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Homogenizer assisted dispersive liquid-phase microextraction for the extraction-enrichment of phenols from aqueous samples and determination by gas chromatography
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Homogenizer assisted dispersive liquid-phase microextraction for the extraction-enrichment of phenols from aqueous samples and determination by gas chromatography Tahmineh Javadi , Bahman Farajmand , Mohammad Reza Yaftian , Abbasali Zamani PII: DOI: Reference:
S0021-9673(19)31165-3 https://doi.org/10.1016/j.chroma.2019.460733 CHROMA 460733
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
6 August 2019 23 November 2019 25 November 2019
Please cite this article as: Tahmineh Javadi , Bahman Farajmand , Mohammad Reza Yaftian , Abbasali Zamani , Homogenizer assisted dispersive liquid-phase microextraction for the extractionenrichment of phenols from aqueous samples and determination by gas chromatography, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460733
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Highlights ●A homogenizer assisted DLPME abbreviated as HA-DLPME is introduced for the first time. ●The method was applied for the preconcentration, followed by GC analysis of phenols. ●The method was successfully used for the determination of phenols in water samples. ●The developed method is very rapid, inexpensive, simple and environmentally friendly.
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Homogenizer assisted dispersive liquid-phase microextraction for the extraction-enrichment of phenols from aqueous samples and determination by gas chromatography
Tahmineh Javadi,1 Bahman Farajmand,1 Mohammad Reza Yaftian,1*Abbasali Zamani2 1
Department of Chemistry, Faculty of Science, The University of Zanjan, Postal Code 45371– 38791, Zanjan, Iran. 2 Department of Environmental Science, Faculty of Science, The University of Zanjan, Postal Code 45371–38791, Zanjan, Iran.
Abstract In this research, dispersive liquid-phase microextraction has been used for the extraction of some phenols including phenol, 3-methylphenol, 4-nitrophenol, 2-chlorophenol, tert-buthylphenol from aqueous samples, and then the analysis was done by the gas chromatography-flame ionization detector technique. For the first time, a laboratory homogenizer has been applied for dispersing of extracting organic solvent. To improve the chromatographic behavior, acetic anhydride was used as a derivatization reagent of the analytes. The effective parameters on the extraction and derivation process such as extraction solvent type and volume, amount and time of derivatization, sample pH and ionic strength, homogenization time and speed were investigated and optimized. The analytical performances of the method, such as linear dynamic range, repeatability, and detection limit were evaluated under the optimum condition. Under the optimal experimental conditions, the calibration plots were linear the range of 1 - 500 μg L−1 with the detection limits between 0.1 - 0.9 μg L−1, and the repeatability in the range of 2.6 to 10.0%. These values vary depend on the compounds. The proposed method was evaluated for the determination of the studied phenolic compounds in different real samples such as river water, tap water and industrial wastewater. The relative recoveries were between 90 and 111%. Keywords: Homogenizer, Dispersive liquid phase microextraction, Phenols, Gas chromatography
*Corresponding author: Mohammad Reza Yaftian 2
E-mail address: [email protected] (Mohammad Reza Yaftian)
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1. Introduction The sample preparation step is crucial in trace chemical analysis, particularly for those are concerned on the samples with complex matrix. Among the modern sample preparation methods, liquid phase microextraction (LPME) methods with different geometries are known as potential preparation techniques [1]. Among these techniques, the dispersive liquid-phase microextraction (DLPME) method has become one of the most considerable research fields [2]. In this method, the extraction solvent is dispersed as tiny droplets in the sample matrix, and an emulsion is formed. After a proper time, separation of extraction solvent was generally done by centrifugation. Two main advantages of DLPME in comparison with other LPME methods are: (1) the shorter extraction time due to the fast establishing of the equilibrium state, and (2) the higher preconcentration factor due to the high phase ratio of aqueous sample to the extraction solvent [2]. Initial configuration of DLPME, which has been developed by Assadi et al. [3], was based on a ternary component system in which a solution of extraction and disperser solvents were rapidly inserted into the aqueous sample. The mixture was stirred, and an emulsion (include of the fine droplet of extraction solvent) was formed. In this configuration, a large volume of a solvent which is miscible with both extraction solvent and the aqueous sample was used as a dispersing element. However, reduction or elimination of disperser solvents is highly appropriate, because most of them are toxic and environmentally harmful. Additionally, the use of organic disperser solvent usually decreases partition of analytes into the extraction solvent and reduces the extraction efficiency [4, 5]. Therefore, various efforts have been made for the reduction or exchange of organic solvent with other dispersers. Manual shaking was the easiest approach which could help to emulsion formation. Tsai and Huang proposed this procedure for the extraction of some organochlorine pesticides in water samples [6]. Very low volume of organic solvent has been used in this method, and good enrichment factors have been achieved. Perhaps, the most important weakness of the manual shaking is the large size of solvent droplets, which reduces the contact surface area between two phases. Application of ultrasound waves for emulsion formation is an alternative way for reduction of droplet size [7]. Li et al. have shown that the application of vortex mixer has better extraction recovery than manual shaking or ultrasound [8].
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Application of homogenizer for dispersing of the organic solvent can be considered as a new concept in liquid phase microextraction method. Homogenizers are typically used for liquidliquid emulsification. One of the usual types of homogenizers is batch rotor-stator mixer. Ika laboratory technology is one of the producers of this type of homogenizer. The system involves a rotor which is placed within a stationary stator, and some slots are embedded in the stator head (Figure 1a). Due to the high circular speed (typical ranges from 10 to 50 m s-1), the liquid mixture is sucked axially inside the dispersion head and then driven radially out through the slots of the stator. There is small proximity between stator and rotor (Figure 1b); it varies from 100 to 3000 μm. This narrow gap produces an enormously strong shear force that expels the liquid mixture through the slots of the stator and the dispersion was formed. The shear rates of this type of mixer are ranging from 20,000 to 100,000 s−1, and consequently, they are usually called high shear mixers (HSMs). High kinetic energy provided by the rotor, dissipates mostly inside the stator and therefore the local energy dissipation rate in a rotor-stator mixer is in the range of 1000 - 100,000 m2 s−3 that can be at least two orders of magnitude higher than in a conventional mechanical stirrer (10 - 1000 m2 s−3) and agitators (0.1 - 100 m2 s−3) [9]. However, the HSMs have lower energy dissipation than ultrasound mixer, but the higher energy density is required for an ultrasonic mixer. HSMs have more uses in food, cosmetics, pharmaceutical, and chemical industries because they produce narrow droplet size distribution. The typical droplet sizes, which are generated by different types of HSMs varies from 0.5 to 100 μm [10]. The droplet size reduces when the energy dissipation rate and rotor speed increase; however, a critical rotor speed was found above which no significant droplet size reduction was observed [12]. Rotor-stator mixers also generate a high intensity of turbulence. Otomo and coworkers simulate the flow patterns of a fluid for a batch rotor-stator mixer with different geometries [11]. Figure 1c shows the simulation of the flow patterns. The flow inside the head is principally tangential but, when the fluids hit the surface of the leading edge of the slot almost all the tangential momentum is converted into radial momentum, and the jet emerges from the slot and behind the jet, the circulation loops are shaped. Theoretical and experimental studies reveal that the jet velocity is proportional to the rotor speed while the energy dissipation rate is scale with the cube of rotor speed. Another application of rotor-stator mixers is designing of the new efficient chemical reactor due to their excellent micro-mixing capability. Visscher et al. studied the liquid-liquid mass 5
transfer rates in a rotor-stator reactor (Figure 1d) [13]. They showed that the mass transfer rates were at least 10 - 25 times higher compared to conventional reactors, which will contribute to better control over selectivity and yield, and a shorter time to process. The velocity gradient over the gap between the rotor and the stator causes an intense shear force to act on the reactor contents. The resulting turbulence intensity leads to a strong dispersion of the reactor contents, and thus a high surface area, which causes high liquid−liquid mass transfer rates. Phenols are present in the environment due to their wide applications in industry as intermediates for the production of dyes, explosives, pesticides, and pharmaceuticals. Because of their moderate bioaccumulation and high toxicity, they can represent serious health hazards. European Community (EU) legislation has agreed a maximum allowed phenol concentration of 0.5 μg L−1 in tap water [14]. Therefore, many efforts have been done for the introducing of new rapid and efficient quantification methods for trace analysis of phenols in the aquatic environment [15-20]. This paper intends to introduce for the first time the application of a homogenizer as the dispersing element for DLPME purposes. The proposed technique has been used for the extraction-preconcentration of phenol,
3-methylphenol (MP),
4-nitrophenol (NP),
2-
chlorophenol (CP), tert-buthylphenol (tert-BP) as phenolic model compounds. 2. Experimental 2.1. Reagents and standards Analytical grade of
phenolic compounds include phenol (P), 3-methylphenol (MP), 4-
nitrophenol (NP), 2-chlorophenol (CP), tert-buthylphenol (tert-BP) were purchased from SigmaAldrich (St. Louis, USA). Acetone (gas chromatographic grade), 2,4-dibromoethane, dibromomethane, carbon tetrachloride, 1,2-dichloroethane, acetic anhydride, sodium chloride and sodium hydroxide (98%) were obtained from Merck (Hohenbrunn, Germany). Deionized water provided by Zolalan purification system (model, m-uv-3+; Iran) was used throughout the study. The initial stock solutions of individual phenolic compounds were prepared by dissolving an appropriate amount of each analyte in acetone to obtain total concentration of 1000 mg L-1. The mixture stock solution was obtained by diluting of the initial stock solutions to 50 mg L-1 in acetone. The standard working solution was prepared by diluting of mixture solution to 1 mg L-1 with deionized water. The individual concentrations for each phenol were 0.1 mg L -1 for P and 6
tert-BP, 0.2 mg L-1 for CP and MP, and 0.4 mg L-1 for NP. This solution has been used for optimization of the parameters affecting the procedure. 2.2. Instrumentation The homogenizer T 18 basic ULTRA-TURRAX with S18N-10G dispersing element (Ika laboratory technology, Germany) was employed for dispersion the organic solvent. Some specifications of this homogenizer are summarized in Table 1. The analysis of phenolic compounds was performed by an Agilent gas chromatograph GC-FID system model 6890N (USA) with split/splitless injector carried out at 260 ºC. The GC was equipped with OV-101 capillary column with 10 m length, 0.25 mm i.d., and 0.1 µm film thickness (SGE, UK). The oven temperature program was as follows: the initial temperature 80 ºC held for 1 min increased at a rate of 25 ºC min-1, and then to 280 ºC hold for 5 min. Nitrogen gas (99.999%) was used as the carrier and make-up gas at flow rates 1 and 30 mL min-1, respectively. The flow rates of air and hydrogen gas for the detector (FID) were correspondingly set at 400 and 40 mL min-1. 2.3. Procedure An aqueous solution (3 mL) containing the phenolic compounds after adjusting the alkalinity and ionic strength was placed in a conical-bottom glass centrifuge tube. The amount of 50 µL of acetic anhydride (as derivatization reagent) was added to the sample, and the mixture was shaken for 5 min at room temperature. Afterward, 10 µL of the extraction solvent (2,4-dibromoethane) was injected into the tube, and the solution was homogenized for 50 sec at 12000 rpm, immediately a cloudy solution appeared in the tube, then two phases were separated by centrifugation for 5 min at 2000 rpm. Ultimately, 1 µL of sediment extracting phase was removed by a 5-µL microsyringe for analysis by gas chromatography. The peak areas were used as analytical signals. Between each determination the homogenizer shaft for next samples was inserted in 0.05 mol L-1 sodium hydroxide solution for 30 sec. Then it was washed by putting the shaft in distilled water for 30 sec, and finally in acetone for further 30s. This procedure allowed removing the possible memory effect due to the presence of the analyte on the homogenizer shaft. The homogenizing speed was set at 8000 rpm for cleaning step. 2.4. Calculation of enrichment factors and relative recoveries
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The enrichment factors (EFs) were calculated according to equation 1, where Cs,initial and Ca,final are the concentrations of the analyte in the initial aqueous sample and final organic solution, respectively. The relative recoveries (RR (%)) for real sample analysis also were obtained by the equation 2, where Cfounded, Creal, and Cadded are the concentration of the real sample after spiking the standard, the real sample concentration and the spiked concentration, respectively. (1) ( )
(2)
2.5. Real samples analysis Three environmental water samples were collected; tap water (Zanjan, Iran), river water was obtained from Zanjan-Rood river (Zanjan, Iran) and industrial wastewater (Paint and resin manufacturing company, Qazvin, Iran). All of the water samples were filtered by 0.45 µm nylon membrane filter (Whatman, USA) before the analysis. All water samples were stored in the refrigerator at 4 ºC until their analysis. In this study, multiple standard addition method was used for determination of target compounds in different real samples. The standard solution was added at the concentration levels of 10, 20, 50 and 100 µg L-1 for P, CP, MP and tert-BP compounds, and 20, 40, 100 and 200 µg L-1 for NP. 3. Results and discussion 3.1. Homogenizer as a dispersing device Application of homogenizer rather than other emulsification methods can have some advantages. Firstly, homogenizer can produce smaller solvent droplet size than manual and agitator shaking. It has also narrower droplet size distribution [10]. Therefore, the extraction of the analytes to the organic solvent can be modified based on the increasing the surface area between two phases. Dispersion of the organic solvent can be formed in less than one minute without the help of surfactant or auxiliary solvents. Perhaps, the homogenizer generates bigger droplet size than ultrasonic dispersion, but by this device, emulsion forms in a shorter time without any harmful ultrasonic waves [22]. Turbulent flows and shear forces inside the homogenizer head can increase the mass transfer of the analyte for extraction and derivatization 8
processes. These could reduce the extraction time and increase the extraction efficiency and makes the homogenizer assisted dispersive liquid-phase microextraction (HA-DLPME) turn to the rapid and efficient sample preparation method. 3.2. Optimization of the HA-DLPME conditions In order to optimize the method for the determination of the studied phenols, effective parameters were evaluated and optimized thoroughly. These factors are essentially divided into four sections include type and volume of extraction solvents, sample alkalinity and ionic strength, derivatization conditions, and homogenization conditions which have discussed in detail in the following section. 3.2.1. Extraction solvent and its volume Selection of a suitable extraction solvent is the essential part of microextraction procedures, because the physicochemical properties of the solvent play decisive role in the extraction efficiency and compatibility of the method with the final analysis instrument. An appropriate solvent must have a low boiling point, low miscibility with water, a high extraction efficiency, and a good chromatographic behavior [17]. Besides, the density of the solvent is important which determine the quality of the phase separation. In recent years, low-density solvents such as 1decanol and 1-undecanol are preferred. In fact, these solvents have typically lower toxicity than high-density solvents, although they have some technical disadvantages. Firstly, after the extraction, picking up the solvent is very difficult. Secondly, they have usually high boiling points, and hence they are not eligible for gas chromatographic analysis; because their peaks can overlap with those of the target analytes. Therefore a set of experiments were conducted to screen different organic solvents having a density higher than the water and good chromatographic performances. The investigated solvents were carbon tetrachloride, 1,2-dichloroethane, 1,2-dibromoethane, and dibromomethane. The results are shown in Figure 2a. It can be seen that 1,2-dibromoethane and 1,2-dichloroethane have good extraction efficiency, but 1,2-dibromoethane has better performance for the extraction of 4-nirtophenol and 2-chlorophenol. Hence, 1,2-dibromoethane was selected as the proper extraction solvent. Further, the volume of 1,2-dibromoethane was optimized by taking different volumes of it ranges 10 - 75 µL. The peak areas of phenols were gradually decreased from 10 -
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75 µL; this may be due to the dilution of the sediment phase at larger volumes (Figure 2b). Therefore, 10 µL of the 1,2 dibromoethane was selected for further experiments. 3.2.2. Sample ionic strength and alkalinity To investigate the effect of ionic strength on the extraction efficiency of the HA-DLPME procedure, NaCl was added in the range of 0 - 0.35 g mL-1 to the sample solutions. The results showed that the peak area increased slowly by the ionic strength increases to 0.3 g mL -1. This revealed the salt concentration did not affect significantly the extraction efficiency of the method (Figure 3a). Therefore, 0.3 g ml-1 of salt was nominated for following the experiments. To find out the effect of sample pH on the proposed HA-DLPME procedure, the analytical renounces of phenols as a function of the aqueous phase pH was evaluated. Initial tests were done on the sample and it became clear that in acidic and neutral medium, analytical signals are very low. Therefore, a set of experiments were conducted in which the alkalinity of the aqueous sample was adjusted by the use of different concentration of NaOH in the range from 0.00001 to 1 mol L-1. The experiment revealed that the NaOH concentration of 0.1 mol L-1 was found to be optimum. The results shown in Figure 3b reveal that the peak areas were suddenly improved when the alkalinity increased. This improvement may be due to the increase in the derivatization yield at alkaline media. Reduction of responses at 1 mol L-1 of sodium hydroxide, may also be due to the hydrolysis of the derivatives formed. 3.2.3. Derivatization conditions Derivatization step usually is performed for modification of chromatographic behavior of polar compounds such as phenols [24]. Different derivatization reagents are usually applied for this purpose; acetic anhydride is one the affordable reagent [23]. It is an inexpensive reagent and can be added directly into the aqueous sample to perform the reaction with high efficiency in only a few minutes. These advantages stimulate us to use acetic anhydride as the derivatization reagent. Acetic anhydride exchanges the hydroxyl group of phenols to ethyl ester functional group. The amount of reagent, temperature, and time of derivatization were studied and optimized. The results are reported in Supplementary Material (Figures S1-S3). Briefly, the amount of 50 µL of acetic anhydride, in 5 min at room temperature provided the maximum responses.
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3.2.4. Homogenization conditions Homogenization time and speed are the parameters may affect the efficiency of the extraction step. In fact, the extraction procedures are generally equilibrium processes and they need enough time for establishing the equilibrium state. For this purpose, a series of the extraction experiments using different homogenization time from 5 to 100 s were performed. The results (Figure 4) confirmed that an increase of the homogenization time up to 50 s, leads to the enhancement of the peak areas. Beyond this time a reduction of the peak areas was observed. An explanation for these observations can be presented by considering that the equilibrium state can be attained after 50 s of homogenization, and the evaporation of the organic solvent or analytes causes the reduction in the extraction efficiency beyond this time of mixing the phases. On the other hands, homogenization speed can affect the sizes of droplets of the organic solvent. Reducing the droplet size will increase the contact surface area between the two phases. Figure 5 describes the effect of homogenization speed on the peak areas. The results showed that by increasing the homogenization speed in the range 4000 - 12000 rpm, the extraction efficiency improves. However, a further increase in the homogenization speed did not affect the process efficiency, which states that a further decrease in droplet size at the higher speeds is not provided [12]. These results conduct us to select the speed of 12000 rpm as a critical speed. 3.3. Figures of merit To evaluate the analytical performance of the HA-DLPME method, its figures of merit were determined under optimal conditions (Table 2). All the solutions were prepared by deionized water. The linear ranges of the calibration curves obtained by the standard calibration method were 1.0 - 500 µg L-1 for P, MP, CP and tert-BP, and 2.0 - 500 µg L-1 for NP. The correlation coefficients were in the range of 0.9914-0.9979. The limit of detections (LODs) based on S/N = 3 of the phenols P, MP, CP, tert-BP and NP were 0.3, 0.2, 0.1, 0.3 and 0.7 µg L-1, respectively. The limit of quantifications (LOQs) based on S/N=10 were calculated too. The repeatability and reproducibility of the method were estimated by evaluating the intra- and inter-day relative standard deviations (RSDs). These evaluations were performed by analyzing five replicates experiments at high and low concentration levels. Intra-day precisions for the target phenols were found to be in the range 4.1-9.5%, and intra-day precisions also were in the range 5.2 10%. Based on the results obtained, the EFs were 127 for P, 153 for MP, 136 for CP, 144 for
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tert-BP and 115 for NP. A comparison of the proposed method with previous methods reported in literature has been summarized in Table 4. This comparison confirmed that the characteristics of the proposed method are close to those of the compared methods. However, it is seen that the extraction time of the method presented in this study is significantly shorter than the compared methods. 3.4. Real sample analysis In order to investigate the applicability of the proposed method, it was applied for the determination of target phenols in water samples collected from three different sources. A tap water (University of Zanjan Campous, Zanjan-Iran), a river water (Zanjan Rood river, Zanjan province, Iran), and an industrial wastewater samples from a paint and resin manufacturing company (Qazvin-Iran) were obtained for this study. The results indicated that there was no evidence of the target analytes present in the examined tap and river water samples, but all of the phenolic compounds except tert-BP were found in the industrial wastewater sample. The concentrations were 3.2 µg L-1 for P, 1.8 µg L-1 for MP, 7.1 µg L-1 for CP and 2.0 µg L-1 for NP which all of them were more than allowable levels (0.5 µg L-1). Table 4 shows the relative recoveries (RRs) for studied real samples spiked with different concentrations of phenols. The relative recoveries ranged from 96 to 109% for the tap water, 90-110% for river water, and 94114% for wastewater samples. Figure 6 reveals the chromatograms for different real samples before and after spiking with analytes. 4. Conclusion A novel, very rapid, inexpensive, simple and environmentally friendly method based on the application of a homogenizer, as a dispersing device, for performing a DLPME procedure of extraction-enrichment of phenols has been described. Rotor-stator configuration of homogenizer generates emulsion of organic solvent in a few second without the assistance of surfactant or disperser solvent. The high rotational speed of the rotor can produce a turbulent flow and accelerate the mass transfer of analytes to the organic solvent, and it could increase the extraction efficiency. By use of homogenizer, the droplet size of the organic solvent is smaller than manual and agitator shaking. In comparison with ultrasound devices, homogenizer forms emulsion in a shorter time without any harmful waves. For evaluation of this application, the proposed method was used for microextraction of some phenolic compounds from water samples. After 12
optimization of different effective parameters, the results showed that the method has good analytical performances for the determination of phenols in water. The method is potential for the determination of phenols in different real samples. As a perspective for developing of this study, and based on the recent research on the automation of the dispersive liquid phase microextraction [26,27], we will continue this study by applying the results for automation of the phenols monitoring. Acknowledgment The authors are grateful to the research council of the University of Zanjan for the financial support of this project. References [1] A. Sarafraz-Yazdi, A. Amiri, Liquid-phase microextraction, TrAC, Trends Anal. Chem. 29 (2010) 1-14. https://doi.org/ 10.1016/j.trac.2009.10.003 [2] H. Yan, H. Wang, Recent development and applications of dispersive liquid–liquid microextraction, J. Chromatogr. A 1295 (2013) 1-15. http://dx.doi.org/10.1016/j.chroma.2013.04.053. [3] M. Rezaee, Y. Assadi, M.R.M. Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, Determination of organic compounds in water using dispersive liquid–liquid microextraction, J. Chromatogr. A 1116 (2006) 1–9. https://doi.org/10.1016/j.chroma.2006.03.007. [4] W. Ahmad, A.A. Al-Sibaai, A.S. Bashammakh, H. Alwael, M.S. El-Shahawi, Recent advances in dispersive liquid-liquid microextraction for pesticide analysis, TrAC, Trends Anal. Chem. 72 (2015) 181-192. https://doi.org/10.1016/j.trac.2015.04.022. [5] M. Saraji, M.K. Boroujeni, Recent developments in dispersive liquid–liquid microextraction, Anal. Bioanal.Chem. 406 (2014) 2027–2066. https://doi.org/10.1007/s00216-013-7467-z. [6] W.C. Tsai, S.D. Huang, Dispersive liquid–liquid microextraction with little solvent consumption combined with gas chromatography–mass spectrometry for the pretreatment of organochlorine pesticides in aqueous samples, J. Chromatogr. A 1216 (2009) 5171-5175. https://doi.org/10.1016/j.chroma.2009.04.086. [7] J. Regueiro, M. Llompart, C. Garcia-Jares, J.C. Garcia-Monteagudo, R. Cela, Ultrasoundassisted emulsification–microextraction of emergent contaminants and pesticides in environmental waters, J. Chromatogr. A 1190 (2008) 27-38. https://doi.org/10.1016/j.chroma.2008.02.091. [8] Y. Li, Y. Jiao, Y. Guo, Y. Yang, Determination of bisphenol-A, 2,4-dichlorophenol, bisphenol-AF and tetrabromobisphenol-A in liquid foods and their packaging materials by
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Figure captions Figure 1. a) Image of an S18-10G dispersing element of a homogenizer and schematic image of its internal components. b) Close view of a dispersing head. c) Flow patterns (radial and tangential velocities) at different regions around the dispersing head [11] (with permission under license number 4678630497689). d) Rotor-stator spinning disc reactor. Figure 2. a) Effect of solvent type on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols at pH=9; no salt addition; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent volume, 50 µL; homogenization speed, 4000 rpm; homogenization time, 1 min. b) Effect of solvent volume on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols at pH=9; no salt addition; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent type, 1,2-dibromoethane; homogenization speed, 4000 rpm; homogenization time, 1 min. Figure 3. a) Effect of ionic strength on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols at pH=9; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization speed, 4000 rpm; homogenization time, 1 min. b) Effect of the sample alkalinity on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols; salt concentration, 0.3 g/mL; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization speed, 4000 rpm; homogenization time, 1 min. Figure 4. Effect of homogenization time on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols and 0.1 mol/L NaOH; salt concentration, 0.3 g/mL; derivatization reagent, 50 µL acetic anhydride; shaking time, 5 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization speed, 4000 rpm. Figure 5. Effect of homogenization speed on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols and 0.1 mol/L NaOH; salt concentration, 0.3 g/mL; derivatization reagent, 50 µL acetic anhydride; shaking time, 5 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization time, 50 s.
16
Figure 6. a) Chromatograms obtained for tap water and spiked tap water at a concentration level of 10 µg/L for P, MP, CP and tert-BP and 20 µg/L for NP after extraction at optimal conditions. b) Chromatograms obtained for river water and spiked tap water at a concentration level of 20 µg/L for P, MP, CP and tert-BP and 40 µg/L for NP after extraction at optimal conditions. c) Chromatograms obtained for tap water and spiked tap water at a concentration level of 10 µg/L for P, MP, CP and tert-BP and 20 µg/L for NP after extraction at optimal conditions.
17
Table 1. Specifications of the homogenizer used in this study [25]. Model Disperser Volume range Stator diameter Rotor diameter The gap between rotor and stator Allowable speed Circumferential speed Immersion depth range Shaft length pH range Working temperature (max) Droplet size range The material in contact with the medium Suitable for solvent
T18 basic S18-10G 1-100 mL 10 mm 7.5 mm 0.35 mm 25000 rpm 9.8 m/s 25-70 mm 108 mm 2-13 180 ºC 1-10 µm AISI 316L and PTFE yes
Table 2. Figures of merit for HA-DLPME method. Compound
P MP CP tert-BP NP
RSD (%) (n=5) Intra-day Inter-day * * High Low High Low 6.6 9.5 9.7 9.8 7.8 4.1 10.0 5.2 4.9 2.6 7.5 5.8 4.7 5.8 5.6 6.3 9.2 7.8 9.7 8.7
Linear range (µg/L) 1-500 1-500 1-500 1-500 2-500
R2
0.9979 0.9941 0.9952 0.9914 0.9961
Limit of Limit of quantitation detection (µg/L) (µg/L) 1 0.3 0.6 0.2 0.4 0.1 1 0.3 2 0.7
EF
127 153 136 144 115
* High and low concentration level were considered 200 and 2 µg L-1 respectively.
Table 3. Relative recoveries of phenolic compounds in different water samples. Compound
P
MP
Added (µg/L)
0 2 5 10 20 50 100 0 2 5 10
Tap water Found (µg/L) Relative recovery (%) 1 ND 1.9 95 5.1 102 9.9 99 21.4 107 54.5 109 109.1 109 ND 1.8 90 4.8 96 10.1 101 18
River water Found Relative (µg/L) recovery (%) ND 2.1 105 4.8 96 9.5 95 22.0 110 49.2 98 100.3 100 ND 2 100 5.1 102 9.6 96
Wastewater Found Relative (µg/L) recovery (%) 3.2 4.9 85 8.0 96 14.1 109 23.0 99 50.2 94 104.3 101 1.8 3.6 90 6.7 98 12.9 111
CP
tert-BP
NP
20 50 100 0 2 5 10 20 50 100 0 2 5 10 20 50 100 0 4 10 20 40 100 200
20.2 53.5 108.4 ND 2 4.7 9.6 20.8 51.1 97.8 ND 2.1 4.6 9.8 21.6 52.0 102.8 ND 3.5 9.1 21.1 39.6 97.2 205.9
101 107 108 100 94 96 104 102 98 105 92 98 108 104 103 87 91 105 99 97 103
19.4 46.5 99.1 ND 2.1 4.8 10.9 20.6 48.5 97.3 ND 2.2 5.3 10.3 19.1 44.9 92.3 ND 3.8 10.2 20.6 44.2 101.0 214.3
97 93 99 105 96 109 103 97 97 110 106 103 95 90 92 95 102 103 110 101 107
20.6 58.8 106.2 7.1 9.2 11.6 16.5 29.2 60.0 111.8 ND 1.7 4.4 11.0 19.8 51.5 104.4 2.0 5.7 11.6 21.8 40.7 106.3 198.3
94 114 104 105 90 94 110 106 105 85 88 110 99 103 104 92 96 99 97 104 98
1. Not detected Table 4. Comparison of the HA-DLPME with other microextraction methods for the determination of phenols. Method
LOD LOQ (µg/L (µg/L ) ) DLLME 2.5-LC10 DAD2
RS D (%) 113
TSP30.01DLLME 1 -GCMS Purge & 0.06trap0.12 GC-MS SDME4- 0.004 GC-MS -0.06 HA0.1DLPME 0.7
0.043.5
< 17
-
0.4-2
Organic solvent type & volume 1octanol/aceto ne 1000 µL Acetone/nhexane 200 µL
Extractio n time (min) -
EF
Real sample
DR 1
Referenc e
-
wastewat er
2.5
[20]
5
3551
River water
2.5
[21]
1.37
-
20
-
2.5
[23]
Hexyl acetate 2.5 µL 1,2dibromoethan
20
86102 115 -
River & wastewat er River water Tap, river and
4.812 4.110
2
[24]
2.5
Proposed method
19
0.8
-GCFID
e, 10 µL
153
wastewat er
1. Dynamic range based on order of magnitude. 2. Dispersive liquid-liquid microextraction followed by liquid chromatography diode array detector. 3. Temperature sensitive polymer. 4. Single drop microextraction
Author Contribution Section Author Miss Tahmineh Javadi Dr. Bahman Farajmand Prof. Mohammad Reza Yaftian Dr. Abbasali Zamani
Role MSc student who performed the experiments Supervisor, designating the study, interpretation of the results and writing the paper Supervisor, designating the study, interpretation of the results and writing the paper Advisor, interpretation of the results
20
Figure 1.
21
22
Figure 2.
Figure 3. 23
Figure 4.
24
Figure 5.
25
26
Figure 6.
Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Homogenizer assisted dispersive liquid-phase microextraction for the extraction-enrichment of phenols from aqueous samples and determination by gas chromatography Tahmineh Javadi , Bahman Farajmand , Mohammad Reza Yaftian , Abbasali Zamani PII: DOI: Reference:
S0021-9673(19)31165-3 https://doi.org/10.1016/j.chroma.2019.460733 CHROMA 460733
To appear in:
Journal of Chromatography A
Received date: Revised date: Accepted date:
6 August 2019 23 November 2019 25 November 2019
Please cite this article as: Tahmineh Javadi , Bahman Farajmand , Mohammad Reza Yaftian , Abbasali Zamani , Homogenizer assisted dispersive liquid-phase microextraction for the extractionenrichment of phenols from aqueous samples and determination by gas chromatography, Journal of Chromatography A (2019), doi: https://doi.org/10.1016/j.chroma.2019.460733
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Highlights ●A homogenizer assisted DLPME abbreviated as HA-DLPME is introduced for the first time. ●The method was applied for the preconcentration, followed by GC analysis of phenols. ●The method was successfully used for the determination of phenols in water samples. ●The developed method is very rapid, inexpensive, simple and environmentally friendly.
1
Homogenizer assisted dispersive liquid-phase microextraction for the extraction-enrichment of phenols from aqueous samples and determination by gas chromatography
Tahmineh Javadi,1 Bahman Farajmand,1 Mohammad Reza Yaftian,1*Abbasali Zamani2 1
Department of Chemistry, Faculty of Science, The University of Zanjan, Postal Code 45371– 38791, Zanjan, Iran. 2 Department of Environmental Science, Faculty of Science, The University of Zanjan, Postal Code 45371–38791, Zanjan, Iran.
Abstract In this research, dispersive liquid-phase microextraction has been used for the extraction of some phenols including phenol, 3-methylphenol, 4-nitrophenol, 2-chlorophenol, tert-buthylphenol from aqueous samples, and then the analysis was done by the gas chromatography-flame ionization detector technique. For the first time, a laboratory homogenizer has been applied for dispersing of extracting organic solvent. To improve the chromatographic behavior, acetic anhydride was used as a derivatization reagent of the analytes. The effective parameters on the extraction and derivation process such as extraction solvent type and volume, amount and time of derivatization, sample pH and ionic strength, homogenization time and speed were investigated and optimized. The analytical performances of the method, such as linear dynamic range, repeatability, and detection limit were evaluated under the optimum condition. Under the optimal experimental conditions, the calibration plots were linear the range of 1 - 500 μg L−1 with the detection limits between 0.1 - 0.9 μg L−1, and the repeatability in the range of 2.6 to 10.0%. These values vary depend on the compounds. The proposed method was evaluated for the determination of the studied phenolic compounds in different real samples such as river water, tap water and industrial wastewater. The relative recoveries were between 90 and 111%. Keywords: Homogenizer, Dispersive liquid phase microextraction, Phenols, Gas chromatography
*Corresponding author: Mohammad Reza Yaftian 2
E-mail address: [email protected] (Mohammad Reza Yaftian)
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1. Introduction The sample preparation step is crucial in trace chemical analysis, particularly for those are concerned on the samples with complex matrix. Among the modern sample preparation methods, liquid phase microextraction (LPME) methods with different geometries are known as potential preparation techniques [1]. Among these techniques, the dispersive liquid-phase microextraction (DLPME) method has become one of the most considerable research fields [2]. In this method, the extraction solvent is dispersed as tiny droplets in the sample matrix, and an emulsion is formed. After a proper time, separation of extraction solvent was generally done by centrifugation. Two main advantages of DLPME in comparison with other LPME methods are: (1) the shorter extraction time due to the fast establishing of the equilibrium state, and (2) the higher preconcentration factor due to the high phase ratio of aqueous sample to the extraction solvent [2]. Initial configuration of DLPME, which has been developed by Assadi et al. [3], was based on a ternary component system in which a solution of extraction and disperser solvents were rapidly inserted into the aqueous sample. The mixture was stirred, and an emulsion (include of the fine droplet of extraction solvent) was formed. In this configuration, a large volume of a solvent which is miscible with both extraction solvent and the aqueous sample was used as a dispersing element. However, reduction or elimination of disperser solvents is highly appropriate, because most of them are toxic and environmentally harmful. Additionally, the use of organic disperser solvent usually decreases partition of analytes into the extraction solvent and reduces the extraction efficiency [4, 5]. Therefore, various efforts have been made for the reduction or exchange of organic solvent with other dispersers. Manual shaking was the easiest approach which could help to emulsion formation. Tsai and Huang proposed this procedure for the extraction of some organochlorine pesticides in water samples [6]. Very low volume of organic solvent has been used in this method, and good enrichment factors have been achieved. Perhaps, the most important weakness of the manual shaking is the large size of solvent droplets, which reduces the contact surface area between two phases. Application of ultrasound waves for emulsion formation is an alternative way for reduction of droplet size [7]. Li et al. have shown that the application of vortex mixer has better extraction recovery than manual shaking or ultrasound [8].
4
Application of homogenizer for dispersing of the organic solvent can be considered as a new concept in liquid phase microextraction method. Homogenizers are typically used for liquidliquid emulsification. One of the usual types of homogenizers is batch rotor-stator mixer. Ika laboratory technology is one of the producers of this type of homogenizer. The system involves a rotor which is placed within a stationary stator, and some slots are embedded in the stator head (Figure 1a). Due to the high circular speed (typical ranges from 10 to 50 m s-1), the liquid mixture is sucked axially inside the dispersion head and then driven radially out through the slots of the stator. There is small proximity between stator and rotor (Figure 1b); it varies from 100 to 3000 μm. This narrow gap produces an enormously strong shear force that expels the liquid mixture through the slots of the stator and the dispersion was formed. The shear rates of this type of mixer are ranging from 20,000 to 100,000 s−1, and consequently, they are usually called high shear mixers (HSMs). High kinetic energy provided by the rotor, dissipates mostly inside the stator and therefore the local energy dissipation rate in a rotor-stator mixer is in the range of 1000 - 100,000 m2 s−3 that can be at least two orders of magnitude higher than in a conventional mechanical stirrer (10 - 1000 m2 s−3) and agitators (0.1 - 100 m2 s−3) [9]. However, the HSMs have lower energy dissipation than ultrasound mixer, but the higher energy density is required for an ultrasonic mixer. HSMs have more uses in food, cosmetics, pharmaceutical, and chemical industries because they produce narrow droplet size distribution. The typical droplet sizes, which are generated by different types of HSMs varies from 0.5 to 100 μm [10]. The droplet size reduces when the energy dissipation rate and rotor speed increase; however, a critical rotor speed was found above which no significant droplet size reduction was observed [12]. Rotor-stator mixers also generate a high intensity of turbulence. Otomo and coworkers simulate the flow patterns of a fluid for a batch rotor-stator mixer with different geometries [11]. Figure 1c shows the simulation of the flow patterns. The flow inside the head is principally tangential but, when the fluids hit the surface of the leading edge of the slot almost all the tangential momentum is converted into radial momentum, and the jet emerges from the slot and behind the jet, the circulation loops are shaped. Theoretical and experimental studies reveal that the jet velocity is proportional to the rotor speed while the energy dissipation rate is scale with the cube of rotor speed. Another application of rotor-stator mixers is designing of the new efficient chemical reactor due to their excellent micro-mixing capability. Visscher et al. studied the liquid-liquid mass 5
transfer rates in a rotor-stator reactor (Figure 1d) [13]. They showed that the mass transfer rates were at least 10 - 25 times higher compared to conventional reactors, which will contribute to better control over selectivity and yield, and a shorter time to process. The velocity gradient over the gap between the rotor and the stator causes an intense shear force to act on the reactor contents. The resulting turbulence intensity leads to a strong dispersion of the reactor contents, and thus a high surface area, which causes high liquid−liquid mass transfer rates. Phenols are present in the environment due to their wide applications in industry as intermediates for the production of dyes, explosives, pesticides, and pharmaceuticals. Because of their moderate bioaccumulation and high toxicity, they can represent serious health hazards. European Community (EU) legislation has agreed a maximum allowed phenol concentration of 0.5 μg L−1 in tap water [14]. Therefore, many efforts have been done for the introducing of new rapid and efficient quantification methods for trace analysis of phenols in the aquatic environment [15-20]. This paper intends to introduce for the first time the application of a homogenizer as the dispersing element for DLPME purposes. The proposed technique has been used for the extraction-preconcentration of phenol,
3-methylphenol (MP),
4-nitrophenol (NP),
2-
chlorophenol (CP), tert-buthylphenol (tert-BP) as phenolic model compounds. 2. Experimental 2.1. Reagents and standards Analytical grade of
phenolic compounds include phenol (P), 3-methylphenol (MP), 4-
nitrophenol (NP), 2-chlorophenol (CP), tert-buthylphenol (tert-BP) were purchased from SigmaAldrich (St. Louis, USA). Acetone (gas chromatographic grade), 2,4-dibromoethane, dibromomethane, carbon tetrachloride, 1,2-dichloroethane, acetic anhydride, sodium chloride and sodium hydroxide (98%) were obtained from Merck (Hohenbrunn, Germany). Deionized water provided by Zolalan purification system (model, m-uv-3+; Iran) was used throughout the study. The initial stock solutions of individual phenolic compounds were prepared by dissolving an appropriate amount of each analyte in acetone to obtain total concentration of 1000 mg L-1. The mixture stock solution was obtained by diluting of the initial stock solutions to 50 mg L-1 in acetone. The standard working solution was prepared by diluting of mixture solution to 1 mg L-1 with deionized water. The individual concentrations for each phenol were 0.1 mg L -1 for P and 6
tert-BP, 0.2 mg L-1 for CP and MP, and 0.4 mg L-1 for NP. This solution has been used for optimization of the parameters affecting the procedure. 2.2. Instrumentation The homogenizer T 18 basic ULTRA-TURRAX with S18N-10G dispersing element (Ika laboratory technology, Germany) was employed for dispersion the organic solvent. Some specifications of this homogenizer are summarized in Table 1. The analysis of phenolic compounds was performed by an Agilent gas chromatograph GC-FID system model 6890N (USA) with split/splitless injector carried out at 260 ºC. The GC was equipped with OV-101 capillary column with 10 m length, 0.25 mm i.d., and 0.1 µm film thickness (SGE, UK). The oven temperature program was as follows: the initial temperature 80 ºC held for 1 min increased at a rate of 25 ºC min-1, and then to 280 ºC hold for 5 min. Nitrogen gas (99.999%) was used as the carrier and make-up gas at flow rates 1 and 30 mL min-1, respectively. The flow rates of air and hydrogen gas for the detector (FID) were correspondingly set at 400 and 40 mL min-1. 2.3. Procedure An aqueous solution (3 mL) containing the phenolic compounds after adjusting the alkalinity and ionic strength was placed in a conical-bottom glass centrifuge tube. The amount of 50 µL of acetic anhydride (as derivatization reagent) was added to the sample, and the mixture was shaken for 5 min at room temperature. Afterward, 10 µL of the extraction solvent (2,4-dibromoethane) was injected into the tube, and the solution was homogenized for 50 sec at 12000 rpm, immediately a cloudy solution appeared in the tube, then two phases were separated by centrifugation for 5 min at 2000 rpm. Ultimately, 1 µL of sediment extracting phase was removed by a 5-µL microsyringe for analysis by gas chromatography. The peak areas were used as analytical signals. Between each determination the homogenizer shaft for next samples was inserted in 0.05 mol L-1 sodium hydroxide solution for 30 sec. Then it was washed by putting the shaft in distilled water for 30 sec, and finally in acetone for further 30s. This procedure allowed removing the possible memory effect due to the presence of the analyte on the homogenizer shaft. The homogenizing speed was set at 8000 rpm for cleaning step. 2.4. Calculation of enrichment factors and relative recoveries
7
The enrichment factors (EFs) were calculated according to equation 1, where Cs,initial and Ca,final are the concentrations of the analyte in the initial aqueous sample and final organic solution, respectively. The relative recoveries (RR (%)) for real sample analysis also were obtained by the equation 2, where Cfounded, Creal, and Cadded are the concentration of the real sample after spiking the standard, the real sample concentration and the spiked concentration, respectively. (1) ( )
(2)
2.5. Real samples analysis Three environmental water samples were collected; tap water (Zanjan, Iran), river water was obtained from Zanjan-Rood river (Zanjan, Iran) and industrial wastewater (Paint and resin manufacturing company, Qazvin, Iran). All of the water samples were filtered by 0.45 µm nylon membrane filter (Whatman, USA) before the analysis. All water samples were stored in the refrigerator at 4 ºC until their analysis. In this study, multiple standard addition method was used for determination of target compounds in different real samples. The standard solution was added at the concentration levels of 10, 20, 50 and 100 µg L-1 for P, CP, MP and tert-BP compounds, and 20, 40, 100 and 200 µg L-1 for NP. 3. Results and discussion 3.1. Homogenizer as a dispersing device Application of homogenizer rather than other emulsification methods can have some advantages. Firstly, homogenizer can produce smaller solvent droplet size than manual and agitator shaking. It has also narrower droplet size distribution [10]. Therefore, the extraction of the analytes to the organic solvent can be modified based on the increasing the surface area between two phases. Dispersion of the organic solvent can be formed in less than one minute without the help of surfactant or auxiliary solvents. Perhaps, the homogenizer generates bigger droplet size than ultrasonic dispersion, but by this device, emulsion forms in a shorter time without any harmful ultrasonic waves [22]. Turbulent flows and shear forces inside the homogenizer head can increase the mass transfer of the analyte for extraction and derivatization 8
processes. These could reduce the extraction time and increase the extraction efficiency and makes the homogenizer assisted dispersive liquid-phase microextraction (HA-DLPME) turn to the rapid and efficient sample preparation method. 3.2. Optimization of the HA-DLPME conditions In order to optimize the method for the determination of the studied phenols, effective parameters were evaluated and optimized thoroughly. These factors are essentially divided into four sections include type and volume of extraction solvents, sample alkalinity and ionic strength, derivatization conditions, and homogenization conditions which have discussed in detail in the following section. 3.2.1. Extraction solvent and its volume Selection of a suitable extraction solvent is the essential part of microextraction procedures, because the physicochemical properties of the solvent play decisive role in the extraction efficiency and compatibility of the method with the final analysis instrument. An appropriate solvent must have a low boiling point, low miscibility with water, a high extraction efficiency, and a good chromatographic behavior [17]. Besides, the density of the solvent is important which determine the quality of the phase separation. In recent years, low-density solvents such as 1decanol and 1-undecanol are preferred. In fact, these solvents have typically lower toxicity than high-density solvents, although they have some technical disadvantages. Firstly, after the extraction, picking up the solvent is very difficult. Secondly, they have usually high boiling points, and hence they are not eligible for gas chromatographic analysis; because their peaks can overlap with those of the target analytes. Therefore a set of experiments were conducted to screen different organic solvents having a density higher than the water and good chromatographic performances. The investigated solvents were carbon tetrachloride, 1,2-dichloroethane, 1,2-dibromoethane, and dibromomethane. The results are shown in Figure 2a. It can be seen that 1,2-dibromoethane and 1,2-dichloroethane have good extraction efficiency, but 1,2-dibromoethane has better performance for the extraction of 4-nirtophenol and 2-chlorophenol. Hence, 1,2-dibromoethane was selected as the proper extraction solvent. Further, the volume of 1,2-dibromoethane was optimized by taking different volumes of it ranges 10 - 75 µL. The peak areas of phenols were gradually decreased from 10 -
9
75 µL; this may be due to the dilution of the sediment phase at larger volumes (Figure 2b). Therefore, 10 µL of the 1,2 dibromoethane was selected for further experiments. 3.2.2. Sample ionic strength and alkalinity To investigate the effect of ionic strength on the extraction efficiency of the HA-DLPME procedure, NaCl was added in the range of 0 - 0.35 g mL-1 to the sample solutions. The results showed that the peak area increased slowly by the ionic strength increases to 0.3 g mL -1. This revealed the salt concentration did not affect significantly the extraction efficiency of the method (Figure 3a). Therefore, 0.3 g ml-1 of salt was nominated for following the experiments. To find out the effect of sample pH on the proposed HA-DLPME procedure, the analytical renounces of phenols as a function of the aqueous phase pH was evaluated. Initial tests were done on the sample and it became clear that in acidic and neutral medium, analytical signals are very low. Therefore, a set of experiments were conducted in which the alkalinity of the aqueous sample was adjusted by the use of different concentration of NaOH in the range from 0.00001 to 1 mol L-1. The experiment revealed that the NaOH concentration of 0.1 mol L-1 was found to be optimum. The results shown in Figure 3b reveal that the peak areas were suddenly improved when the alkalinity increased. This improvement may be due to the increase in the derivatization yield at alkaline media. Reduction of responses at 1 mol L-1 of sodium hydroxide, may also be due to the hydrolysis of the derivatives formed. 3.2.3. Derivatization conditions Derivatization step usually is performed for modification of chromatographic behavior of polar compounds such as phenols [24]. Different derivatization reagents are usually applied for this purpose; acetic anhydride is one the affordable reagent [23]. It is an inexpensive reagent and can be added directly into the aqueous sample to perform the reaction with high efficiency in only a few minutes. These advantages stimulate us to use acetic anhydride as the derivatization reagent. Acetic anhydride exchanges the hydroxyl group of phenols to ethyl ester functional group. The amount of reagent, temperature, and time of derivatization were studied and optimized. The results are reported in Supplementary Material (Figures S1-S3). Briefly, the amount of 50 µL of acetic anhydride, in 5 min at room temperature provided the maximum responses.
10
3.2.4. Homogenization conditions Homogenization time and speed are the parameters may affect the efficiency of the extraction step. In fact, the extraction procedures are generally equilibrium processes and they need enough time for establishing the equilibrium state. For this purpose, a series of the extraction experiments using different homogenization time from 5 to 100 s were performed. The results (Figure 4) confirmed that an increase of the homogenization time up to 50 s, leads to the enhancement of the peak areas. Beyond this time a reduction of the peak areas was observed. An explanation for these observations can be presented by considering that the equilibrium state can be attained after 50 s of homogenization, and the evaporation of the organic solvent or analytes causes the reduction in the extraction efficiency beyond this time of mixing the phases. On the other hands, homogenization speed can affect the sizes of droplets of the organic solvent. Reducing the droplet size will increase the contact surface area between the two phases. Figure 5 describes the effect of homogenization speed on the peak areas. The results showed that by increasing the homogenization speed in the range 4000 - 12000 rpm, the extraction efficiency improves. However, a further increase in the homogenization speed did not affect the process efficiency, which states that a further decrease in droplet size at the higher speeds is not provided [12]. These results conduct us to select the speed of 12000 rpm as a critical speed. 3.3. Figures of merit To evaluate the analytical performance of the HA-DLPME method, its figures of merit were determined under optimal conditions (Table 2). All the solutions were prepared by deionized water. The linear ranges of the calibration curves obtained by the standard calibration method were 1.0 - 500 µg L-1 for P, MP, CP and tert-BP, and 2.0 - 500 µg L-1 for NP. The correlation coefficients were in the range of 0.9914-0.9979. The limit of detections (LODs) based on S/N = 3 of the phenols P, MP, CP, tert-BP and NP were 0.3, 0.2, 0.1, 0.3 and 0.7 µg L-1, respectively. The limit of quantifications (LOQs) based on S/N=10 were calculated too. The repeatability and reproducibility of the method were estimated by evaluating the intra- and inter-day relative standard deviations (RSDs). These evaluations were performed by analyzing five replicates experiments at high and low concentration levels. Intra-day precisions for the target phenols were found to be in the range 4.1-9.5%, and intra-day precisions also were in the range 5.2 10%. Based on the results obtained, the EFs were 127 for P, 153 for MP, 136 for CP, 144 for
11
tert-BP and 115 for NP. A comparison of the proposed method with previous methods reported in literature has been summarized in Table 4. This comparison confirmed that the characteristics of the proposed method are close to those of the compared methods. However, it is seen that the extraction time of the method presented in this study is significantly shorter than the compared methods. 3.4. Real sample analysis In order to investigate the applicability of the proposed method, it was applied for the determination of target phenols in water samples collected from three different sources. A tap water (University of Zanjan Campous, Zanjan-Iran), a river water (Zanjan Rood river, Zanjan province, Iran), and an industrial wastewater samples from a paint and resin manufacturing company (Qazvin-Iran) were obtained for this study. The results indicated that there was no evidence of the target analytes present in the examined tap and river water samples, but all of the phenolic compounds except tert-BP were found in the industrial wastewater sample. The concentrations were 3.2 µg L-1 for P, 1.8 µg L-1 for MP, 7.1 µg L-1 for CP and 2.0 µg L-1 for NP which all of them were more than allowable levels (0.5 µg L-1). Table 4 shows the relative recoveries (RRs) for studied real samples spiked with different concentrations of phenols. The relative recoveries ranged from 96 to 109% for the tap water, 90-110% for river water, and 94114% for wastewater samples. Figure 6 reveals the chromatograms for different real samples before and after spiking with analytes. 4. Conclusion A novel, very rapid, inexpensive, simple and environmentally friendly method based on the application of a homogenizer, as a dispersing device, for performing a DLPME procedure of extraction-enrichment of phenols has been described. Rotor-stator configuration of homogenizer generates emulsion of organic solvent in a few second without the assistance of surfactant or disperser solvent. The high rotational speed of the rotor can produce a turbulent flow and accelerate the mass transfer of analytes to the organic solvent, and it could increase the extraction efficiency. By use of homogenizer, the droplet size of the organic solvent is smaller than manual and agitator shaking. In comparison with ultrasound devices, homogenizer forms emulsion in a shorter time without any harmful waves. For evaluation of this application, the proposed method was used for microextraction of some phenolic compounds from water samples. After 12
optimization of different effective parameters, the results showed that the method has good analytical performances for the determination of phenols in water. The method is potential for the determination of phenols in different real samples. As a perspective for developing of this study, and based on the recent research on the automation of the dispersive liquid phase microextraction [26,27], we will continue this study by applying the results for automation of the phenols monitoring. Acknowledgment The authors are grateful to the research council of the University of Zanjan for the financial support of this project. References [1] A. Sarafraz-Yazdi, A. Amiri, Liquid-phase microextraction, TrAC, Trends Anal. Chem. 29 (2010) 1-14. https://doi.org/ 10.1016/j.trac.2009.10.003 [2] H. Yan, H. Wang, Recent development and applications of dispersive liquid–liquid microextraction, J. Chromatogr. A 1295 (2013) 1-15. http://dx.doi.org/10.1016/j.chroma.2013.04.053. [3] M. Rezaee, Y. Assadi, M.R.M. Hosseini, E. Aghaee, F. Ahmadi, S. Berijani, Determination of organic compounds in water using dispersive liquid–liquid microextraction, J. Chromatogr. A 1116 (2006) 1–9. https://doi.org/10.1016/j.chroma.2006.03.007. [4] W. Ahmad, A.A. Al-Sibaai, A.S. Bashammakh, H. Alwael, M.S. El-Shahawi, Recent advances in dispersive liquid-liquid microextraction for pesticide analysis, TrAC, Trends Anal. Chem. 72 (2015) 181-192. https://doi.org/10.1016/j.trac.2015.04.022. [5] M. Saraji, M.K. Boroujeni, Recent developments in dispersive liquid–liquid microextraction, Anal. Bioanal.Chem. 406 (2014) 2027–2066. https://doi.org/10.1007/s00216-013-7467-z. [6] W.C. Tsai, S.D. Huang, Dispersive liquid–liquid microextraction with little solvent consumption combined with gas chromatography–mass spectrometry for the pretreatment of organochlorine pesticides in aqueous samples, J. Chromatogr. A 1216 (2009) 5171-5175. https://doi.org/10.1016/j.chroma.2009.04.086. [7] J. Regueiro, M. Llompart, C. Garcia-Jares, J.C. Garcia-Monteagudo, R. Cela, Ultrasoundassisted emulsification–microextraction of emergent contaminants and pesticides in environmental waters, J. Chromatogr. A 1190 (2008) 27-38. https://doi.org/10.1016/j.chroma.2008.02.091. [8] Y. Li, Y. Jiao, Y. Guo, Y. Yang, Determination of bisphenol-A, 2,4-dichlorophenol, bisphenol-AF and tetrabromobisphenol-A in liquid foods and their packaging materials by
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vortex-assisted supramolecular solvent microextraction/high performance liquid chromatography, Anal. Methods 5 (2013) 5037-5043. https://doi.org/10.1039/C3AY40586A. [9] V.A. Atiemo-Obeng, R.V. Calabrese, Handbook of industrial mixing: Science and Practice: rotor-stator mixing devices, John Wiley & Sons, Hoboken, New Jersey, 2004. [10] J. Zhang, S. Xu, W. Li, High shear mixers: A review of typical applications and studies on power draw, flow pattern, energy dissipation and transfer properties, Chem. Eng. Process. Process Intensif. 57-58 (2012) 25– 41. https://doi.org/10.1016/j.cep.2012.04.004. [11] A. Utomo, M. Baker, A.W. Pacek, The effect of stator geometry on the flow pattern and energy dissipation rate in a rotor–stator mixer, Chem. Eng. Res. Des. 87 (2009) 533-542. https://doi.org/10.1016/j.cherd.2008.12.011 [12] N. Thapar, Liquid–liquid dispersions from in-line rotor–stator mixers, Ph.D. Dissertation, Cranfield University, Cranfield Bedfordshire, 2004. [13] F. Visscher, J. vander Schaaf, M.H.J.M. de Croon, J.C. Schouten, Liquid–liquid mass transfer in a rotor–stator spinning disc reactor, Chem. Eng. J. 185-186 (2012) 267-273. https://doi.org/10.1016/j.cej.2012.01.002. [14] Decision No. 2455/2001/EC of the European parliament and of the council of 20 November 2001 establishing the list of priority substances in the field of water policy and amending directive 2000/60/EC (last updated, August 2009). Official Journal of the European Communities. [15] K.D. Wang, P.S. Chen, S.D. Huang, Simultaneous derivatization and extraction of chlorophenols in water samples with up-and-down shaker-assisted dispersive liquid–liquid microextraction coupled with gas chromatography/mass spectrometric detection, Anal. Bioanal. Chem. 406 (2014) 2123-2131. https://doi.org/10.1007/s00216-013-7044-5. [16] A. González, J. Avivar, V. Cerdà, Determination of priority phenolic pollutants exploiting an in-syringe dispersive liquid–liquid microextraction–multisyringe chromatography system, Anal. Bioanal. Chem. 407 (2015) 2013-2022. https://doi.org/10.1007/s00216-015-8464-1. [17] E. Mirparizi, M. Rajabi, M. Bazregar, A. Asghari, Centrifugeless dispersive liquid-liquid microextraction based on salting-out phenomenon as an efficient method for determination of phenolic compounds in environmental samples, Anal. Bioanal. Chem. 409 (2017) 3007-3016. https://doi.org/10.1007/s00216-017-0246-5. [18] A. Jain, S. Soni, K. Reddy-Noone, A.K.K.V. Pillai, K.K. Verma, Combined headspace single-drop microextraction and solid-phase microextraction for the determination of phenols as their methyl ethers by gas chromatography-mass spectrometry, Anal. Methods 9 (2017) 65906598. https://doi.org/10.1039/C7AY02275A [19] Y.T. Chong, M.M. Ariffin, N.M. Tahir, S.H. Loh, A green solvent holder in electromediated microextraction for the extraction of phenols in water, Talanta 176 (2018) 558-564. https://doi.org/10.1016/j.talanta.2017.08.068.
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[20] G.M. Salcedo, L. Kupski, L. Degang, L.C. Marube, S.S. Caldas, E.G. Primel, Determination of fifteen phenols in wastewater from petroleum refinery samples using a dispersive liquid-liquid microextraction and liquid chromatography with a photodiode array detector, Microchem. J. 146 (2019) 722-728. https://doi.org/10.1016/j.microc.2019.01.075. [21] X. Chen, Z. Guo, Y. Wang, Y. Liu, Y. Xu, J. Liu, Z. Li, J. Zhao, Temperature sensitive polymer-dispersive liquid-liquid microextraction with gas chromatography-mass spectrometry for the determination of phenols, J. Chromatogr. A 1592 (2019) 183-187. https://doi.org/10.1016/j.chroma.2019.01.052. [22] B. Smagowska, M. Pawlaczyk-Łuszczyńska, Effects of Ultrasonic Noise on the Human Body-A Bibliographic Review, Int. J. Occup. Saf. Ergo. (JOSE) 19 (2013) 195–202. https://doi.org/10.1080/10803548.2013.11076978 [23] Y. Yu, S. Zhong, G. Su, H. Liu, X. Dai, R. Wang, H. Cai, H. Yu, Trace analysis of phenolic compounds in water by in situ acetylation coupled with purge and trap-GC/MS, Anal. Methods 4 (2012) 2156-2161. https://doi.org/10.1039/C2AY05887A [24] M. Saraji, M. Bakhshi, Determination of phenols in water samples by single-drop microextraction followed by in-syringe derivatization and gas chromatography–mass spectrometric detection, J. Chromatogr. A 1098 (2005) 30-36. https://doi.org/10.1016/j.chroma.2005.08.063. [25] https://www.ika.com/ika/pdf/flyer-catalog/201901_POV_2019_IWS_EUR_EN_screen.pdf (accessed 27 July 2019) [26] L. Guo, S. Hua, C. Hian, K. Lee, Automated agitation-assisted demulsification dispersive liquid–liquid microextraction, Anal. Chem. 88 (2016) 2548-2552. https://doi.org/10.1021/acs.analchem.5b03919 [27] M. Alexovič, B. Horstkotte, I. Šrámková, P. Solich, J. Sabo, Automation of dispersive liquid–liquid microextraction and related techniques. Approaches based on flow, batch, flowbatch and in-syringe modes, TrAC Trends Anal. Chem. 86 (2017) 39-55. https://doi.org/10.1016/j.trac.2016.10.003
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Figure captions Figure 1. a) Image of an S18-10G dispersing element of a homogenizer and schematic image of its internal components. b) Close view of a dispersing head. c) Flow patterns (radial and tangential velocities) at different regions around the dispersing head [11] (with permission under license number 4678630497689). d) Rotor-stator spinning disc reactor. Figure 2. a) Effect of solvent type on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols at pH=9; no salt addition; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent volume, 50 µL; homogenization speed, 4000 rpm; homogenization time, 1 min. b) Effect of solvent volume on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols at pH=9; no salt addition; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent type, 1,2-dibromoethane; homogenization speed, 4000 rpm; homogenization time, 1 min. Figure 3. a) Effect of ionic strength on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols at pH=9; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization speed, 4000 rpm; homogenization time, 1 min. b) Effect of the sample alkalinity on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols; salt concentration, 0.3 g/mL; derivatization reagent, 20 µL acetic anhydride; shaking time, 2 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization speed, 4000 rpm; homogenization time, 1 min. Figure 4. Effect of homogenization time on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols and 0.1 mol/L NaOH; salt concentration, 0.3 g/mL; derivatization reagent, 50 µL acetic anhydride; shaking time, 5 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization speed, 4000 rpm. Figure 5. Effect of homogenization speed on the peak areas of phenols. Extraction conditions: 3 mL sample containing 1 mg/L of phenols and 0.1 mol/L NaOH; salt concentration, 0.3 g/mL; derivatization reagent, 50 µL acetic anhydride; shaking time, 5 min at room temperature; solvent, 10 µL 1,2-dibromoethane; homogenization time, 50 s.
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Figure 6. a) Chromatograms obtained for tap water and spiked tap water at a concentration level of 10 µg/L for P, MP, CP and tert-BP and 20 µg/L for NP after extraction at optimal conditions. b) Chromatograms obtained for river water and spiked tap water at a concentration level of 20 µg/L for P, MP, CP and tert-BP and 40 µg/L for NP after extraction at optimal conditions. c) Chromatograms obtained for tap water and spiked tap water at a concentration level of 10 µg/L for P, MP, CP and tert-BP and 20 µg/L for NP after extraction at optimal conditions.
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Table 1. Specifications of the homogenizer used in this study [25]. Model Disperser Volume range Stator diameter Rotor diameter The gap between rotor and stator Allowable speed Circumferential speed Immersion depth range Shaft length pH range Working temperature (max) Droplet size range The material in contact with the medium Suitable for solvent
T18 basic S18-10G 1-100 mL 10 mm 7.5 mm 0.35 mm 25000 rpm 9.8 m/s 25-70 mm 108 mm 2-13 180 ºC 1-10 µm AISI 316L and PTFE yes
Table 2. Figures of merit for HA-DLPME method. Compound
P MP CP tert-BP NP
RSD (%) (n=5) Intra-day Inter-day * * High Low High Low 6.6 9.5 9.7 9.8 7.8 4.1 10.0 5.2 4.9 2.6 7.5 5.8 4.7 5.8 5.6 6.3 9.2 7.8 9.7 8.7
Linear range (µg/L) 1-500 1-500 1-500 1-500 2-500
R2
0.9979 0.9941 0.9952 0.9914 0.9961
Limit of Limit of quantitation detection (µg/L) (µg/L) 1 0.3 0.6 0.2 0.4 0.1 1 0.3 2 0.7
EF
127 153 136 144 115
* High and low concentration level were considered 200 and 2 µg L-1 respectively.
Table 3. Relative recoveries of phenolic compounds in different water samples. Compound
P
MP
Added (µg/L)
0 2 5 10 20 50 100 0 2 5 10
Tap water Found (µg/L) Relative recovery (%) 1 ND 1.9 95 5.1 102 9.9 99 21.4 107 54.5 109 109.1 109 ND 1.8 90 4.8 96 10.1 101 18
River water Found Relative (µg/L) recovery (%) ND 2.1 105 4.8 96 9.5 95 22.0 110 49.2 98 100.3 100 ND 2 100 5.1 102 9.6 96
Wastewater Found Relative (µg/L) recovery (%) 3.2 4.9 85 8.0 96 14.1 109 23.0 99 50.2 94 104.3 101 1.8 3.6 90 6.7 98 12.9 111
CP
tert-BP
NP
20 50 100 0 2 5 10 20 50 100 0 2 5 10 20 50 100 0 4 10 20 40 100 200
20.2 53.5 108.4 ND 2 4.7 9.6 20.8 51.1 97.8 ND 2.1 4.6 9.8 21.6 52.0 102.8 ND 3.5 9.1 21.1 39.6 97.2 205.9
101 107 108 100 94 96 104 102 98 105 92 98 108 104 103 87 91 105 99 97 103
19.4 46.5 99.1 ND 2.1 4.8 10.9 20.6 48.5 97.3 ND 2.2 5.3 10.3 19.1 44.9 92.3 ND 3.8 10.2 20.6 44.2 101.0 214.3
97 93 99 105 96 109 103 97 97 110 106 103 95 90 92 95 102 103 110 101 107
20.6 58.8 106.2 7.1 9.2 11.6 16.5 29.2 60.0 111.8 ND 1.7 4.4 11.0 19.8 51.5 104.4 2.0 5.7 11.6 21.8 40.7 106.3 198.3
94 114 104 105 90 94 110 106 105 85 88 110 99 103 104 92 96 99 97 104 98
1. Not detected Table 4. Comparison of the HA-DLPME with other microextraction methods for the determination of phenols. Method
LOD LOQ (µg/L (µg/L ) ) DLLME 2.5-LC10 DAD2
RS D (%) 113
TSP30.01DLLME 1 -GCMS Purge & 0.06trap0.12 GC-MS SDME4- 0.004 GC-MS -0.06 HA0.1DLPME 0.7
0.043.5
< 17
-
0.4-2
Organic solvent type & volume 1octanol/aceto ne 1000 µL Acetone/nhexane 200 µL
Extractio n time (min) -
EF
Real sample
DR 1
Referenc e
-
wastewat er
2.5
[20]
5
3551
River water
2.5
[21]
1.37
-
20
-
2.5
[23]
Hexyl acetate 2.5 µL 1,2dibromoethan
20
86102 115 -
River & wastewat er River water Tap, river and
4.812 4.110
2
[24]
2.5
Proposed method
19
0.8
-GCFID
e, 10 µL
153
wastewat er
1. Dynamic range based on order of magnitude. 2. Dispersive liquid-liquid microextraction followed by liquid chromatography diode array detector. 3. Temperature sensitive polymer. 4. Single drop microextraction
Author Contribution Section Author Miss Tahmineh Javadi Dr. Bahman Farajmand Prof. Mohammad Reza Yaftian Dr. Abbasali Zamani
Role MSc student who performed the experiments Supervisor, designating the study, interpretation of the results and writing the paper Supervisor, designating the study, interpretation of the results and writing the paper Advisor, interpretation of the results
20
Figure 1.
21
22
Figure 2.
Figure 3. 23
Figure 4.
24
Figure 5.
25
26
Figure 6.
Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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