Introduction

Phenolic compounds are important contaminants in waste-water due to wide use in many industrial processes, such as pesticides, dyes, pharmaceuticals, and plastics [1–3]. Because of high toxicity, most of phenols and substituted phenols are listed as priority pollutants in many countries.

Several procedures have been reported for measurement of phenolic compounds in environmental water samples, including liquid-liquid extraction (LLE) [4–6] and solid phase extraction (SPE) [1, 7–9] and final analysis by high performance liquid chromatography (HPLC), gas chromatography (GC), or capillary electrophoresis (CE). However, in addition to time consuming and low sensitivity, LLE requires large amount of toxic organic solvents. SPE uses considerably less solvent, but it can be relatively expensive. Hence, a more sensitive, fast, and simple method is necessary for measurement of phenolic compounds.

Dispersion of droplets of a one liquid in another (two immiscible liquids) is defined as an emulsion. The emulsion has been a powerful technology for many separation processes including removal and recovery of various metals such as zinc, silver, cobalt and cadmium [10–12] and successfully used in cosmetic, foods and pharmaceutical industries [13, 14].

The present study described an oil-in-water emulsion (OWE) method for the extraction of four phenolic pollutants in environmental water samples. This method is very fast, flexible in selection of extraction solvents, simple, low cost and high preconcentration factor. In order to show the performances of the proposed method, extraction of phenolic compounds as case study in environmental water samples by gas chromatography and flame ionization detection (GC-FID) were investigated.

Experimental

Reagents

Phenol (Ph), 2,4-dimethylphenol (24DMPh), 2,4,6-trichlorophenol (246TCPh), 1-naphtol (1 Np), salicylaldeyade (internal standard, I.S.), ethanol (HPLC grade), carbon tetrachloride, chloroform, dichloromethane, benzene, toluene, cyclohexane, n-hexane, ethyl acetate, sodium chloride and hydrochloric acid were purchased from Merck (Darmstadt, Germany, www.merck.com). Butyl acetate was acquired from Acros (Geel, Belgium, www.acros.com). Methyl isobutyl keton was supplied from Scharlau (Barcelona, Spain, www.scharlau.com).

Apparatus

The measurements were performed with a gas chromatograph (model GC-17 Shimadzu, www.shimadzu.com) equipped with a flame ionization detector (GC-FID) and a BP21-FFAP capillary column (25-m length and 0.52-mm i.d.). Helium was used as the carrier gas at a total flow rate of 29 mL min−1. After injection of samples, the temperature of the column oven was kept constant for 1 min (100°C), linearly increased to 270°C (30°C min−1) and kept constant at this temperature for the remaining time of analysis. Injector and detector temperatures were set at 280 and 300°C, respectively. Retention times of the peaks were as follows: Ph, 1.69 min; 24DMPh, 2.68 min; 246TCPh, 4.30 min; 1 Np, 5.27 min; I.S., 2.10 min. Quantitative data obtained using the internal standard method based on the peak area of the standard solutions and I.S.

The pH measurements were made with a 780 pH meter (Metrohm, Switzerland, www.metrohm.com) equipped with a combine Ag/AgCl glass electrode. The centurion scientific centrifuge (model K280R, UK, www.centurionscientific.co.uk) was used for centrifuging.

OWE preparation

The OWE system components are schematically shown in Fig. 1. An aliquot (5 mL) of solution (pH value 2, adjusted with hydrochloric acid) containing 4 mg L−1 of phenolic compounds and I.S. was placed in a 10-mL volumetric flask. Carbon tetrachloride (20.2 µL), as heavier solvent, and butyl acetate (79.8 µL), as lighter solvent, were added at the bottom and at the top of sample solution using a 250-µL eppendorf micropipette sampler, respectively, (extraction solvents can be mixed preliminarily and add to sample). The mixture was stirred by means of a 19.5 mm × 6 mm stir bar (1 min, 700 rpm), a stable emulsion or a milky solution was formed (it was stable at least for 5 h). In order to break down emulsion, 10 µL heavier solvent was added. The milky solution was transferred to a test tube with conical bottom and separation of the phases was accomplished by centrifuged at 4000 rpm for 3 min. Then the dispersed fine droplets of organic phase were sedimented in the bottom of test tube. The 2 µL of sedimented phase was removed using a 10 µL microsyringe (Hamilton, USA, www.hamiltoncompany.com) and injected onto GC-FID system.

Fig. 1
figure 1

Schematic illustration of OWE device

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Calibration curve

Individual solutions of Ph, 24DMPh and I.S. were prepared in double distilled water. Due to the limited solubility of 246TCPh and 1 Np in water, these analytes were initially dissolved in ethanol. Stock standard solutions of phenolic compounds and internal standard were prepared in ethanol at the concentration level of 1000 mg L−1. Working solutions were obtained by appropriate dilution. Calibration standards were made at different concentration ranges. Each one was prepared in five replicates. Equations were obtained by least squares linear regression of the peak area ratio of standard to internal standard versus analyte concentration.

Sample preparation

Tap, mineral, river and waste water samples were collected in glass bottles from Ilam province (Ilam, Iran). The samples were filtered using Whatman No. 42 filter paper before analysis and stored in refrigerator in the dark. The pH of samples was adjusted by hydrochloric acid and the preconcentration procedure was applied based on the above mention method.

Results and discussion

Optimization of the OWE method

In this study the effect of several important parameters influencing the extraction efficiency including the heavier and lighter organic solvents, volume of organic phase and its density, salting-out, pH of aqueous sample, temperature, time of extraction and stirring rate was investigated. Chromatographic peak area of analyte and that of I.S. was used to assess the extraction efficiency under experimental conditions tested. A solution 4 mg L−1 each analyte and I.S. was used for optimization of OWE procedure.

The most important step in the development of the proposed method is the selection of appropriate extraction solvents. The selection of extraction solvents was based on (a) immiscibility with aqueous phase; (b) the density of heavier and lighter solvents must be higher and lower than aqueous phase, respectively (c) good chromatographic analysis. Based on these considerations dichloromethane (CH2Cl2), chloroform (CHCl3) and carbon tetrachloride (CCl4) as heavier extraction solvents and methyl isobutyl ketone (MIBK), cyclohexane, ethyl acetate, butyl acetate, benzene, toluene, and n-hexane as lighter extraction solvents were tested. It was found that except for carbon tetrachloride-butyl acetate system, all other combinations of heavier and lighter solvents did not show stable cloudy solution. Therefore, CCl4 and butyl acetate were selected as the extraction solvents for subsequent experiments.

As previously mentioned, stability of this kind of OWE is based on variation of density of binary extraction solvent to approach density of sample. The effect of density of the binary extraction solvent (CCl4 and butyl acetate) was studied in the range 0.881–1.591 g L−1. The desired density (density of binary organic phase) was calculated as follows [15]:

$$ {\rho_{mixture}} = \frac{{{\rho_l} \times {v_l} + {\rho_h} \times {v_h}}}{{{v_T}}} $$
(1)
$$ {v_T} \cong {v_l} + {v_h} $$
(2)

where ρ l and ρ h represent the density of lighter and heavier organic solvents and v l and v h represent the volume of lighter and heavier organic solvents, respectively. It was found that response gradually increased with an increase in the binary extraction solvent density from 0.881 to 1.024 g L−1. Beyond this point, the response was decreased. Hence, the optimum density of the binary extraction solvent was selected by considering density of aqueous sample.

The effects of ionic strength were extensively evaluated in the traditional liquid-liquid extraction, because salt addition can increase the partition of analytes to the organic phase. The effect of salting-out was investigated by adding NaCl (0–10% w/v). Peak areas of all analytes slightly increase and gradually when amounts of NaCl increase up to 2% and became constant. Hence, a salt concentration of 2% was chosen for further experiments.

The effect of pH in the range from 1 to 7 was evaluated. The analytical signal as a function of pH of aqueous sample is demonstrated in Fig. 2. As it can be observed, the extraction efficiency of analytes at low pH value was superior to that at higher pH value. Based on the results, pH value of 2 was adopted for subsequent tests.

Fig. 2
figure 2

Effect of pH of aqueous solution on the extraction efficiency. Extraction conditions: sample volume: 5 mL; NaCl concentration: 2% (w/v); heavier phase volume: 30.3 µL (CCl4); lighter phase volume: 119.7 µL (butyl acetate); extraction time: 1 min; stirring rate: 800 rpm; extraction temperature: 23°C

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The effect of volume of binary extraction solvent (CCl4 and butyl acetate) was examined in the range 40–200 µL. The results of Fig. 3 show that for all analytes, a similar pattern is observed in the volume range between 90 µL and 130 µL. With less than 40 µL of binary extraction solvent no two phase system was observed. However, a further increase in the volume of binary extraction solvent has no significant consequence on the extraction efficiency. This may be because of formation of large droplets of binary extraction solvent [16, 17]. Therefore, all further experiments were carried out at the optimum volume of 100 µL.

Fig. 3
figure 3

Effect of binary organic phase volume on the extraction efficiency. Extraction conditions: sample volume: 5 mL; NaCl concentration: 2% (w/v); pH: 2 (0.01 mol L−1 HCl); heavier phase: CCl4; lighter phase: butyl acetate; extraction time: 1 min; stirring rate: 800 rpm; extraction temperature: 23°C

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Stirring rate is one of the most important parameters that can reduce the time required to attain thermodynamic equilibrium and enhanced the extraction efficiency. In order to generate fine and a stable emulsion formation fast agitation was used. The results indicated that the extraction efficiency increased with increasing of stirring rate from 100 to 700 rpm and then approximately remained constant across the range of 800–1000 rpm. Therefore, all further experiments were performed with a stirring rate of 700 rpm.

The extraction efficiency increases with longer extraction time in traditional liquid-liquid extraction (LLE). When the influence of extraction time was studied in the range 0 to 30 min, it was found that this parameter had no influence on the extraction efficiency for the analytes under study. This may be attributed to the quickly equilibrium reaching and independency of proposed method to the time. Thus, in this investigation an extraction time of 1 min was selected for subsequent experiments.

The effect of temperature was investigated in the range 23–65°C. It was found that an increase in the solution temperature reduced the extraction efficiency. This may be attributed to decreases in surface tension of immiscible liquids with increasing temperature [18, 19]. Therefore, a temperature of 23°C was finally selected for subsequent experiments.

Analytical performance of the developed OWE method

Under the above optimum experimental conditions, the proposed method was validated by linearity, precision and limit of detection. The calibration plots were found to be linear in the range of 0.1–20 , 0.025–20, 0.05–20 and 0.05–20 mg L−1, for Ph, 24DMPh, 246TCPh and 1 Np, respectively, with a coefficient of determination (r2) more than 0.9994 (n = 11). For each concentration level, three replicate extractions were performed. The limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were in the range 19.2–76.0 and 64.1–251.0 µg L−1, respectively. As it can be seen, the proposed method has low LOD and LOQ and can be used for trace analysis of phenolic pollutants in different water samples. The intra-day and inter-day precisions of the assay were evaluated by analyses of quality control samples at three concentration levels (0.2, 8.0, and 18.0 mg L−1) on the same day (n = 10) and the five consecutive days. It was found that the intra-day and inter-day precisions for all analytes were in the range of 1.2–5.0 and 1.5–5.0%, respectively.

Application to real sample analysis

In order to investigate the recovery of the proposed method, four water samples were collected, spiked with standards at three concentration levels (0.1, 5.0 and 15.0 mg L−1) and analyzed. The results are listed in Table 1. The recoveries of the samples ranged from 96.0–102.0% and relative standard deviations (RSDs) were in the range of 1.0 to 2.5%, respectively. The preceding results demonstrated that the proposed method was feasible for quantitative the determination of phenolic pollutions in environmental water samples.

Table 1 Recovery of OWE method in environmental waters unspiked and spiked with phenolic compounds by standard addition method (n = 3)
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The proposed method was applied to determination of phenolic pollutions in tap, mineral, river and waste water samples. Representative chromatograms of different water sample extracts are shown in Fig. 4. Average concentrations of phenolic compounds are summarized in Table 1. As it can be seen, tap, river and mineral water samples were free of phenolic compounds and the phenolic pollutions (except 24DMPh) were found in waste water sample.

Fig. 4
figure 4

GC-FID chromatograms of OWE extracts of unspiked a standard, b waste water, c tap water, d river water, e mineral water

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Table 2 indicates the limit of detection (LOD), relative standard deviation (RSD), coefficient of determination (r2), linear range, extraction time and recovery using solid-phase microextraction-gas chromatography-flam ionization detection (SPME-GC-FID) [8], phase transfer catalysis-gas chromatography-mass spectrometric detection (PTC-GC-MS) [20], micro-liquid chromatography-ultraviolet absorption spectrometry (µLC-UV) [21], single-drop microextraction-gas chromatography-mass spectrometric detection (SDME-GC-MS) [3], solvent microextraction-gas chromatography-mass spectrometric detection (SME-GC-MS) [22] and OWE methods for the determination of phenolic compounds in water samples. The proposed method provides similar quantification extraction efficiency, with advantages of being faster and using smaller volume of organic solvents.

Table 2 Comparison of different methods for the determination of phenolic compounds in pure waters
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Conclusions

In this research work a simple OWE method was developed and applied for the determination for the determination of phenolic compounds in water samples. The method was based on ternary component solvents emulsion formation. The present method is attractive due to its simplicity, analytical precision, considerable saving in terms of time of analysis, and organic solvent used. The results from validation indicate the proposed method can be used for the determination of phenolic pollutants in water samples.