The phenolic compounds include phenol (PN), 3-chlorophenol (3CP), 2,4-dimethylphenol (24DMP), 2,4-dichlorophenol (24DCP), 2,4,6-trichlorophenol (246TCP), 2,3,4,6-tetrachlorophenol (2346TeCP) and pentachlorophenol (PCP) were obtained from Merck (Darmstadt, Germany). Standard solutions (2000 mg L^-1) from each individual compounds were prepared in methanol. The organic solvents (HPLC-grade or suprasolv for chromatography), sodium chloride and potassium chloride (analytical reagent grade) were purchased from Merck (Darmstadt, Germany) and Fluka (Buchs, Switzerland). Ultrapure water was prepared by a Milli-Q system (Bedford, MA, USA). Individual stock standard solution of 1000 mg L^-1 for each compound was prepared in methanol and stored at -20 °C. Stock standard mixture of the phenolic compounds was weekly prepared by diluting the stock solutions with methanol. The working standard solutions were daily made by diluting the mixed stock solutions with Milli-Q water to the required concentration. A pH 2 buffer was prepared using 25 mL of 0.2 M KCl and 6.5 mL of 0.2 M HCl in 100 mL of water, and saturated salt solutions were prepared with NaCl.

Tap water sample was freshly collected from our laboratory and mineral water was obtained from supermarket. The tap and mineral water samples were collected in glass bottles. The tap water sample was filtered before the analysis by a 0.45 µm membrane filter (MSI, Westboro, MA, USA). The water samples were stored in refrigerator at 4 °C until their analysis.

The residual analysis was carried out using an Agilent 1100 HPLC equipped with a manual injector and diode array detector. A Zorbax Eclipse XDB-C18 column (150 mm ( 4.6 mm, 5 µm particle sizes) was used and all injections were performed manually with 20.0 µL sample loop. The operating conditions were as follows: mobile phase, methanol/water, 95:5 v/v; flow rate, 0.8 mL/min; column temperature, 25 ± 1°C; and the wavelength of detector, 220 nm. Screw cap glass test tubes (10 mL) with conical bottoms (used as extraction vessels) were heated at 500 °C in a furnace (model CWF 1200, Carbolite, UK) to remove any organic compounds. Stirring the solution was carried out with a magnetic heater-stirrer (Heidolph MR 3001K, Germany). A simple water bath placed on the heater-stirrer was for controlling the temperature of the sample solutions.

Extractions were carried out according to the following procedure: (1) a 20-mL aqueous solution containing the phenols was added into the sample vial with a 8 mm × 4 mm magnetic stirring bar; (2) the sample vial was put on a MR 3001K hot plate stirrer (Heidolph, Germany); (3) desired volume of the organic solvent was placed on the surface of the solution using a 25 µL Hamilton syringe (Bondaduz, Switzerland); (4) the vial was sealed and the stirrer turned on; (5) after the extraction, sample vial was transferred into an ice beaker, and the organic solvent was solidified nearly 5 min afterwards; (6) the solidified organic solvent was transferred into the conical vial by a simple spatula where, it started to melt; (7) the whole melted solvent was injected into the HPLC for quantification.

The selection of a proper extraction solvent is of great importance for the optimization of the LPME process. To choose an appropriate organic solvent, the following points should be considered. Firstly, the chosen solvent should illustrate a high boiling point and a low vapor pressure in order to reduce the risk of evaporation [22]. Secondly, it should exhibit a good chromatographic behavior [23] and, thirdly, the partitioning coefficient of the analyte should be high. Furthermore, the solvent must have a good affinity for the target compounds [24] and, finally, it should demonstrate a melting point near the room temperature (in the range of 10–30 °C) [21]. According to these considerations, several extracting solvents, including 1-undecanol, 1-dodecanol, 2-dodecanol and n-hexadecane were considered. Among the tested extracting solvents, 1-undecanol presented the best extraction efficiency, while its chromatographic peak was easily separated from the analyte peaks. Also because of its low vapor pressure at the extraction conditions, the extract was stable at the extraction period (Fig. 1). Thus, 1-undecanol was chosen as the extracting solvent in this investigation. It is noteworthy that a 20 mL aqueous solution spiked into with the phenols (at the concentration level of 5.0 µg L^-1) was used in the extraction studies.

Fig. (1) Effect of organic solvent type on extraction efficiency.

The effect of micro drop volume on the analytical signal was studied in the range of 10.0-20.0 µL. Fig. (2) shows that there was a corresponding increase in the analytical signal of phenolic compounds from 10.0 until 20.0 µL of solvent volumes. Based on LLE equations, rate of the analytes migration into micro drop is directly related to the surface area between the two liquid phases and is inversely related to the organic-phase volume. Thus, increase in the drop volume raises the interfacial area following the analytical signals. But further increase in the micro drop volume leads to decrease in the analytical signals. Hence, the volume of 14.0 µL was chosen as the optimal solvent volume.

Fig. (2) Extraction efficiencies obtained for different organic solvent volume.

Agitation of the sample solution enhances the rate of extraction. The stirring speed has a direct influence on extraction efficiency in limited times due to increasing mass transfer into the organic drop. In this work, the samples with a volume of 20 mL were stirred at different stirring speed (400, 600, 800, 1000 and 1200 rpm) on a stirrer plate. Based on Fig. (3), 1200 rpm was chosen as the suitable stirring speed.

Fig. (3) Effect of stirring rate on the relative peak area.

The addition of acid and salt, singularly and in combination, was investigated as a means of enhancing the amount extracted by the solvent. Fig. (4)shows signal intensity obtained for all conditions using a 20 min extraction time, which was also used for control samples of the same concentration at neutral pH and with no salt added. The positive effects of both acid and salt can be realized when they are used in combination. Thus, according to the results, the positive effects of both acid and salt can be realized when they are used in combination. With pH 2 and saturated salt conditions, the amount extracted for every analyte in the mixture was greater than the control sample at pH 7 and with no salt added. Under these conditions, all phenolic compounds are in their neutral form and are salted out of solution and into the solvent.

Fig. (4) Effect of acid and salt on extraction

The effect of sample temperature on extraction efficiency was also investigated between 26 and 55 ºC. Generally, by increasing temperature, higher enrichment factors can be obtained in LPME experiments. This process facilitates mass transfer of the analytes from the sample into the organic solvent and thus increases the efficiency of the extraction. Results showed that by increasing the temperature, the extraction efficiency was increased (Fig. 5). At high temperatures (>55 °C), the over-pressurization of the sample vial made the extraction system unstable. Thus, in further experiments the sample vial temperature was held at 55 °C.

Fig. (5) Influence of temperature on the extraction efficiencies.

To increase the precision and sensitivity of the LPME method, it is necessary to select an exposure time that guarantees the equilibrium between the aqueous and organic phases. Therefore, the extraction time plays a very essential role in the whole process. The range of extraction time investigated was 5-30 min with other extraction conditions being constant. Fig. (6) shows that there was a corresponding increase in analytical signal from 5 to 15 min, followed by a period of stability. An extraction time of 15 min was selected as a reasonable compromise between enrichment factor and analysis time.

Fig. (6) Time profiles obtained for the studied analytes.

Quantitative parameters of the purposed method, such as linearity ranges (LR) and limit of detections (LOD) were calculated by pre-concentration 20 mL of water sample, spiked by standard solution of phenolic compounds as summarized in Table 1. As it is illustrated, that the LR were obtained at concentration range of 0.3-870 µg L^-1 for phenolic compounds, (RSD < 10.1%). The coefficient of determinations (r²) was satisfactory in the range of 0.9973-0.9992 for all the analytes studied. Limit of detections, based on a signal to noise ratio of S/N=3 [25], were ranged from 0.05 to 3.0 µg L^-1.

Table 1.

Figures of Merit of the Proposed LPME-HPLC-DAD Method for Pre-Concentration and Determination of Phenols

In order to evaluate efficiency of the proposed method in monitoring of low levels of phenols, their levels in tap and mineral water were investigated. Results are summarized in Table 2. The chromatograms obtained by HPLC-DAD of unspiked mineral water and that spiked at two concentrations of each analyte after the developed method at optimum conditions is shown in Fig. (7).

Table 2.

Figures of Merit of the Proposed LPME-HPLC-DAD Method for Pre-Concentration and Determination of Phenols

Fig. (7) Chromatograms of mineral water sample, (a) unspiked; (b)spiked at 4.0 µg L-1and (c) spiked at 10.0 µg L-1 phenols. (1) PN,(2) 3CP, (3) 24DMP, (4) 24DCP, (5) 234TCP, (6) 2346TeCP, (7)PCP.