Optimization of dispersive liquid-liquid microextraction for preconcentration and spectrophotometric determination of phenols in Chabahar Bay seawater after derivatization with 4-aminoantipyrine.

Marine Pollution Bulletin xxx (2014) xxx–xxx

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Optimization of dispersive liquid–liquid microextraction for preconcentration and spectrophotometric determination of phenols in Chabahar Bay seawater after derivatization with 4-aminoantipyrine Mir Mahdi Zahedi a,⇑, Mahmoud Nassiri a, Seied Mahdi Pourmortazavi b,⇑, Mehdi Yousefzade a a b

Department of Marine Chemistry, Faculty of Marine Sciences, Chabahar Maritime University, Chabahar, Iran Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, Tehran, Iran

a r t i c l e

i n f o

Keywords: Phenol determination Dispersive liquid–liquid microextraction (DLLME) Spectrophotometry Seawater Chabahar Bay 5530 APHA standard method

a b s t r a c t We have optimized dispersive liquid–liquid microextraction to preconcentrate trace phenolic compounds after derivatization with 4-aminoantipyrine in artificial sea water for spectrophotometric determination. Factors such as reaction time (7.5 min), pH (9.5), solvent (chloroform), dispersing solvent (ethanol), and volume ratio of dispersing to organic phase (11:1) were optimized. Under optimum conditions, the limit of detection was 0.18 lg/L and the linearity range 1–900 lg/L. The relative standard deviation and enrichment factor were 6% (n = 7) and 920, respectively. The results demonstrate the efficiency of coupling the 5530 APHA standard for derivation and dispersive liquid–liquid microextraction of phenolic compounds from seawater samples. Using this method, total phenol content in seawater from several locations in Chabahar Bay (southeast Iran) was estimated at 27.8–74.8 lg/L. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The world faces enormous environmental problems due to contamination of water and the marine environment with hazardous industrial waste. Contamination of the marine environment with organic pollutants is becoming increasingly serious. Organic pollutants are often toxic to aquatic organisms and can affect food webs (Patnaik, 2007). Phenols (especially chloro- and nitrophenols) are toxic and potentially carcinogenic, affecting the taste and odor of water at very low concentrations. These compounds may be present in wastes from production of various materials, including petroleum, paper, plastics and pharmaceuticals (Dimou et al., 2004; Ni et al., 2011). Several techniques are commonly used for the analysis of phenols in environmental samples: liquid chromatography (LC) Fiamegos et al., 2002, capillary electrophoresis (CE) Wang et al., 2012, ⇑ Corresponding authors. Address: Department of Marine Chemistry, Faculty of Marine Sciences, Chabahar Maritime University, P.O. 99717-56499, Chabahar, Iran. Tel.: +98 545 2220020; fax: +98 545 2224264 (M.M. Zahedi). Address: Faculty of Material and Manufacturing Technologies, Malek Ashtar University of Technology, P.O. Box 16765-3454, Tehran, Iran. Fax: +98 212 2936578 (S.M. Pourmortazavi). E-mail addresses: [email protected] (M.M. Zahedi), pourmortazavi@ yahoo.com (S.M. Pourmortazavi).

high-performance liquid chromatography (HPLC) Guo and Lee, 2011, gas chromatography (GC) Padilla-Sanchez et al., 2011; Pizarro et al., 2012; Faraji et al., 2009; Fariña et al., 2007, and kinetic spectrophotometry (Ni et al., 2011; Mitic and Zivanoic, 2002). Analysis using these methods usually involves complex sample preparation, making them expensive and time-consuming. Moreover, phenols commonly occur at low concentrations in complex environmental sample matrices. Thus, effective extraction and preconcentration of phenols is necessary prior to analysis (Zhang et al., 2011; Santana et al., 2009). Several preconcentration methods have been developed, such as solid-phase extraction (SPE) Padilla-Sánchez et al., 2011; Liu et al., 2004, liquid-phase microextraction (LPME) Zhang et al., 2011, dispersive liquid–liquid microextraction (DLLME) Fariña et al., 2007, ultrasound-assisted emulsification–microextraction (USAEME) Pizarro et al., 2012; Regueiro et al., 2009; Villar-Navarro et al., 2013, and solidification of floating organic droplets (UAEM–SFO) Wang et al., 2012. However, given the importance of the analysis of phenols in water samples, seeking new methods is still essential. Derivatization of phenols with 4-aminoantipyrine for spectrophotometric determination was developed by Emerson in 1943 (Standard Methods for the Examination of Water and Waste Water, 1989). This procedure is still commonly used due to its speed, costeffectiveness and absence of laborious steps. This method is based

http://dx.doi.org/10.1016/j.marpolbul.2014.03.037 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Zahedi, M.M., et al. Optimization of dispersive liquid–liquid microextraction for preconcentration and spectrophotometric determination of phenols in Chabahar Bay seawater after derivatization with 4-aminoantipyrine. Mar. Pollut. Bull. (2014), http://dx.doi.org/ 10.1016/j.marpolbul.2014.03.037

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M.M. Zahedi et al. / Marine Pollution Bulletin xxx (2014) xxx–xxx

on the oxidative coupling of phenols with 4-aminoantipyrine (4-AAP) in the presence of an oxidant to form antipyrine dyes, the concentration of which can be readily measured via spectrophotometry (Fiamegos et al., 2002). DLLME is a simple, rapid and inexpensive extraction method providing a high enrichment factor along with excellent accuracy and precision (Rezaee et al., 2006; Rahimi-Nasrabadi et al., 2012). Therefore, this technique has attracted great attention in environmental analysis and is used successfully for analysis of organic compounds in environmental water samples. In this study, dispersive liquid–liquid microextraction coupled with UV–Vis spectrophotometry was optimized for the determination of phenols in seawater. Although the dispersive liquid–liquid microextraction combined with micro-volume spectrophotometry was optimized for the determination of phenols in drinking water and wastewater (as a replacement for the 5530 APHA standard method) (Lavilla et al., 2012), there are no reports on DLLME and spectrophotometric determination of phenols in seawater. It should be noted that due to the complexity of the seawater sample matrix and the fact that it is fundamentally different from other types of water samples, the previously reported procedure is not applicable for successful extraction of phenol derivatives from seawater samples. Thus, in order to ensure the best simulation of real samples, dispersive liquid–liquid microextraction was optimized for preconcentration and spectrophotometric determination of phenols derivatized with 4-aminoantipyrine using artificial seawater. Finally, the optimized method was used to measure the total concentration of phenols in Chabahar Bay seawater (southeast Iran) for the determination of environmental hazards.

purposes as received. Dichloromethane, trichloromethane, chlorobenzene, and carbon tetrachloride, which were used as extraction solvents, were from Merck (Darmstadt Germany) or Fluka (Buchs, Switzerland) and were used after cleaning up with ultrapure water. Analytical grade methanol, ethanol, acetone, and acetonitrile (Merck and Fluka) were used as received. Other compounds were of analytical grade; solutions were prepared in ultra-pure water. Artificial seawater was prepared by dissolving the following salts in 1 L of deionized water: NaF (3 mg), KBr (100 mg), Na2SiO39H2O (20 mg), SrCl2H2O (20 mg), KCl (3 mg), MgCl26H2O (10.780 g), NaHCO3 (200 mg), H3BO3 (30 mg), CaCl22H2O (1.470 g) and NaCl (23.500 g) López-Darias et al., 2010. All salts, which were of analytical grade, were purchased from Merck, Fluka and Sigma–Aldrich (Steinheim, Germany) and used without further purification. 2.3. DLLME procedure Using a 15 mL conical glass sample tube, 10 mL sample of buffered (pH adjusted to 9.5 by addition of 250 lL of 0.5 M hydrogen phosphate/ammonia solution) seawater was mixed with 150 lL of 1% w/v 4-aminoantipyrine (4-AAP) and 100 lL of 1.5% w/v potassium peroxodisulfate. The mixture was kept at room temperature until reddish-brown (approximately 10 min). Then 980 lL of chloroform:ethanol (volume ratio 1:11) was injected into the sample tube using a 2 mL syringe. A cloudy dispersion formed, which was centrifuged for 5 min at 3000 rpm. Finally, 40 lL of the organic phase was removed by micro-syringe and placed in a microcell for determination of total phenols by UV–Vis spectrophotometry.

2. Experimental methods 3. Results and discussion 2.1. Instrumentation 3.1. Derivatization reaction Spectrophotometry was carried out on a UNICO S2100 UV–Vis spectrophotometer equipped with a 10 lL quartz microcell (model Q-01701). A Centurion Scientific K3 series K241R centrifuge was used to accelerate phase separation. A TPS WP-80 digital pH meter was used for pH adjustments. A 100 lL Hamilton syringe (Hamilton Company, Nevada) was used for phase separation of collected sediments. 2.2. Reagents A 1000 mg/L 1 standard solution of phenol (Merck, Darmstadt, Germany) was prepared by dissolving in water. Working solutions were prepared daily by dilution of this stock solution. 4-Aminoantipyrine, potassium peroxodisulfate, potassium hexacyanoferrate (III), hydrogen peroxide, ammonium hydroxide and potassium hydrogen phosphate were purchased from Merck (Darmstadt, Germany) or Fluka (Buchs, Switzerland) and used for derivation

The use of colorimetric methods (Fiamegos et al., 2002) has been suggested for following the reaction of 4-aminoantipyrine with phenols, either unsubstituted or ortho- or meta-substituted with halogens or carboxy, methoxy or sulfonate groups. This reaction at pH 9.5 leads to the formation of stable reddish-brown colored antipyrine dyes (Fig. 1). Dye generation is carried out in the presence of suitable oxidants. Our experiments showed that potassium peroxodisulfate is superior to other suggested reagents. The results of spectrophotometric studies in aqueous solution or chloroform are shown in Fig. 2. As seen there, the dye exhibits kmax of 510 nm and 460 nm, respectively, in water and chloroform. Therefore, all absorbance measurements were performed at these wavelengths. Our kinetic study of dye formation showed that in the presence of potassium peroxodisulfate as oxidant, the reaction of phenol with 4-aminoantipyrine is first order (with a rate constant of 0.836). This rate gives a stable color within 10 min. Fig. 3

Fig. 1. Reaction scheme for phenol treated with 4-AAP.



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extraction efficiency and reduces the analysis time. Fig. 4(B) shows the absorbance of the sedimented phase vs. injection time. The absorption reached its maximum value at 7 min and then remained constant. 3.3. Effects of 4-aminoantipyrine (4-AAP) concentration The effect of 4-aminoantipyrine (4-AAP) concentration was investigated because the amount of dye associated with derivative substances may affect the extraction. As seen in Fig. 5, increasing the concentration of 4-AAP in seawater (in the range 0.006– 0.02% w/v) enhanced absorbance. Based on these results, a 4-AAP concentration of 0.014% w/v was optimal. However, the amount of 4-AAP required depends on the concentration of phenol as well as sample volume. Fig. 2. Absorption spectra for derivatization of phenol with 4-AAP.

3.4. Selection of extraction and dispersing solvents shows a plot of Ln([dye]) vs. time (min), confirming the reported order of the antipyrine dye formation reaction. The combination of derivatization with dispersive liquid–liquid microextraction (DLLME), beyond the known advantages of DLLME itself, gives several additional advantages, such as increased sensitivity, smaller sample volume and reduced reagent consumption (Lavilla et al., 2012). Because seawater is a complex matrix, optimization of parameters on the DLLME of phenols from seawater is necessary to achieve high sensitivity. 3.2. Effects of pH and injection time The pH of the sample solution is a very important factor in the colorimetric derivatization/extraction process. Here the effect of pH on derivatization and DLLME was investigated in the pH range 4–11. Other variables were kept constant and standard seawater was used as the main component of the working sample matrix. The results shown in Fig. 4(A) demonstrate that the maximum signal intensity for antipyrine dye was observed at a pH of 9.5. Therefore, a pH of 9.5 was selected for subsequent works and real sample analysis. Because the DLLME procedure is carried out after derivatization, the time of solvent injection into the sample is of considerable importance. Several factors (type of oxidant, pH, etc.) may affect this time. Optimization of the injection time enhances the

The extraction and dispersing solvents are important factors in DLLME. In this study, five organic solvents (dichloromethane, trichloromethane, chlorobenzene, chloroform, and carbon tetrachloride) were tested for extraction (Fig. 6). The results showed that chloroform in the presence of methanol as dispersing solvent formed a more stable two-phase system; as a result, higher signals could be observed. Dispersing solvents should have appropriate miscibility with both extraction solvent and sample solution in order to form a distinct cloudy solution. Thus, four organic solvents (methanol, ethanol, acetonitrile and acetone) were investigated as dispersers. As seen in Fig. 7, ethanol gave the highest absorbance for antipyrine dyes and possessed a relatively high enrichment factor. Compared with the previous report on DLLME (Lavilla et al., 2012), the optimal extraction and dispersing solvents depend on the composition of the water sample, differing between drinking water and seawater samples. This difference may be due to the salting-out effect of the sample matrix on the miscibility of the dispersing solvent with water and by the dielectric constant of the extraction solvent (Wang and Anderko, 2001), both of which influence DLLME yield. 3.5. Effect of extraction solvent volume Volumes of extraction and dispersing solvents, as well as the ratio, strongly affect the efficiency of extraction of antipyrine dyes.

Fig. 3. Plot of Ln([dye]) vs. time (min) emphasizing the first-order behavior of the antipyrine dye of phenol.


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Fig. 6. Effect of extraction solvent on the DLLME procedure. Conditions: 10 mL water, 900 lL methanol, 80 lL extraction solvent, pH = 9.5, temperature = 25 °C.

Fig. 4. Effect of (A) pH, and (B) injection time on the derivatization and DLLME procedure. Conditions: 10 mL water, 900 lL ethanol, 80 lL chloroform, pH = 9.5, temperature = 25 °C.

Fig. 7. Effect of dispersing solvent on the DLLME procedure. Conditions: 10 mL water, 900 lL dispersing solvent, 80 lL chloroform, pH = 9.5, temperature = 25 °C.

3.6. Effect of dispersing solvent volume The volume of dispersing solvent affects the cloudiness of the solution (water/dispersing solvent/extraction solvent), as well as the degree of solvent dispersion in the aqueous phase. Volume can therefore affect the extraction efficiency and enrichment factor. Consequently, when using ethanol as dispersing solvent, optimization of volume is necessary. To determine the optimal volume of ethanol, experiments using different volumes (i.e., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.00 and 1.2 mL) were performed. The volume of chloroform was fixed at 80 lL. Based on the results, 0.9 mL was found to be the optimal ethanol volume to achieve maximal extraction efficiency.

3.7. Effect of salt amount Fig. 5. Effect of 4-aminoantipyrine (4-AAP) concentration. Conditions: 10 mL water, 900 lL methanol, 80 lL extraction solvent, pH = 9.5, temperature = 25 °C.

The volume of chloroform for extraction was optimized using 0.9 mL ethanol and different volumes of chloroform (20, 30, 40, 50, 60, 70, 80, 90 and 100 lL). Ultimately, a chloroform volume of 80 lL was found to be optimal.

Adding a salt in dispersive liquid–liquid microextraction usually improves the yield of most analytes due to the salting-out effect. In this study, the occurrence of the salting-out effect was evaluated by measuring the extraction efficiency in the presence of different concentrations of NaCl (0–1.5 mol/L).As shown in Fig. 8, addition of salt had no significant effect on the extraction efficiency. However, observing such trend was predictable, due to the high salt concentration in the seawater matrix. Thus, the remaining extraction experiments were performed without added salt.


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Table 1 Analysis of total phenols using optimized DLLME in several water samples obtained from different locations of Chabahar Bay (Iran). Sampling location

Phenol found (lg/L) May 2013

Phenol found (lg/L) September 2013

Mean recovery(%) for 50 lg/L added phenol

1 2 3 4 5

3.2 ± 62.9 2.7 ± 66.7 2.2 ± 74.1 4.1 ± 74.8 1.9 ± 73.5

2.1 ± 27.8 1.7 ± 28.5 1.9 ± 31.0 1.3 ± 30.2 1.8 ± 29.5

97.8 99.4 98.2 95.6 99.00

Table 2 Comparison of the selected and the reported method based on the 5530 APHA standard method for the determination of phenols.

Fig. 8. Effect of NaCl concentration (mol/L procedure.

Parameter

Optimized DLLME method for seawater

Optimized medium Sample volume Extraction solvent

Artificial sea water 10 mL 80 lL of chloroform

Disperser Extraction time (min) Oxidant 4-Aminoantipyrine (4AAP) Enrichment factor (EF) Relative standard deviation, (RSD) LOD (lg/L 1) LOQ (lg/L 1) Dynamic linear range (lg/L 1)

900 lL of methanol 7 (25 °C) Same 1% w/v; 150 lL 920 %6 (n = 7)

5 mL 50 lL of trichloromethane 200 lL of acetonitrile 10 (27 °C) Same 1% w/v; 50 lL 700 %5.2 (n = 6)

0.18 0.6 1–900

0.8 2.5 Up to 150

1

) on absorbance in the DLLME

3.8. Analytical performance To evaluate the efficiency of DLLME coupled with UV–Vis spectrophotometry, linear range, repeatability and limit of detection (LOD) were investigated under optimal conditions. The calibration curve was linear in the concentration range 1–900 lg/L, with a determination coefficient of 0.998. The EF of the method for total phenol content was high (920). The repeatability of DLLME was studied by extracting spiked seawater samples at a concentration of 50 lg/L; the relative standard deviation (RSD) was 6% (n = 7). The method gave a limit of detection (LOD) of 0.18 lg/L, and a limit of quantification (LOQ) of 0.6 lg/L for phenol. These results confirmed the potential of the method to quantify trace phenols in seawater with adequate sensitivity and repeatability. 3.9. Sampling and analysis of real seawater samples The applicability of the method was evaluated by extracting phenols from seawater samples. Sampling was carried out in the surface layer (25 cm depth) from 5 different locations of Chabahar

Reported method (Lavilla et al., 2012)

Bay (Fig. 9). Sampling stations were 50–100 m from the coast. Samples were collected in dark glass bottles (sampling time were May and September 2013). Seawater samples were pretreated by filtering through glass microfibers (GF/C Whatman). To prevent degradation of analytes, the samples were collected and analyzed during daytime. The salinity, temperature and pH of the seawater samples varied between 27–39, 27–30 °C, and 7.8–8.3, respectively. Analytical results are presented in Table 1. There are several sources of phenol pollution in Chabahar Bay, including the industrial and city wastes, effluents from marine transportation, and maintenance of boats and ships in coastal areas. Our results showed a significant difference between two times of sampling (Table 1). This difference may be due to the drastic climate change during the sampling period, caused by the seasonal monsoon that reduces phenol accumulation. Comparison of optimized DLLME with the previously reported method for drinking water (Lavilla et al., 2012) revealed considerable differences (Table 2). This is due to the high concentrations of salts in seawater, which affect the yield and optimal extraction conditions. These results suggest that analysis of seawater requires special conditions compared to drinking water. 4. Conclusions

Fig. 9. Locations of coastal Chabahar sampling sites (Site 1: N 25° 18 39, E 60° 36 00; Site 2: N 25° 18 28, E 60° 37 26; Site 3: N 25° 18 54 E 60° 36 47; Site 4: N 25° 35 58 E 60° 59 99; Site 5: N 25° 36 63, E 60° 60 77).

Dispersive liquid–liquid microextraction was optimized for spectrophotometric determination of total phenols in seawater samples. Our results show the analytical performance of DLLME under optimal conditions combined with the 5530 APHA standard method for derivatization and extraction of phenolic compounds in seawater samples. Comparison of this DLLME method, optimized for seawater samples, with the previously reported method for


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drinking water shows considerable differences. This new method gives simple and rapid preconcentration of total phenols from seawater, a higher enrichment factor, a lower limit of detection, and a wide dynamic range.

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