochemicals, plastic, coke, petroleum, pesticide and paper.³ Due to the high toxicity, persistence, and unpleasant organoleptic properties of chlorophenols, both the European Union and US Environmental Protection Agency have classified a number of chlorophenols as priority pollutants.⁴ Primarily, the research has been focused on environmental contamination, especially the contamination of water. However, the migration and transformation of chlorophenols to foods has drawn attention during recent years.^5,6 The direct determination of CPs in environmental samples is usually difficult because of their low concentration (sub μg L⁻¹). Therefore, it is vital to develop simple and sensitive methods to determine the presence of CPs in water samples. Modern trends in analytical chemistry are geared toward simplification, miniaturization of the sample preparation system, and minimization of organic solvent and sample volumes. Since solid phase microextraction (SPME) was introduced by Pawliszyn and Arthur⁷ in the early 1990s, it has been widely accepted and applied as a sample preparation technique. SPME has been developed in a number of different ways, including coated fibers and in-tube devices.⁸ To further enhance the extraction capacity and improve the stability of the fibers, in-tube solid-phase microextraction (IT-SPME) was developed.⁹ This method is simple and can be easily coupled online to high performance liquid chromatography (HPLC),^10,11 LC/MS^12,13 and LC/MS/MS.^14,15 However, IT-SPME showed poor selectivity and a low extraction efficiency to ionic compounds. Recently, in order to overcome this difficulty, a new method based on coupling electrochemically controlled solid phase microextraction (EC-SPME) with IT-SPME method, namely the electrochemically controlled in-tube SPME (EC-IT-SPME) method was developed for the extraction of ionic components.^16–18 In this method, as with the IT-SPME method, the selection of a suitable adsorbent is necessary.

Organic polymers are the most widely used coatings for the extraction of organic compounds and can be applied to a variety of supports. Since the first report of electrical conductivity in a conjugated polymer in 1977 by Shirakawa et al.,¹⁹ conducting polymers, particularly polypyrrole (Ppy) and its derivatives have received much attention due to their multifunctional properties such as hydrophobicity, their acid–base character, electroactivity, polar functional groups, ion exchange property, hydrogen bonding, and ability to establish π–π interactions.^20,21 Although conductive polymers were successfully employed as IT-SPME coatings, there are some limitations in certain applications. For example, the connection between Ppy and stainless steel wire is not very strong, and its extraction capacity and mechanical stability are still not large enough.^22,23

Nafion (Naf) is a sulfonated tetrafluoroethylene based fluoro copolymer. In 1998, Naf was applied as a fiber coating material for the extraction of alcohols from hexane and gasoline samples and exhibited better extraction efficiency than all of the commercially available selected coatings.²⁴ Naf has a high mechanical stability, excellent protonic conductivity, high ion exchange capacity,²⁵ and good affinity towards polar analytes.²⁶ These features leads to increased use of Naf as a binder and the extraction phase in SPME.^26–28 Hence, the use of Naf as an anion dopant in the electrochemical synthesis of Ppy and the formation of a composite form of Ppy–Naf presents new opportunities to enhance the extraction efficiency, mechanical stability and number of SPME coatings. In this process, Naf not only plays the role of anion dopant but it also acts as a sorbent and binder too.

In continuing our previous studies on developing the EC-in-tube SPME method,^16,17,29 a new nanostructured copolymer coating consisting of Ppy doped with Naf was electrochemically synthesized on the inner surface of a stainless steel capillary tube by cyclic voltammetry. Then, it was utilized for on-line EC-IT-SPME coupled with HPLC for the determination of chlorophenols in rain, river and tap water samples. Combining the properties of Ppy and Naf, the Ppy/Naf coated capillary was thought to exhibit high extraction efficiency for chlorophenols from different water matrices.

Experimental

Chemicals and reagents

All chemicals were analytical reagent grade. Standards of 2-chlorophenol (2-CP), 4-chlorophenol (4-CP), 2,3-dichlorophenol (2,3-DCP), 2,4-dichlorophenol (2,4-DCP), 2,3,6-trichlorophenol (2,3,6-TCP), and 2,4,6-trichlorophenol (2,4,6-TCP) were obtained from Sigma-Aldrich (Milwaukee, WI, USA). The stock solutions of the chlorophenols (1000 mg L⁻¹) were prepared by dissolving 10 mg of the compounds in 10 mL methanol. Lower concentrations were prepared by diluting the stock solution with methanol. Synthetic pyrrole (98% pure) was obtained from Sigma-Aldrich. Nafion (a 5% (w/w) in a mixture of a lower aliphatic alcohol and water) was obtained from Sigma–Aldrich. Sodium hydroxide and hydrochloric acid (95% pure) were obtained from Merck (Darmstadt, Germany). Ultra-pure water was produced using a Youngling ultrapure water purification system model Aqua MaxTM-ultra (Seoul, South Korea). Other chemicals used were of analytical reagent grade or of the highest available purity.

Apparatus

Chromatographic analysis was performed with an HPLC instrument including a Varian 9012 HPLC pump (Walnut Creek, CA, USA), a six-port Cheminert HPLC valve from Valco (Houston, TX, USA) with a 20 μL sample loop and equipped with a Varian 9050 UV-vis detector. Chromatographic data were recorded and analyzed using Chromana software (version 3.6.4). The separations were run on an ODS-3 column (250 mm × 4.6 mm, with a 5 μm particle size) from Hector Company (Daejeon, Korea). All the pH measurements were performed with a WTW Inolab pH meter (Weilheim, Germany). A GPFA1-380 peristaltic pump from Ultra-Voltammetry Company (Tehran, Iran) was used to pass the solutions through the stainless-steel capillary tube. The particle size and morphology of the synthesized NPs were determined by scanning electron microscopy (SEM), model EM3200 from KYKY Zhongguancun (Beijing, China).

Chromatographic separation

The chromatographic separation was performed by using a mobile phase consisting of 10 mmol L⁻¹ phosphate buffer (adjusted by nitric acid to pH 3.0) and acetonitrile, and delivered at a flow rate of 1.2 mL min⁻¹. The gradient program was as follows: starting with 20% acetonitrile, then increasing to 55% in 15 min, keeping constant until 18 min, increasing to 80% in 18–23 min, keeping constant until 26 min, thereafter restored to 20% in 2 min, followed by 5 min equilibration time. Detection of the analytes was achieved at 280 nm.

Preparation of polymer-coated capillary tubes

The Ppy/Naf coating was prepared electrochemically using a cyclic voltammetry instrument. A stainless steel tube (10 cm length and 0.75 mm diameter) was employed as the working electrode; Pt and saturated Ag/AgCl electrodes were used as the counter and reference electrodes, respectively. A peristaltic pump was used to deliver the monomer solution from the inner surface of the stainless steel tube. Before electrochemical deposition, the steel tubes were cleaned with acetone and HPLC-grade water and finally air dried at room temperature. As pyrrole has different roles in polymeric films including anion dopant, binder to immobilize the Ppy/Naf film on the inner surface of the stainless steel capillary and extraction phase, it seems that the volume of Nafion is a critical parameter in the polymer synthesis and optimization of the pyrrole volume is necessary. The Ppy/Naf film was electrodeposited directly on the inner surface of a stainless steel tube electrode from a 40 mL aqueous solution containing 0.2 M pyrrole and a different volume of Nafion as the anion dopant (0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 mL) using cyclic voltammetry. The cyclic voltammetry instrument was operated at a scan rate of 50 mV s⁻¹ within the potential range of −0.6 to 0.6 V and the number of scans was set at 25. Fig. S1† shows the multi sweep cyclic voltammograms for 0.2 M pyrrole monomer and 1 mL Nafion in water solution. The slight increase in the peak currents with the increase of the number of cycles indicates the growth of an electroactive Ppy/Naf film inside the electrode surface. After electrochemical deposition, the steel tube coated with the Ppy/Naf film was washed with methanol, acetone, and water sequentially to remove all excess pyrrole and Nafion from the film, then dried under nitrogen gas flow. Fig. S1† shows the multi sweep cyclic voltammograms taken during the electropolymerization of pyrrole in the presence of Naf. In order to select the best conditions for the electrochemical synthesis of the Ppy/Naf coating, the volume of Naf was optimized in the range of 0.2–1.4 mL. Result showed that when 1.0 mL of Naf was used as the electrolyte, maximum extraction efficiency was obtained. So, 1.0 mL of Naf was used for electrochemical synthesis of Ppy/Naf coating.

On-line EC-IT-SPME procedure

A schematic diagram of the complete assembly and operation mode of the instrument is shown in Fig. 1. The Ppy/Naf coated stainless-steel tube was mounted on valve 1 (V1) in the position where the loop was originally positioned. Capillary connections were facilitated by using a 2.5 cm sleeve of 1/16-in polyether ether ketone (PEEK) tubing at each end of the capillary. Both V₁ and valve 2 (V₂) were initially set at load position (red arrows). Pump A is on to flow the sample solution through the tube at 3.3 mL min⁻¹ and pump B is off. The effluent of V₁ was poured back into sample compartment after passing through the coated tube. In summary, this procedure was carried out in a circulating path. The platinum electrode was connected to the negative potential and used as the cathode electrode. By passing the sample solution through the Ppy/Naf electrode, the extraction of chlorophenols (anionic form) occurred by applying a positive potential (0.8 V) under flow conditions. After extraction for a given time interval, the Pt electrode was connected to the positive potential and used as the anode electrode. V₁ and V₂ were directed to the inject position, and pump A was turned off while pump B was turned on to flow the desorption solvent (0.1 mol L⁻¹ NaCl in water) through the tube at 3.0 mL min⁻¹. By passing 80 μL of the desorption solvent over the inner surface of the Ppy/Naf electrode, desorption of chlorophenols occurred by applying a negative potential (−0.7 V). Finally after a given desorption time, V₂ was directed to the load position and the desorption solvent was collected in HPLC loop. Then pump B was turned off, V₂ was returned to the injection position and the extracted analytes collecting in the loop of V₂ were eluted by the mobile phase into the HPLC column for analysis.

Fig. 1 Schematic representation of the EC-in-tube SPME followed by on-line HPLC analysis.

Results and discussion

In the present study, the applicability of the Ppy/Naf coating capillary in the EC-IT-SPME method was investigated for the extraction of chlorophenols from different water samples. Based on the literature and the previous experience of our research group,^16,17,29 in order to optimize the extraction efficiency of chlorophenols by EC-IT-SPME procedure, seven independent variables, namely extraction voltage, desorption voltage, pH of sample solution, extracting flow rate, desorption flow rate, extraction time and desorption time should be considered. The effect of extraction voltage and desorption voltage on the extraction of chlorophenols by the suggested method was optimized using a “one-variable at-a-time” (OVAT) process. Optimizing the effects of the other parameters was performed using the response surface method. The optimization was carried out using working solutions containing 50 μg L⁻¹ of the analytes. The injected volume of the extracted analytes into HPLC/UV was kept constant at 20 μL during the experiments. Initial experimental conditions were as follows: extraction voltage +0.5 V, desorption voltage −0.5 V, extraction time, 15 min; desorption time, 5 min; extraction flow rate, 2.0 mL min⁻¹; desorption flow rate, 2.0 mL min⁻¹ and pH = 8.

Characterization of Ppy/Naf coating

Ppy is the cationic polymer and for making a neutral compound, a counter ion (dopant) is used in the electrochemical synthesis procedure. Also, Nafion contains sulfonate groups with a high ion exchange capacity that can contribute as a dopant for Ppy. Naf is a strong adsorbent and binder and can interact with analytes by dipole–dipole interactions. Hence, a Naf coated fiber could show the best affinity for extracting most of the polar compounds.

During electropolymerization, a dark film formed slowly on the inner surface of the stainless steel capillary. Fig. S1† shows the cyclic voltammograms of the Ppy/Naf electropolymerization process. The increase in the anodic peak current and the negative shift of the initial oxidation potential when increasing the number of scans shows the growth of the Ppy/Naf film occurring on the inner surface of the stainless steel capillary while the scans are run. Hence, a coating with the desired thickness can be easily prepared using appropriate scan numbers.

The coating thickness and surface morphology of the Ppy/Naf coated capillary was characterized by SEM under different magnifications (Fig. S2†). As illustrated in Fig. S2a,† the thickness of the coating was about 55 μm. The high-magnification SEM image (Fig. S2b†) indicated that the coating fiber possesses a homogenous and highly porous structure together with Ppy and Naf nanoparticles on the surface, which should significantly increase the specific surface area of the coating and improve the efficient adsorption/extraction of the analytes. The nano-structured particles are observed in Fig. S2C† with a diameter lower than 70 nm.

In order to investigate the swelling, mechanical stability, acid and alkaline resistance of the present coating, acetone, methanol, acetonitrile, hydrochloric acid (1 mol L⁻¹) and sodium hydroxide (1 mol L⁻¹) were passed directly through the coated capillary for more than 4 h. Finally, the Ppy/Naf coated capillary was used to extract the chlorophenols from the aqueous sample. The result shows no measurable change in the extraction quantities after passing each of the solvents through the coated capillary. This is an indicator of the favorable chemical stability and mechanical strength of the Ppy/Naf coating. Moreover, all extraction processes including optimization, calibration curve and the analysis of real samples were carried out using one coated tube.

Memory effect

After the desorption step, the memory effect in the Ppy/Naf coating was investigated in order to detect the presence of species in the coating. The carry-over of CPs was only 0.06%. This advanced performance is mainly due to two reasons: (a) applying a negative potential in the desorption step, which increases the rate and amount of desorption of the analyte from the coating and decreases the memory effect, (b) selecting suitable desorption time, which decreases the memory effect.

To minimize the carry-over effects after each extraction, the Ppy/Naf coated tube was washed by passing methanol–water (50 : 50 v/v) solution for 5 min.

OVAT optimization of extraction and desorption voltages

It is known that in EC-IT-SPME, the required time to reach the extraction equilibrium is proportional to the extraction voltage. The impact of the extraction voltage upon the results achieved from the EC-IT-SPME of chlorophenols was investigated at seven levels in the range of 0 to +1.0 V. Fig. 2A shows that applying positive voltage to extract the anionic form of the chlorophenols with EC-IT-SPME is the most effective way of improving the extraction efficiency. The results showed that by increasing the extraction voltage from 0 to 0.8 V, the extraction efficiency increases significantly. By increasing the extraction voltage above 0.8 V; no further substantial increase was obtained. Hence, an extraction voltage of 0.8 V was selected for the next experiments.

Fig. 2 Effect of (A) extraction voltage (V), (B) desorption voltage (V) on the extraction efficiency of chlorophenols. Initial experimental conditions: extraction voltage +0.5 V, desorption voltage −0.5, extraction time, 15 min; desorption time, 5 min; extraction flow rate, 2.1 mL min⁻¹; desorption flow rate, 2.1 mL min⁻¹ and pH 8.

In order to ensure the complete desorption of the analytes from the Ppy/Naf coating and avoid the memory effect or carryover, a suitable desorption voltage was optimized by applying different desorption voltages, from 0 to −1.0 V while the sample volume was kept constant at 30 mL. Due to the high solubility of the undissociated chlorophenols in methanol, a low negative voltage was needed for the desorption of chlorophenols from the surface of the Ppy/Naf tube. Fig. 2B shows that the desorption efficiency was slowly increased by a variation of the desorption voltage from 0 to −0.7 V. When desorption voltage was raised from −0.7 to −1.0 V, the extraction efficiency remained relatively constant. Hence, a desorption voltage of −0.7 V was selected for the next experiments.

Optimization of parameters using fractional central composite design

In the next step, a rotatable, orthogonal half-fractional central composite design (FCCD) was employed to determine the optimal conditions for the remaining factors, namely the pH of the sample solution (A), the extracting flow rate (B), the desorption flow rate (C), the extraction time (D) and the desorption time (E). This design is a combination of a two-level half-factorial design (N_f = 2^f−1 = 2⁵⁻¹), axial points (N_a = 2f = 2 × 5), and a set of center points (N₀ = 10) of the experimental region. Center points are usually repeated to get a good estimate of experimental error (pure error). The axial points are located at +α and −α from the center of the experimental domain. An axial distance α was selected with a value of 2.0 in order to establish the rotatability and orthogonality conditions.^28,30

The total number of experiments needed (N) to perform FCCD was determined to be 36 using eqn (1):

N = 2^f−1 + 2f + N₀ (1)

where, f is the number of variables. Table S1† shows the coded and uncoded values of the designed experiments with the HPLC/UV peak area values. In total, the design matrix had 36 runs where the runs were randomly carried out in order to nullify the effect of extraneous or “nuisance” variables. For an experimental design with five factors, the model including quadratic and cross terms can be expressed as eqn (2).

R₁ = +1623.04 + 185.08 × A + 165.96 × B + 122.96 × C + 220.27 × D + 145.96 × E + 88.71 × AD + 138.06 × BC + 147.94 × BE − 234.45 × B² − 271.32 × C² + 97.93 × D² − 154.45 × E² (2)

Table 1 shows the regression coefficients of the proposed accuracy model and their P-values (probability of error), which are used to determine the significant parameters. The analysis of the results from the analysis of variance (ANOVA) showed all factors were statistically significant for this microextraction procedure at a 95% confidence level (P-value > 0.05). The obtained ANOVA results in Table 1 demonstrated that the F value of 23.15 is significant, and there is only a 0.01% chance that a large “model F-value” could occur by accident. Also, ANOVA was performed (Table 1) and showed that the model was significant (P-value < 0.0001) and the “lack of fit” was not significant (P-value = 0.2525), which implied that the model was fitted. The quality of the model was expressed by the coefficient of determination (R² and adjusted-R²).

Table 1 Analysis of variance table (ANOVA) for response surface quadratic model

Source	Sum of square	d.f.^a	Mean square	F-Ratio	P-Value	Effect
a Degree of freedom.
Model	1.119 × 10^7	12	9.324 × 10^5	23.15	<0.0001	Significant
A-pH	1.850 × 10^6	1	1.850 × 10^6	45.93	<0.0001
B-Extraction flow rate	6.610 × 10^5	1	6.610 × 10^5	16.41	0.0005
C-Desorption flow rate	3.629 × 10^5	1	3.629 × 10^5	9.01	0.0064
D-Extraction time	9.981 × 10^5	1	9.981 × 10^5	24.78	<0.0001
E-Desorption time	5.113 × 10^5	1	5.113 × 10^5	12.69	0.0017
AD	2.833 × 10^5	1	2.833 × 10^5	7.03	0.0142
BC	3.050 × 10^5	1	3.050 × 10^5	7.57	0.0114
BE	3.502 × 10^5	1	3.502 × 10^5	8.69	0.0072
B^2	1.759 × 10^6	1	1.759 × 10^6	43.67	<0.0001
C^2	2.356 × 10^6	1	2.356 × 10^6	58.49	<0.0001
D^2	3.069 × 10^5	1	3.069 × 10^5	7.62	0.0111
E^2	7.633 × 10^5	1	7.633 × 10^5	18.95	0.0002
Residual	9.264 × 10^5	23	40 278.08
Lack of fit	6.567 × 10^5	14	46 903.98	1.56	0.2525	Not significant
Pure error	2.697 × 10^5	9	29 971.12
Cor total	1.212 × 10^7	35

---

The R² and adjusted-R² were calculated according to the following equations:

SS_res and SS_tot are the sum of squares of the residual and core total, respectively. N_res and N_tot are the degrees of freedom of the residual and core total, respectively. R² is a measure of the amount of variations around the mean explained by the model and is equal to 0.9235. The adjusted-R² is adjusted for the number of terms in the model and it decreases as the number of terms in the model increases if those additional terms do not add value to the model. It is equal to 0.8836. In the present study, the adjusted R² is well within the acceptable limits of R² > 0.8 and is similar to R² which reveals that the experimental data shows a good fit with the second-order polynomial equations (Table 1).

Fig. 3 illustrates the relationship between the explanatory and response variables in a three-dimensional representation of the response surface. After an analysis of the results, the following conditions were selected to evaluate the performance of the extraction procedure: (a) a sample pH value of 8.5; (b) an extraction time of 25 min (c) a desorption time of 8 min (d) an extraction flow rate of 3.3 mL min⁻¹ (e) a desorption flow rate of 3.0 mL min⁻¹. Through the use of Design-Expert 8.0.3, polynomial equations, response surface and central plots for a particular response are produced.

Fig. 3 Response surfaces for: (a) extraction time (min) vs. pH of sample solution; (b) desorption flow rate (mL min⁻¹) vs. extraction flow rate (mL min⁻¹); (c) desorption time (min) vs. extraction flow rate (mL min⁻¹) and counter plot for (d) desorption time (min) vs. extraction flow rate (mL min⁻¹).

Method validation

Analytical performances for on-line EC-IT-SPME–HPLC analysis of chlorophenols in the water sample were investigated. Parameters including the calibration curves, establishing linearity, extraction recovery (ER%), limits of detection (LODs), limit of quantification (LOQs), intra- and inter-assay precision (RSD%), and accuracy (relative recovery%) were performed under the optimized conditions according to the EPA forum on environmental measurements (FEM) and the results are listed in Table 2.

Table 2 Figures of merit for the EC-IT-SPME of chlorophenols

Chlorophenols	Linearity			Precision^a (RSD%, n = 3)						Tube-to-tube RSD% (n = 3)
				Inter-day			Intra-day			Intra-day	Inter-day	ER (%)
	LDR^a	R^2	LOD^a	2	10	20	2	10	20	10	10
a All concentrations are in μg L^−1.
2-CP	0.5–500.0	0.9984	0.2	6.4	6.3	5.8	5.2	4.7	4.7	5.0	6.5	54.6
4-CP	0.5–500.0	0.9987	0.2	6.8	6.4	6.2	5.9	5.7	5.4	6.0	6.4	53.2
2,3-DCP	0.4–500.0	0.9990	0.1	6.2	6.0	5.7	5.5	5.0	4.8	5.3	6.0	59.9
2,4-DCP	0.4–500.0	0.9986	0.1	6.5	6.1	5.9	5.3	5.1	4.6	5.9	6.8	59.0
2,3,6-TCP	0.2–500.0	0.9991	0.07	6.1	5.9	5.6	5.0	4.6	4.2	4.5	5.7	61.0
2,4,6-TCP	0.2–500.0	0.9987	0.07	5.8	5.7	5.4	4.8	4.4	4.0	4.8	5.9	62.5

---

The ER% was calculated according to the following equation:

where n_elu and n₀ are the mole numbers of analyte in the eluent phase and the initial mole numbers of analyte in the sample solution, respectively. C_elu and C₀ are the concentration of analyte in the eluent phase and the initial concentration of analyte in the sample solution, respectively. PF is the preconcentration factor and V_elu and V_aq are the volumes of the receiving and the source phases, respectively. Under optimum conditions, an ER% in the range of 53.2–62.5% was obtained. As can be seen, the proposed EC-IT-SPME method shows an ER% that is better or comparable to the previously reported methods in most cases. Better extraction recoveries can effectively improve the linear dynamic ranges and limits of detection. The linear dynamic range was evaluated by plotting the calibration curve based on the peak areas versus the concentration of the chlorophenols using ten concentration levels over a range of 0.2–500 μg L⁻¹. Desirable linearity was achieved for all of the analytes with a coefficients of determination more than 0.9984 in the studied range. The values of LOD (S/N = 3/1) were in the range of 0.07–0.2 μg L⁻¹ and LOQs (S/N = 10/1) were between 0.2–0.5 μg L⁻¹. Intra-day (n = 3) and inter-day standard deviations were calculated by extracting the analytes from water samples at levels of 2, 10, and 20 μg L⁻¹ and RSDs% of lower than 6.0% and 6.8% were obtained, respectively. The tube-to-tube reproducibility was assessed by calculating the relative standard deviation (RSD%) for chlorophenols extraction. The intra-day and inter-day RSDs% were in the range of 4.5–6.0% and 5.7–6.8%, respectively.

The extraction capabilities of the Ppy/Naf coating for the extraction of chlorophenols in water samples were compared with other coating materials in the literature.^31–34 As Table 3 shows, our results are comparable with the data obtained by other researchers using GC and LC, although in most cases the results show rather lower detection limits for chlorophenols. Moreover, the linearity and precision of this method were comparable to (or better than) those of the other techniques reported for the extraction and determination of chlorophenols. This comparison shows that the proposed extraction method is very simple and does not need any complex and expensive instruments. It is a sensitive and rapid method with low consumption of extraction solvents and consequently less organic waste. It also shows an enhanced sensitivity and short analysis time.

Table 3 Comparison of the proposed method with other microextraction techniques for determination of chlorophenols in different samples

Extraction technique^a	Type of coating	Extraction time (min)	Linear range^a (μg L^−1)	r^2^a	LOD^a (μg L^−1)	RSD%	Ref.
a The data reported are related to water samples.
SPME-GC/MS	γ-Fe2O3@SiO2-PW	30	1–200 (4-CP) 0.1–200 (2,4-DCP, 2,4,6-TCP)	>0.9960	0.005–0.06	<10.1	31
SPME-GC/MS	PIL	60	5–20 (2-CP) 4–20 (2,4-DCP)	>0.990	1.5–4.0	<12.0	32
SPME-GC/FID	UiO-66	50	1–1000 (2,4-DCP)	0.995	0.15	<5.9	33
SPME–HPLC/UV	MWCNTs-COOH	30	26.25–1050 (2,4-DCP) 20.0–500 (2,4,6-TCP)	>0.9960	1.43–1.58	<6.38	34
EC-IT-SPME–HPLC/UV	Ppy/Naf	25	1.0–100 (2-CP, 4-CP) 0.6–50 (2,3-DCP, 2,4-DCP) 0.3–50 (2,3,6-TCP, 2,4,6-TCP)	>0.9984	0.1–0.7	<6.8	The present method

---

Analysis of rain, tap and river water

To evaluate the applicability and accuracy of the suggested method for the determination of CP in real water samples, three kinds of water samples (tap water, rain water and river water) were analyzed using the proposed method. The tap water sample was collected fresh from our laboratory. Rain water with numbers of I, II, III and IV were collected from different parts of Tehran (Iran). River water was obtained from Aras River (Azarbaijan Sharghi, Iran). The results of the real sample analysis are shown in Table 4, and indicate a satisfactory agreement among the obtained results with spiked values. Accuracy was calculated as the relative recovery (RR%) for the analysis of known amounts of the target analyte added into the real samples using the proposed method (Table 4). The RR% was acquired from the equation below:³⁵wherein C_initial, C_found, and C_added are the concentrations of analyte in the real sample, the concentration of analyte after the addition of a known amount of the standard into the real sample, and the concentration of a known amount of the spiked standard into the real sample, respectively. The RR% for samples obtained by EC-IT-SPME–HPLC method were between 94–105.3%, indicating that the process is not influenced by the matrix of the samples containing the studied chlorophenols. Precision, defined as the relative standard deviation (RSD%), was determined by three repeated determinations in each concentration level in the range of expected calibrations. The RSD% for chlorophenol determination in the examined real water samples were located in the range of 5.5–7.2%. The results confirmed that the tap water sample was free of these chlorophenols. However, the target analytes were detected in all of the rain water and river water samples. Analysis of the rain water shows that the concentrations of chlorophenols in Amirabad and Valiasr rain waters were higher than the Tajrish and Ekbatan rain water. Based on this result it can be concluded that air pollution in these areas is higher than in Tajrish and Ekbatan. Fig. 4 depicts the EC-IT-SPME–HPLC/UV-vis chromatograms of chlorophenols in the rain water III and river water, before and after spiking with 15 μg L⁻¹ and 10 μg L⁻¹ of chlorophenols, respectively. It can be concluded that the matrices of natural water samples have no significant effect on the extraction efficiency of the present method.

Table 4 Analytical results for extraction and determination of chlorophenols in real samples^a

Sample	Cadded		2-CP	4-CP	2,3-DCP	2,4-DCP	2,3,6-TCP	2,4,6-TCP	ME%
a All concentrations in this table are in μg L^−1.b n.d.: not detected.c n = 3.
Rain water I (Tajrish-Tehran)	0		1.5	n.d.^b	n.d.	0.8	n.d.	n.d.
	10.0	RR%	103.0	99.0	98.0	97.0	102.0	104.0	97.6
	75.0	RR%	101.0	97.1	100.0	95.0	103.0	102.0	98.9
	200.0	RR%	105.2	98.3	103.6	104.3	99.4	102.6	99.8
	10.0	RSD%^c	6.6	6.3	6.0	6.4	5.8	5.5
Rain water II (Valiasr-Tehran)	0		3.6	2.7	3.2	5.3	6.0	4.0
	15.0	RR%	101.3	102.0	98.0	101.3	99.3	100.7	98.0
	75.0	RR%	104.2	101.3	99.6	104.2	101.8	100.5	98.5
	200.0	RR%	99.6	101.9	99.2	102.5	99.7	99.9	98.3
	10.0	RSD%	7.0	7.0	6.6	6.0	5.6	5.8
Rain water III (Amirabad-Tehran)	0		7.2	5.6	6.4	4.0	10.3	6.9
	15.0	RR%	98.7	98.0	102.7	105.3	97.3	97.3	97.9
	75.0	RR%	99.4	98.6	102.1	102.2	99.2	99.6	99.1
	200.0	RR%	101.0	98.6	100.3	105.5	98.9	99.3	98.6
	10.0	RSD%	7.2	6.8	6.1	6.3	5.9	6.0
Rain water IV (Ekbatan-Tehran)	0		3.0	2.5	n.d.	n.d.	4.5	1.0
	10.0	RR%	97.0	96.0	97.0	105.0	103.0	99.0	97.9
	75.0	RR%	98.6	96.9	97.6	103.7	102.0	99.5	98.3
	200.0	RR%	99.0	98.3	98.8	103.1	99.7	98.6	99.4
	10.0	RSD%	6.5	6.7	6.4	6.0	6.1	5.7
River water	0		0.9	n.d.	n.d.	n.d.	0.5	n.d.
	10.0	RR%	102.0	98.0	95.0	98.0	103.0	94.0	97.3
	75.0	RR%	101.8	98.7	96.2	97.5	102.8	97.3	98.8
	200.0	RR%	102.2	100.4	98.4	99.6	102.0	97.8	98.7
	10.0	RSD%	6.6	6.4	6.3	6.5	6.2	5.9
Tap water	0		n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
	10.0	RR%	96.0	96.0	99.0	95.0	97.0	98.0	98.9
	75.0	RR%	95.9	97.7	99.9	97.8	99.1	98.3	99.4
	200.0	RR%	98.0	97.6	99.4	98.5	101.0	98.2	99.9
	10.0	RSD%	6.4	6.2	6.5	6.0	5.8	5.5

---

Fig. 4 The HPLC-UV chromatograms of non-spiked and spiked samples where (A) 15 μg L⁻¹ of chlorophenols in rain water III (B) 10 μg L⁻¹ of chlorophenols in river water samples.

Conclusions

The feasibility of EC-IT-SPME for the analysis of CPs in water has been demonstrated. In this study, a novel polypyrrole/Nafion coating was fabricated by a simple electrochemical deposition method on the inner surface of a stainless steel tube as a working electrode for on-line EC-IT-SPME of trace amounts of chlorophenols followed by HPLC-UV. The Ppy/Naf coating was found to be the most effective coating for the analysis of CPs. In the Ppy/Naf coating, Naf plays the roles of anion dopant, binder and sorbent. Combining the properties of Ppy and Naf, the Ppy/Naf coated capillary exhibited a high extraction efficiency for chlorophenols. In addition, the fiber proved to be highly stable in organic solvents (acetone, methanol and acetonitrile), hydrochloric acid (1 mol L⁻¹) and sodium hydroxide (1 mol L⁻¹). Based on the obtained results, it can be predicted that the EC-IT-SPME technique based on Ppy/Naf coating could be used for the efficient extraction of anionic species in complex aqueous solution matrices without the need to modify the fiber coating. The method was successfully applied for the evaluation of chlorophenol concentrations in some water samples and satisfactory results were obtained. Also, this method can be used for the analysis of CPs in water samples at levels even lower than 0.2 μg L⁻¹, which is the European Community legislation limit for individual phenols in drinking water.⁴

Acknowledgements

The authors gratefully acknowledge financial support from Tarbiat Modares University.

References

1. S. Morales, P. Canosa, I. Rodríguez, E. Rubí and R. Cela, J. Chromatogr. A, 2005, 1082, 128–135.
2. J. B. Quintana, R. Rodil, S. Muniategui-Lorenzo, P. López-Mahía and D. Prada-Rodríguez, J. Chromatogr. A, 2007, 1174, 27–39.
3. G. Busca, S. Berardinelli, C. Resini and L. Arrighi, J. Hazard. Mater., 2008, 160, 265–288.
4. L. Wennrich, P. Popp and M. Möder, Anal. Chem., 2000, 72, 546–551.
5. L. Maggi, A. Zalacain, V. Mazzoleni, G. L. Alonso and M. R. Salinas, Talanta, 2008, 75, 753–759.
6. N. Campillo, P. Viñas, J. I. Cacho, R. Peñalver and M. Hernández-Córdoba, J. Chromatogr. A, 2010, 1217, 7323–7330.
7. C. L. Arthur and J. Pawliszyn, Anal. Chem., 1990, 62, 2145–2148.
8. H. Kataoka, Anal. Bioanal. Chem., 2002, 373, 31–45.
9. R. Eisert and J. Pawliszyn, Anal. Chem., 1997, 69, 3140–3147.
10. Y. Li and H. Xu, J. Chromatogr. A, 2015, 1395, 23–31.
11. L. P. Melo, R. H. C. Queiroz and M. E. C. Queiroz, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., 2011, 879, 2454–2458.
12. Y. Nonaka, K. Saito, N. Hanioka, S. Narimatsu and H. Kataoka, J. Chromatogr. A, 2009, 1216, 4416–4422.
13. Y. Yang, H. Lord and J. Pawliszyn, J. Chromatogr. A, 2013, 1318, 12–21.
14. J. H. Dahl and R. B. Van Breemen, Anal. Biochem., 2010, 404, 211–216.
15. C. Cavaliere, P. Foglia, C. Guarino, M. Nazzari, R. Samperi and A. Laganà, Anal. Chim. Acta, 2007, 596, 141–148.
16. H. Asiabi, Y. Yamini, S. Seidi, A. Esrafili and F. Rezaei, J. Chromatogr. A, 2015, 1397, 19–26.
17. H. Asiabi, Y. Yamini, F. Rezaei and S. Seidi, Microchim. Acta, 2015, 182, 1941–1948.
18. S. H. Ahmadi, A. Manbohi and K. T. Heydar, Anal. Chim. Acta, 2015, 853, 335–341.
19. H. Shirakawa, E. J. Louis, A. G. MacDiarmid, C. K. Chiang and A. J. Heeger, J. Chem. Soc., Chem. Commun., 1977, 578–580.
20. H. Ge, K. J. Gilmore, S. A. Ashraf, C. O. Too and G. G. Wallace, J. Liq. Chromatogr., 1993, 16, 1023–1044.
21. J. Zou, X. Song, J. Ji, W. Xu, J. Chen, Y. Jiang, Y. Wang and X. Chen, J. Sep. Sci., 2011, 34, 2765–2772.
22. S. Zhao, M. Wu, F. Zhao and B. Zeng, Talanta, 2013, 117, 146–151.
23. X. Rong, F. Zhao and B. Zeng, Talanta, 2012, 98, 265–271.
24. T. Gorecki, P. Martos and J. Pawliszyn, Anal. Chem., 1998, 70, 19.
25. C. M. Bautista-Rodríguez, A. Rosas-Paleta, J. A. Rivera-Márquez and O. Solorza-Feria, Int. J. Electrochem. Sci., 2009, 4, 60–76.
26. J. Zeng, C. Zhao, J. Chen, F. Subhan, L. Luo, J. Yu, B. Cui, W. Xing, X. Chen and Z. Yan, J. Chromatogr. A, 2014, 1365, 29–34.
27. W. Chen, J. Zeng, J. Chen, X. Huang, Y. Jiang, Y. Wang and X. Chen, J. Chromatogr. A, 2009, 1216, 9143–9148.
28. J. Dron, R. Garcia and E. Millan, J. Chromatogr. A, 2002, 963, 259–264.
29. H. Asiabi, Y. Yamini, S. Seidi, M. Safari and M. Shamsayei, RSC Adv., 2016, 6, 14049–14058.
30. H. Sereshti, V. Khojeh and S. Samadi, Talanta, 2011, 83, 885–890.
31. M. M. Abolghasemi, S. Hassani, E. Rafiee and V. Yousefi, J. Chromatogr. A, 2015, 1381, 48–53.
32. J. López-Darias, V. Pino, J. L. Anderson, C. M. Graham and A. M. Afonso, J. Chromatogr. A, 2010, 1217, 1236–1243.
33. H. Shang, C. Yang and X. Yan, J. Chromatogr. A, 2014, 1357, 165–171.
34. X. Liu, Y. Ji, Y. Zhang, H. Zhang and M. Liu, J. Chromatogr. A, 2007, 1165, 10–17.
35. Y. Fan, Y. Q. Feng, Z. G. Shi and J. B. Wang, Anal. Chim. Acta, 2005, 543, 1–8.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13768g

---