2.1 Reagents and samples

All the reagents and solvents used in this study were of the highest analytical purity grade. The studied compounds include 2-chlorophenol (2-CP, 99%) and 2,4-dichlorophenol (2,4-DCP, 99%) were purchased from Sigma Aldrich GmbH (Steinheim, Germany). The main physico-chemical properties of these molecules are presented in Table 1.

Acetonitrile and formic acid were purchased from J.T. Baker (Deventer, The Netherlands). Methanol and dichloromethane were obtained from Fischer Scientific-Bioblock (Illkirch, France). Acetic acid and ethyl acetate were supplied by Acros Organics (Noisy-le-Grand, France).

2.2 Preparation of standard solutions and water samples

Individual stock standard solutions of 1000 mg L⁻¹ of each chlorophenol were prepared in methanol. A stock standard mixture of the target compounds was weekly prepared by diluting the individual stock solutions in methanol. The working solutions were freshly prepared in acetonitrile/ultrapure water (55/45, v/v) by appropriate dilutions of the stock standard solution to reach the working concentration range. This composition ensured good stability of the chlorophenols in water samples. The ultrapure water used for the preparation of the samples was produced by an Elga Option-Q DV-25 system (Antony, France). All solutions were stored in glass bottles in the dark at −20 °C. Standard mixtures were preserved two months and working solutions three weeks. To demonstrate the applicability of the developed method real water samples including river water and drinking water were used in this work. The samples were collected in October 2013 and January 2014, from different locations (from Brittany region, France). All samples were collected in baked glass 10 L amber bottles with Teflon lined caps to ensure sample integrity, filtered through a 0.45 μm cellulose membrane and then stored in the dark at 4 °C until their analysis (within one week of collection).

2.3 Apparatus and analytical conditions

The quantification of chlorophenols was achieved using an Acquity™ ultra-high pressure liquid chromatography system coupled to a photodiode array detector (UHPLC/PDA), purchased from Waters (Saint-Quentin en Yvelines, France).

The chromatographic analyses were carried out using an Acquity UHPLC H-Class system, containing a binary pump, an auto-sampler and a thermostated column compartment (Waters, Saint-Quentin en Yvelines, France). Chromatographic conditions that directly affect chromatographic separation such as chromatographic column, elution mode, mobile phase composition and additives, temperature and flow rate were studied and optimized in this work. Separation of chlorophenols was carried out using an Ethylene Bridged Hybrid (BEH) C18 column (100 × 2.1 mm, 1.7 μm; Waters, Ireland). The column in the chromatographic system was protected by an in-line filter unit purchased from Waters (Saint-Quentin en Yvelines, France). The analytical column compartment was maintained at 45 °C. The auto-sampler was conditioned at 5 °C. The flow rate was maintained at 0.4 mL min⁻¹ and the injection volume was set to 5 μL. Acetonitrile (A) and ultrapure water (B) (55/45 v/v) containing 0.1% (v/v) formic acid (pH = 5) were used as mobile phase. Elution was done under isocratic mode. Under optimized analytical conditions the total run time was 4.5 min. In addition, blanks were periodically run during the analysis to confirm the absence of contamination. Details on the optimized UHPLC conditions are presented in Section 3.1. Furthermore, the detection wavelength was optimized. The PDA scanning range was between 200 and 350 nm. Multiple wavelength UV detection was used for the quantification of analytes: 275 nm for 2-CP and 286 nm for 2,4-DCP. The Empower 2 software (Waters, Saint-Quentin en Yvelines, France) was employed to control the instruments, data acquisition and processing.

2.4 Sample preparation

The sample extraction procedure was performed by using an off-line SPE 12-port Visiprep SPE vacuum manifold obtained from Supelco (Bellefonte, PA, USA). Oasis HLB (Hydrophilic-Lipophilic Balanced) cartridges (6 cc, 200 mg, Waters, Milford, MA) were selected in this study for the preconcentration of analytes. Recovery experiments were designed to evaluate the elution efficiency of different organic solvents for conditioning step, loading rates and elution conditions.

After the extraction step, the organic eluent was collected in a 14 mL, conical graduated glass Pyrex® tube (VWR, Fontenay-sous-Bois, France). The resulting extracts were evaporated in a water batch at 30 °C and concentrated under a high-purity nitrogen stream using an N-Evap system (Organomation, Berlin, MA, USA). Finally, the obtained extracts were reconstituted using acetonitrile/ultrapure water (55/45, v/v).

2.5 Quality parameters

3.1 Optimization of UHPLC/PDA conditions

The primary aim of this work was to evaluate the potential of UHPLC system for the analysis of chlorophenols in water samples. The UHPLC system takes full advantage of chromatographic separation with high resolution and rapid analysis time by using columns packed with smaller particles (1.7 μm). It is well known, that the type of separation column, mobile phase composition, column temperature and flow rate applied during analysis affect the elution of analytes. Indeed, these three parameters, greatly affect the peak shapes, analysis times and thus the chromatographic resolution. It will be noticed, that these parameters were optimized here. These experiments were carried out before the SPE process.

Three types of UHPLC columns, Acquity BEH HSS T3 (100 × 2.1 mm, 1.7 μm), Acquity BEH C8 (100 × 2.1 mm, 1.7 μm) column and Acquity BEH C18 column (100 × 2.1 mm, 1.7 μm) were tested to improve the UHPLC separation of the target analytes. The obtained results showed that C18 column provides a better retention and separation than Acquity BEH HSS T3 column. On the other hand, we compared C8 column with Acquity C18 column, and it was found that C18 column is better for capturing chlorophenols than C8. A C18 column would theoretically provide a better retention of 2-CP and 2,4-DCP than a C8 column, because longer chain lengths may be more appropriate for retention of small compounds. The high retention on C18 made chlorophenols eluted out at higher organic portions in mobile phase. Therefore, their determination is improved, where the signal intensities increased with the portion of organic modifier in mobile phase. Hence, from the view of sensitivity, C18 showed the best performance and it was chosen for this work.

Generally in chromatographic analysis, the separation of analytes is largely dependent on the solvent conditions. Therefore, the effects of mobile phase composition and of additives on sensitivity and separation of the target chlorophenols were also investigated. In this study, different compositions of binary mixtures of acetonitrile, methanol and water were tested as eluting solvents in both isocratic and gradient modes to achieve good separation in minimum run time for all target analytes. A good compromise between high peak shapes and a reasonably analysis time was obtained with acetonitrile/water and with an isocratic elution mode. So, this phase was selected as mobile phase in this work (data not shown). Furthermore, in order to achieve an efficient separation of chlorophenols by using BEH C18 column, we tested and compared acetonitrile/water containing formic acid with acetonitrile/water containing acetic acid by changing their percentage in ultrapure water at 0.05%, 0.1%, 0.2%, 0.3% and 0.4% (data not shown).

The obtained results indicated that the retention of the chlorophenols is affected by the pH of the mobile phase. In addition it was observed that the measured responses using acetonitrile/water, modified with 0.1% formic acid were higher than those using acetonitrile/water containing acetic acid (Fig. 1). Indeed, it is known that the pH of the mobile phase affects the retention of phenols in the column, depending on the degree of dissociation. Partial dissociation could lead to peak broadening and asymmetric peaks due to co-elution of the acid and appearance of its conjugate base. As a consequence, the acidification of the aqueous mobile phase has a favorable effect on the separation, as the dissociation of analytes, retention times are shorter and peak asymmetry is improved (Mahugo Santana et al., 2009). For this reason the separation was performed at low pH and acetonitrile/water modified with 0.1% formic acid was selected as mobile phase due to the good separation and high sensitivity for all compounds. In general, the retention time increases with the concentration in formic acid in solvent A. Therefore, this additive was kept at 0.1% to ensure satisfactory quantification.

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Figure 1

Influence of the presence of acetic acid and formic acid in mobile phase (acetonitrile/ultrapure water (55:45, v/v)) on the chlorophenols measured responses. The mobile phase was modified with 0.1% formic acid and 0.1% acetic acid in water.

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The effect of column temperature and flow rate was also studied. Different column temperatures from 35 °C to 50 °C were evaluated and it was observed that a temperature of 45 °C significantly improves the resolution, peak shape, intensity of the response and retention times of the considered analytes.

Effect of flow rates from 0.2 to 0.5 mL min⁻¹ on the responses of 2-CP and 2,4-DCP was also tested. Results showed that the flow rate significantly affects the signal intensity and therefore the peak area (Fig. 2). It can be observed that the peak areas of chlorophenols are enhanced by the increasing of the flow rate and reached a maximum value when the flow rate was 0.4 mL min⁻¹. Subsequently, it decreases gradually with the increasing of the flow rate. This effect is supposed to be due to the instability of chlorophenols at elevated flow rates.

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Figure 2

Effect of flow rate on the peak area of chlorophenols.

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Therefore, 0.4 mL min⁻¹ was selected as the working flow rate in this study. Under these experimental conditions, the target analytes were eluted rapidly (the retention time of the last eluting peak <3 min) and the separation and resolution of peaks were good. The obtained retention times for 2-CP and 2,4-DCP were 1.35 min and 2.56 min, respectively (Fig. 3). It should be noted that under this analytical conditions the UHPLC separation was six times faster than the conventional HPLC separation with the same analytes. Moreover, it consumes ca. 50% less organic solvents than the corresponding HPLC technique (Li et al., 2007; Opeolu et al., 2007). Finally, the results indicated that optimized UHPLC method developed here for the analysis of cholorophenols has other practical advantages such as faster analysis times and reduced solvent consumption rates, compared to conventional HPLC technique.

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Figure 3

The obtained retention times for 2-2-chlorophenol (2-CP) and 2,4 dichlorophenol (2,4-DCP).

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Since a PDA was used here for the detection of analytes, the absorption wavelengths were also optimized in order to reach the maximum sensitivity for each compound. The wavelengths selected for the simultaneous detection of chlorophenols were: 275 nm for 2-CP and 286 nm for 2,4-DCP. Therefore, this analytical UHPLC/PDA technique can be considered as a promising method that can easily compete with conventional HPLC/PDA systems in this field of application.

3.2 Optimization of SPE process

In order to determine lower concentrations of chlorophenols in water samples, a pre-concentration step is necessary. As stated below in order to decrease the limit of detection and quantification of these analytes an off-line-SPE methodology was developed in this work. SPE experiments were carried out after the optimization of UHPLC/PDA conditions. The optimization of the extraction process was performed in order to attain excellent recoveries for all target compounds in a single extraction step. According to literature data, Oasis HLB cartridges are one of the most used, because they are able to retain a large list of organic pollutants through its unique ratio of hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene sorbent (de Almeida et al., 2000). Thus, Oasis HLB cartridges were selected for this work.

Different parameters of the extraction method, such as organic solvents for conditioning step, loading rates and elution conditions, were investigated and optimized to determine the operating conditions providing the highest recovery in ultrapure water samples. Consequently, the optimal operating conditions for the SPE method consisted of the use of acetonitrile and methanol (6 mL of each) for conditioning the HLB sorbent. Generally, in the SPE methodology, the flow rate of the sample affects the retention of the target molecules on the surface of the adsorbent.

The optimum flow rate used for the extraction of the samples was determined to be between 3 and 5 mL min⁻¹, and there was no effect on recovery. In addition, several organic solvents of variable polarity were tested as eluent solvents, including acetonitrile, ethyl acetate, dichloromethane and methanol (data not shown). Finally, methanol was selected as the most effective solvent for the elution step.

Therefore, the optimized SPE method is established as follows: Oasis HLB cartridges are conditioned using 2 × 3 mL acetonitrile, and 2 × 3 mL methanol. The SPE cartridges were equilibrated with 2 × 5 mL of ultrapure water acidified with formic acid (pH 3). All compounds were spiked into the 250 mL water sample. The sample passed through the SPE cartridges at a flow rate of 4 mL min⁻¹. After the extraction, the cartridges were washed with 2 × 3 mL of ultrapure water adjusted to pH 3 with formic acid. Then, the molecules adsorbed onto the sorbents were eluted successively with 2 × 2 mL of methanol at a flow rate of 3 mL min⁻¹. The eluates were transferred to a clean tube and concentrated by evaporation under a nitrogen stream to a final volume of approximately 0.1 mL (concentration factor of 2500). The obtained extracts are then reconstituted using acetonitrile/ultrapure water (55/45, v/v) and transferred to injection vials. Finally, the extracts were stored at 4 °C until further analysis.

3.3 Analytical performance

The performance characteristics of the proposed method were established by determining the linearity, limit of detection, limit of quantification, precision, accuracy, and extraction recovery.

3.4 Application of the proposed method: analysis of real water samples

In order to investigate the applicability of the proposed SPE–UHPLC/PDA method, the extraction and determination of the target analytes were conducted under the optimum conditions using real environmental water samples, including river waters. Three water samples were collected in October 2013 and January 2014 from different locations situated in the Brittany region (France) and analyzed using the SPE–UHPLC/PDA. Furthermore, the analytes were not detected in the real environmental waters (Table 4). The river samples (n = 6) were firstly spiked with the standard solutions of chlorophenols at two concentration levels in order to assess the matrix effects. The results obtained for extraction recoveries and standard deviations are listed in Table 4. Extraction recoveries higher than 97% were obtained for all compounds from all river water samples, with RSD values <6%. These satisfactory results demonstrate that the matrices of the real water samples analyzed here have no significant effects for the determination of chlorophenols by the developed SPE–UHPLC/PDA method.

The potential applicability of the proposed method was also evaluated by analyzing drinking water samples from four locations in France. They were collected in triplicates from each location and analyzed. The results demonstrated that the collected samples were free of contamination with 2-CP and 2,4-DCP (data not shown). The analysis of real water samples showed that the resulting CPs levels posed no health risk for the population of the Brittany region at the time of sampling.

The overall results presented in this work confirm that the combined use of SPE protocol and UHPLC/PDA technique is suitable for the simultaneous determination of 2-CP and 2,4-DCP in ultrapure water, environmental water and drinking water samples.