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Determination of benzothiazole in untreated wastewater using polar-phase stir bar sorptive extraction and gas chromatography–mass spectrometry
Analytica Chimica Acta 689 (2011) 65–68
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Determination of benzothiazole in untreated wastewater using polar-phase stir bar sorptive extraction and gas chromatography–mass spectrometry Elke Fries ∗ Institute of Environmental Systems Research, University of Osnabrueck, Barbarastraße 12, D-49076 Osnabrueck, Germany
a r t i c l e
i n f o
Article history: Received 25 October 2010 Received in revised form 7 January 2011 Accepted 10 January 2011 Available online 18 January 2011 Keywords: Wastewater influent Wastewater treatment plant Solvent-free extraction technique Tyres Vulcanisation accelerator 2-Mercaptobenzothiazole
a b s t r a c t Stir bar sorptive extraction (SBSE) was applied to extract benzothiazole (BT) from untreated wastewater using a novel polyacrylate (PA)-coated stir bar (PA Twister® ). After extraction, BT was desorbed in a thermal desorption system (TDS) and analysed by GC–MS (gas chromatography–mass spectrometry). The sample contained 30% (w/w) NaCl, the sample temperature was 30 ◦ C and the extraction time was 240 min. Since no filtering or clean-up steps or solvents were necessary SBSE clearly performs better than all previously used extractions techniques for analysing BT in untreated wastewater in terms of easy use, sample throughput and analytical costs. In addition, matrix effects were small. The calibration curve resulting from the standard addition method was linear with a value of the stability index (R2 ) of 0.999 (n = 3). A good inter-day repeatability of the method was observed with a relative standard deviation (RSD) of 9.8% (n = 6). A low limit of detection (LOD) of 0.256 g L−1 was achieved using only a small sample volume of 18 mL. Small sample volumes significantly reduce sample transport costs. The concentration of BT in untreated wastewater was determined to be 1.04 g L−1 . © 2011 Elsevier B.V. All rights reserved.
1. Introduction BT is mainly produced by sunlight photolysis of 2mercaptobenzothiazole (MBT) [1], which is added as a vulcanisation accelerator to rubber composition in the manufacturing process of the tyre industry [2]. MBT is also produced by hydrolysis and/or photolysis of the fungicide 2(thiocyanomethylthio)benzothiazole (TCMTB) used in the leather, pulp and paper, and wastewater industries [3]. BT is also used in antifreeze products, in pesticides and as photo sensitisers in photography [4]. BT was measured in municipal and industrial wastewater influents in both Europe and China [3,5–8]. In Berlin, mean concentrations of BT in untreated municipal wastewater were 0.55 and 0.85 g L−1 , respectively [5]. BT was also measured in household wastewater with mean concentrations of 0.48 and 0.79 g L−1 , respectively [5]. In Beijing, a mean BT concentration of 2.26 g L−1 was measured in untreated municipal wastewater [5]. The BT concentration in a domestic wastewater treatment plant (WWTP) was 0.131 g L−1 , whereas a much higher concentration of 2.629 g L−1 was measured in a WWTP that also receives industrial wastewater [7].
∗ Tel.: +49 541 969 3441; fax: +49 541 969 2599. E-mail address: [email protected] 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.01.015
The determination of BT in wastewater requires a sensitive analytical method suitable for complex matrices. BT is a relatively polar compound with a water solubility of 3000 mg L−1 and an octanol–water partitioning coefficient (log KOW ) of 1.99 at 24 ◦ C [1]; special attention must be paid to its extraction from aqueous samples. Liquid–liquid extraction (LLE) [9] and, more frequently, solid phase extraction (SPE) [6,7,10,11] have been applied to extract BT from industrial and municipal wastewater. In these studies, BT has been analysed by either liquid chromatography (LC) or gas chromatography (GC) coupled to different detectors, including mass spectrometry (MS). Headspace solidphase micro extraction (HS-SPME) has been applied to extract BT from wines [12]. However, extraction efficiencies of SPME for compounds that are partially water soluble are relatively low [13,14]. Stir bar sorptive extraction (SBSE) is another solvent-free sample preparation technique for the extraction and enrichment of organic compounds from aqueous matrices. This method is based on the same principles as SPME. With SBSE, however, the volume of extraction phase is greater, resulting in a higher sample capacity and extracting efficiency [15]. Due to the greater phase volume of the stir bar compared to the SPME-fibre, compounds with values of log KOW below 2.0 were still extracted with SBSE, whereas this was not the case with SPME [16]. Rodil et al. [17] introduced glass fibre foil strips coated with a polyacrylate (PA) for the first time to extract polar compounds from water. However, the polymer used showed considerable thermal decomposition, leading to
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E. Fries / Analytica Chimica Acta 689 (2011) 65–68
Fig. 1. Mass traces m/z 135 and 149 with signals of BT and 2-MBT, respectively, resulting from SBSE/GC–MS analysis of spiked untreated wastewater sample (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT).
an unpleasantly high background. Most crucially, the handling of the foil strips was cumbersome. In this paper, the analysis of BT in untreated wastewater by SBSE followed by thermodesorption–gas chromatography–mass spectrometry (TDS–GC–MS) is reported for the first time. Polarphase SBSE was performed using a novel stir bar coated with a polyacrylate (PA Twister® ). The parameters during extraction were optimised for BT and matrix effects on SBSE were studied. 2. Materials and methods
equipped with PTFE/silicone septa after inserting the stir bar (all purchased from Gerstel, Muelheim an der Ruhr, Germany). Samples were stirred at 1500 rounds per min (rpm) in a water bath on a heated magnetic stirrer. The sample temperature was kept at 30 ◦ C. The effects of extraction time and ionic strength on BT response were studied. After extraction, the stir bar was removed using tweezers, held above a clean paper tissue to remove water droplets and introduced to an empty thermal-desorption glass tube. Subsequently, analytes were thermally desorbed in a thermal desorption system (TDS) (Gerstel, Muelheim an der Ruhr, Germany).
2.1. Standards and reagents
2.3. Instrumentation
BT and 2-methylbenzothiazole (2-MBT) were purchased from Sigma–Aldrich (Seelze, Germany, purity ≥98.0%). 2-MBT was used as an internal standard (IS), after proving its absence in river water and untreated waste water. Phosphoric acid and sodium chloride (NaCl) (both analytical grade) were purchased from Merck (Darmstadt, Germany). Individual stock solutions of BT and 2-MBT were prepared in ultra pure water taken from a Sartorius Arium 611VF system (Goettingen, Germany) and diluted in ultra pure water when necessary. Ultra pure water was used instead of solvents to avoid them having an effect on extraction. A PA Twister® (25 L phase volume, 10 mm length) was purchased as a beta-test kit from Gerstel (Muelheim an der Ruhr, Germany). The centre of the Twister® is a glass-coated stirring magnet, coated externally with an enrichment phase. The enrichment phase comprises a PA with a proportion of polyethylene glycol.
The whole analytical process, including desorption of the stir bar and separation/detection of the desorbed analytes, was fully automated. The TDS device was coupled to a 7890A GC (Agilent Technologies, Santa Clara, USA). The initial temperature of 50 ◦ C was increased to 220 ◦ C at a rate of 70 ◦ C min−1 ; the end temperature was held for 5 min. The helium flow rate was 61 mL min−1 (solvent vent mode). The temperature of the transfer line between the TDS and the GC was set to 250 ◦ C. The GC was equipped with a cryo-injection system (CIS-4) (Gerstel, Muelheim an der Ruhr, Germany), which was operated as a cryotrap cooled by liquid nitrogen for cryogenic refocusing of thermally desorbed analytes. During the thermal desorption process, the CIS-4 temperature was kept at −20 ◦ C. Once the desorption step was completed, the CIS-4 was heated to 250 ◦ C at a rate of 12 ◦ C min−1 . The end temperature was held for 3 min. The CIS-4 was run in splitless mode for 1 min. The GC was equipped with a 30 m DB-Wax capillary column with an ID of 0.25 mm and a film thickness of 0.5 m (Agilent Technologies, Santa Clara, USA). The GC oven programme was as follows: 80 ◦ C for 2 min, then heating at 10 ◦ C min−1 to 240 ◦ C and holding for 10 min. Helium grade 5.0 served as the carrier gas. The column was operated in the constant pressure mode at 1.02 bar; the column flow was set at 1.5 mL min−1 . The transfer line between the GC and the mass selective detector (MSD 5975 inert XL EI, Agilent Technologies, Santa Clara, USA) was set at 240 ◦ C. The MS was run in the electron ionisation positive ion mode (EI+ ) and in the selected ion mode (SIM) mode, where three characteristic ions were selected for BT and 2-MBT, respectively. Quantification was undertaken by relating
2.2. Sampling and extraction procedure One river water grab samples was collected from the River Hase (Germany). One grab sample of untreated wastewater was collected from a municipal WWTP. Sampling was performed on 9 November 2009. All environmental samples were collected in amber glass bottles and stored at 5 ◦ C until analysis. The samples were analysed within one week. An aliquot of 18 mL of water was poured into a 20 mL glass headspace vial, to which NaCl was added. Samples were acidified to pH 2.0 ± 0.03 by adding phosphoric acid, and sealed with screw caps
E. Fries / Analytica Chimica Acta 689 (2011) 65–68
fortified samples of ultra pure water, river water and untreated wastewater under optimum extraction conditions (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). The signals of BT were corrected by the corresponding signals obtained from nonspiked samples. The results are shown in Fig. 3. The responses of BT resulting from SBSE from different matrices were rather similar, but much lower for SBSE of 2-MBT from ultra pure water compared to river water and wastewater. Since the extraction for both compounds was dependent to a different extent on the sample matrix, the standard addition procedure is recommended for quantifying BT in environmental samples.
4.E+05
Abundance
67
2.E+05
0.E+00 240 min, 0% NaCl
60 min, 30% NaCl (w/w)
240 min, 30% NaCl (w/w)
360 min, 30% NaCl (w/w)
Fig. 2. Effects of ionic strength and extraction time on the response of BT.
the base peak areas of m/z 135 (BT) to m/z 149 (2-MBT). Qualifier ions were m/z 108 and 69 for both compounds. Data acquisition, processing and instrument control were performed using Chemstation Software (Agilent Technologies, Santa Clara, USA). Fig. 1 shows the mass traces m/z 135 and m/z 149 with signals of BT and 2-MBT, respectively, resulting from SBSE/TDS–GC–MS analysis of a spiked untreated wastewater sample (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). Retention times were 14.987 min for BT and 14.825 min for 2-MBT. 3. Results and discussion 3.1. Carry over The effectiveness of a bake-out step in between two consecutive desorption steps was studied (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). A spiked wastewater sample was extracted, desorbed once, and the stir bar was conditioned at 220 ◦ C for 20 min and then desorbed again. No signals occurred after the second desorption step, and thus no carry-over occurred under the applied bake-out conditions. In contrast, the bake-out time for the classic polydimethylsiloxane (PDMS) Twister® (24 L phase volume, 10 mm length, 0.5 mm phase thickness) for BT was 300 min. No separate tube conditioner is necessary if PA Twisters® are used, reducing analytical costs.
3.4. Method validation The optimised SBSE/TDS–GC–MS method for the analysis of BT in untreated wastewater was validated considering precision, linear range and limit of detection (LOD). A good inter-day repeatability is necessary because SBSE is not yet automated. For this reason, the method precision was studied by extracting six consecutive wastewater samples within one week. The samples were spiked with BT (addition level 5.7 g L−1 ) and 2-MBT (addition level 29.2 g L−1 ). To eliminate instrument variability, precision for the analysis of BT was determined by the relative standard deviation (RSD) of the ratio of the peak areas of m/z 135 to m/z 149. The RSD was 9.8%, indicating a good inter-day repeatability of the method. A laboratory blank was taken into account when calculating the limit of detection (LOD) according to DIN 32645 [18] (p: 0.05, N = 1, N = 4). With a t-value of 2.353, the LOD was 0.256 g L−1 . The concentration of BT in untreated wastewater was determined using the standard addition procedure. 18 mL-aliquots of an untreated wastewater sample were spiked with BT (addition levels: 5.7 g L−1 and 11.3 g L−1 ). One non-spiked sample was also analysed. The resulting calibration curve is shown in Fig. 4 (the y-axis is labelled with the ratio of m/z 135 to m/z 149). The value of R2 of the calibration curve was 0.999, demonstrating a very good linearity in the studied concentration range. The BT concentration in untreated wastewater was determined to be 1.04 g L−1 . This was rather similar to the mean concentration of 0.85 g L−1 determined in untreated wastewater collected from a municipal WWTP in Berlin [5]. 3.5. Comparison of SBSE to other extraction methods
3.2. Optimisation of extraction conditions
3.3. Matrix effects SBSE can be significantly affected by the composition of the sample matrix. Thus, matrix effects were studied by extracting
A relatively high LOD of 2.5 g L−1 was achieved in the analysis of BT in industrial wastewater after direct injection of 100 L sample volume using LC/ESI-MS/MS [19]. Lower LODs were achieved by applying different extraction methods. The limit of quantification (LOQ) for the analysis of BT in untreated wastewater by SPE/LC–MS was 0.420 g L−1 (LOQ: signal/noise >10) when using a sample volume of 50 mL [6]. A LOD value of 10 ng L−1 was obtained
BT 8.E+05
Abundance
The effects of ionic strength and extraction time on the response of BT were studied and optimised by analysing fortified ultra pure water samples (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). The results are shown in Fig. 2. First, the influence of the extraction time was studied by performing SBSE for 60, 240 and 360 min. NaCl concentration was 30% (w/w). Increasing the extraction time from 60 to 240 min resulted in a BT response that was approximately four times higher. The maximum response was observed for an extraction time of 240 min, indicating that equilibrium was reached between the aqueous phase and the PA coating. Secondly, the influence of ionic strength was studied. The extraction time was 240 min. SBSE was performed without NaCl addition (0%, w/w) and with the addition of NaCl (30%, w/w). An increase in the BT response by a factor of approximately three was observed when NaCl was added. A NaCl concentration of 30% and an extraction time of 240 min were selected as optimum parameters for all further analyses.
2-MBT
4.E+05
0.E+00 Ultra pure water
River water
Untreated wastewater
Fig. 3. Influence of sample matrix on the responses of BT and 2-MBT.
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E. Fries / Analytica Chimica Acta 689 (2011) 65–68
0.6
Ratio m/z 135 to 149
A much lower sample volume was required to reach a similar LOD as methods reported previously. A good precision with an RSD of 9.8% indicated the method’s good inter-day repeatability. Compared to analytical techniques previously reported, the polarphase SBSE/TDS–GC–MS method saves analytical time and costs because no filtering and clean-up steps or solvents were necessary. SBSE/TDS–GC–MS can also have a great potential for the extraction of polar substances with similar physico-chemical properties such as BT from untreated wastewater.
y = 0.0548x + 0.0567 R2 = 0.9999
0.4
0.2
Acknowledgements
0 0
5
10
Concentration [µg L-1] Fig. 4. Calibration curves resulting from standard addition of BT and 2-MBT to untreated waste water.
for the analysis of BT in raw wastewater by SPE coupled to twodimensional GC and time-of-flight MS when a sample volume of 500 mL was used [7]. Compared to other sample preparation techniques reported previously for the analysis of BT in untreated wastewater, the advantage of the SBSE/TDS–GC–MS method presented in this study is a low LOD, although only a few mL of the sample were used. Thus, transport costs are significantly reduced. BT can be volatilised during extraction procedures [6], resulting in low recoveries for LLE and SPE. Thus, one main advantage of SBSE is that losses via volatilisation during extraction are omitted because extraction is performed in sealed vials. Prior LLE and SPE wastewater samples were filtered [5–7,9–19]. A clean-up step is usually applied prior to GC analysis [10]. For the SBSE/TDS–GC–MS method presented in this study, neither filtering nor clean-up was necessary. SBSE is a solvent-free technique that clearly performs better than all previously used extractions techniques for the analysis of BT in wastewater in terms of easy use, sample throughput and analytical costs. 4. Conclusions A solvent-free extraction method based on polar-phase SBSE followed by TDS–GC–MS using a novel PA Twister® has been applied for the first time to analyse BT in untreated wastewater.
The authors are grateful to Dr. Eike Kleine-Benne from the company Gerstel for kindly providing the novel PA Twister® and to Melanie Wissing for her help with the analysis. The financial support granted by the University of Osnabrueck is gratefully acknowledged. References [1] B.G. Brownlee, J.H. Carey, G.A. MacInnis, I.T. Pellizzari, Environ. Toxicol. Chem. 11 (1992) 1153. [2] H.W. Engels, H.J. Weidenhaupt, M. Abele, M. Pieroth, W. Hofmann, in: B. Elvers, S. Hawkins, W. Russey, G. Schulz (Eds.), Ullmann’s Encyclopedia of Industrial Chemistry, vol. AZ3, 5th ed., VCH, Weinheim, 1993, p. 365. [3] T. Reemtsma, O. Fiehn, G. Kalnowski, M. Jekel, Environ. Sci. Technol. 29 (1995) 478. [4] A. Wik, G. Dave, Environ. Pollut. (2009) 1. [5] A. Kloepfer, M. Jekel, T. Reemtsma, Environ. Sci. Technol. 39 (2005) 3792. [6] A. Kloepfer, M. Jekel, T. Reemtsma, J. Chromatogr. A 1058 (2004) 81. [7] E. Jover, V. Matamoros, J.M. Bayona, J. Chromatogr. A 1216 (2009) 4013. [8] M. Matamoros, E. Jover, J.M. Bayonea, Water Sci. Technol. 61 (2010) 191. [9] O. Fiehn, T. Reemtsma, M. Jekel, Anal. Chim. Acta 295 (1994) 297. [10] H.G. Ni, F.H. Lu, X.L. Luo, H.Y. Tian, E.Y. Zeng, Environ. Sci. Technol. 42 (2008) 1892. [11] J. Van Leerdam, A.C. Hogenbooma, M.M.E. van der Kooia, P. de Voogta, Int. J. Mass Spectrom. 282 (2009) 99. [12] V. Bellavia, M. Natangelo, R. Fanelli, D. Rotilio, J. Agric. Food Chem. 48 (2000) 1239. [13] C.L. Arthur, K. Pratt, S. Motlagh, J. Pawliszyn, R.L.J. Belardi, High Resolut. Chromatogr. 15 (1992) 741. [14] J. Beltran, F.J. Lopez, O. Cepria, F. Hernandez, J. Chromatogr. A 808 (1998) 257. [15] E. Baltussen, P. Sandra, F. David, C. Cramers, J. Microcolumn Sep. 11 (1999) 737. [16] A. Hoffmann, R. Bremer, P. Sandra, F. David, Gerstel Aktuell. 24 (2000) 4. [17] R. Rodil, J. von Sonntag, L. Montero, P. Popp, M.R. Buchmeiser, J. Chromatogr. A 1138 (2007) 1. [18] DIN (Deutsches Institut für Normung e.V.—German Institute for Standardisation) Detection limit, reporting limit and quantification limit (in German). DIN 32645, Beuth, Berlin, Germany, 1994. [19] T. Reemtsma, Rapid Commun. Mass Spectrom. 14 (2000) 1612.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Determination of benzothiazole in untreated wastewater using polar-phase stir bar sorptive extraction and gas chromatography–mass spectrometry Elke Fries ∗ Institute of Environmental Systems Research, University of Osnabrueck, Barbarastraße 12, D-49076 Osnabrueck, Germany
a r t i c l e
i n f o
Article history: Received 25 October 2010 Received in revised form 7 January 2011 Accepted 10 January 2011 Available online 18 January 2011 Keywords: Wastewater influent Wastewater treatment plant Solvent-free extraction technique Tyres Vulcanisation accelerator 2-Mercaptobenzothiazole
a b s t r a c t Stir bar sorptive extraction (SBSE) was applied to extract benzothiazole (BT) from untreated wastewater using a novel polyacrylate (PA)-coated stir bar (PA Twister® ). After extraction, BT was desorbed in a thermal desorption system (TDS) and analysed by GC–MS (gas chromatography–mass spectrometry). The sample contained 30% (w/w) NaCl, the sample temperature was 30 ◦ C and the extraction time was 240 min. Since no filtering or clean-up steps or solvents were necessary SBSE clearly performs better than all previously used extractions techniques for analysing BT in untreated wastewater in terms of easy use, sample throughput and analytical costs. In addition, matrix effects were small. The calibration curve resulting from the standard addition method was linear with a value of the stability index (R2 ) of 0.999 (n = 3). A good inter-day repeatability of the method was observed with a relative standard deviation (RSD) of 9.8% (n = 6). A low limit of detection (LOD) of 0.256 g L−1 was achieved using only a small sample volume of 18 mL. Small sample volumes significantly reduce sample transport costs. The concentration of BT in untreated wastewater was determined to be 1.04 g L−1 . © 2011 Elsevier B.V. All rights reserved.
1. Introduction BT is mainly produced by sunlight photolysis of 2mercaptobenzothiazole (MBT) [1], which is added as a vulcanisation accelerator to rubber composition in the manufacturing process of the tyre industry [2]. MBT is also produced by hydrolysis and/or photolysis of the fungicide 2(thiocyanomethylthio)benzothiazole (TCMTB) used in the leather, pulp and paper, and wastewater industries [3]. BT is also used in antifreeze products, in pesticides and as photo sensitisers in photography [4]. BT was measured in municipal and industrial wastewater influents in both Europe and China [3,5–8]. In Berlin, mean concentrations of BT in untreated municipal wastewater were 0.55 and 0.85 g L−1 , respectively [5]. BT was also measured in household wastewater with mean concentrations of 0.48 and 0.79 g L−1 , respectively [5]. In Beijing, a mean BT concentration of 2.26 g L−1 was measured in untreated municipal wastewater [5]. The BT concentration in a domestic wastewater treatment plant (WWTP) was 0.131 g L−1 , whereas a much higher concentration of 2.629 g L−1 was measured in a WWTP that also receives industrial wastewater [7].
∗ Tel.: +49 541 969 3441; fax: +49 541 969 2599. E-mail address: [email protected] 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.01.015
The determination of BT in wastewater requires a sensitive analytical method suitable for complex matrices. BT is a relatively polar compound with a water solubility of 3000 mg L−1 and an octanol–water partitioning coefficient (log KOW ) of 1.99 at 24 ◦ C [1]; special attention must be paid to its extraction from aqueous samples. Liquid–liquid extraction (LLE) [9] and, more frequently, solid phase extraction (SPE) [6,7,10,11] have been applied to extract BT from industrial and municipal wastewater. In these studies, BT has been analysed by either liquid chromatography (LC) or gas chromatography (GC) coupled to different detectors, including mass spectrometry (MS). Headspace solidphase micro extraction (HS-SPME) has been applied to extract BT from wines [12]. However, extraction efficiencies of SPME for compounds that are partially water soluble are relatively low [13,14]. Stir bar sorptive extraction (SBSE) is another solvent-free sample preparation technique for the extraction and enrichment of organic compounds from aqueous matrices. This method is based on the same principles as SPME. With SBSE, however, the volume of extraction phase is greater, resulting in a higher sample capacity and extracting efficiency [15]. Due to the greater phase volume of the stir bar compared to the SPME-fibre, compounds with values of log KOW below 2.0 were still extracted with SBSE, whereas this was not the case with SPME [16]. Rodil et al. [17] introduced glass fibre foil strips coated with a polyacrylate (PA) for the first time to extract polar compounds from water. However, the polymer used showed considerable thermal decomposition, leading to
66
E. Fries / Analytica Chimica Acta 689 (2011) 65–68
Fig. 1. Mass traces m/z 135 and 149 with signals of BT and 2-MBT, respectively, resulting from SBSE/GC–MS analysis of spiked untreated wastewater sample (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT).
an unpleasantly high background. Most crucially, the handling of the foil strips was cumbersome. In this paper, the analysis of BT in untreated wastewater by SBSE followed by thermodesorption–gas chromatography–mass spectrometry (TDS–GC–MS) is reported for the first time. Polarphase SBSE was performed using a novel stir bar coated with a polyacrylate (PA Twister® ). The parameters during extraction were optimised for BT and matrix effects on SBSE were studied. 2. Materials and methods
equipped with PTFE/silicone septa after inserting the stir bar (all purchased from Gerstel, Muelheim an der Ruhr, Germany). Samples were stirred at 1500 rounds per min (rpm) in a water bath on a heated magnetic stirrer. The sample temperature was kept at 30 ◦ C. The effects of extraction time and ionic strength on BT response were studied. After extraction, the stir bar was removed using tweezers, held above a clean paper tissue to remove water droplets and introduced to an empty thermal-desorption glass tube. Subsequently, analytes were thermally desorbed in a thermal desorption system (TDS) (Gerstel, Muelheim an der Ruhr, Germany).
2.1. Standards and reagents
2.3. Instrumentation
BT and 2-methylbenzothiazole (2-MBT) were purchased from Sigma–Aldrich (Seelze, Germany, purity ≥98.0%). 2-MBT was used as an internal standard (IS), after proving its absence in river water and untreated waste water. Phosphoric acid and sodium chloride (NaCl) (both analytical grade) were purchased from Merck (Darmstadt, Germany). Individual stock solutions of BT and 2-MBT were prepared in ultra pure water taken from a Sartorius Arium 611VF system (Goettingen, Germany) and diluted in ultra pure water when necessary. Ultra pure water was used instead of solvents to avoid them having an effect on extraction. A PA Twister® (25 L phase volume, 10 mm length) was purchased as a beta-test kit from Gerstel (Muelheim an der Ruhr, Germany). The centre of the Twister® is a glass-coated stirring magnet, coated externally with an enrichment phase. The enrichment phase comprises a PA with a proportion of polyethylene glycol.
The whole analytical process, including desorption of the stir bar and separation/detection of the desorbed analytes, was fully automated. The TDS device was coupled to a 7890A GC (Agilent Technologies, Santa Clara, USA). The initial temperature of 50 ◦ C was increased to 220 ◦ C at a rate of 70 ◦ C min−1 ; the end temperature was held for 5 min. The helium flow rate was 61 mL min−1 (solvent vent mode). The temperature of the transfer line between the TDS and the GC was set to 250 ◦ C. The GC was equipped with a cryo-injection system (CIS-4) (Gerstel, Muelheim an der Ruhr, Germany), which was operated as a cryotrap cooled by liquid nitrogen for cryogenic refocusing of thermally desorbed analytes. During the thermal desorption process, the CIS-4 temperature was kept at −20 ◦ C. Once the desorption step was completed, the CIS-4 was heated to 250 ◦ C at a rate of 12 ◦ C min−1 . The end temperature was held for 3 min. The CIS-4 was run in splitless mode for 1 min. The GC was equipped with a 30 m DB-Wax capillary column with an ID of 0.25 mm and a film thickness of 0.5 m (Agilent Technologies, Santa Clara, USA). The GC oven programme was as follows: 80 ◦ C for 2 min, then heating at 10 ◦ C min−1 to 240 ◦ C and holding for 10 min. Helium grade 5.0 served as the carrier gas. The column was operated in the constant pressure mode at 1.02 bar; the column flow was set at 1.5 mL min−1 . The transfer line between the GC and the mass selective detector (MSD 5975 inert XL EI, Agilent Technologies, Santa Clara, USA) was set at 240 ◦ C. The MS was run in the electron ionisation positive ion mode (EI+ ) and in the selected ion mode (SIM) mode, where three characteristic ions were selected for BT and 2-MBT, respectively. Quantification was undertaken by relating
2.2. Sampling and extraction procedure One river water grab samples was collected from the River Hase (Germany). One grab sample of untreated wastewater was collected from a municipal WWTP. Sampling was performed on 9 November 2009. All environmental samples were collected in amber glass bottles and stored at 5 ◦ C until analysis. The samples were analysed within one week. An aliquot of 18 mL of water was poured into a 20 mL glass headspace vial, to which NaCl was added. Samples were acidified to pH 2.0 ± 0.03 by adding phosphoric acid, and sealed with screw caps
E. Fries / Analytica Chimica Acta 689 (2011) 65–68
fortified samples of ultra pure water, river water and untreated wastewater under optimum extraction conditions (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). The signals of BT were corrected by the corresponding signals obtained from nonspiked samples. The results are shown in Fig. 3. The responses of BT resulting from SBSE from different matrices were rather similar, but much lower for SBSE of 2-MBT from ultra pure water compared to river water and wastewater. Since the extraction for both compounds was dependent to a different extent on the sample matrix, the standard addition procedure is recommended for quantifying BT in environmental samples.
4.E+05
Abundance
67
2.E+05
0.E+00 240 min, 0% NaCl
60 min, 30% NaCl (w/w)
240 min, 30% NaCl (w/w)
360 min, 30% NaCl (w/w)
Fig. 2. Effects of ionic strength and extraction time on the response of BT.
the base peak areas of m/z 135 (BT) to m/z 149 (2-MBT). Qualifier ions were m/z 108 and 69 for both compounds. Data acquisition, processing and instrument control were performed using Chemstation Software (Agilent Technologies, Santa Clara, USA). Fig. 1 shows the mass traces m/z 135 and m/z 149 with signals of BT and 2-MBT, respectively, resulting from SBSE/TDS–GC–MS analysis of a spiked untreated wastewater sample (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). Retention times were 14.987 min for BT and 14.825 min for 2-MBT. 3. Results and discussion 3.1. Carry over The effectiveness of a bake-out step in between two consecutive desorption steps was studied (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). A spiked wastewater sample was extracted, desorbed once, and the stir bar was conditioned at 220 ◦ C for 20 min and then desorbed again. No signals occurred after the second desorption step, and thus no carry-over occurred under the applied bake-out conditions. In contrast, the bake-out time for the classic polydimethylsiloxane (PDMS) Twister® (24 L phase volume, 10 mm length, 0.5 mm phase thickness) for BT was 300 min. No separate tube conditioner is necessary if PA Twisters® are used, reducing analytical costs.
3.4. Method validation The optimised SBSE/TDS–GC–MS method for the analysis of BT in untreated wastewater was validated considering precision, linear range and limit of detection (LOD). A good inter-day repeatability is necessary because SBSE is not yet automated. For this reason, the method precision was studied by extracting six consecutive wastewater samples within one week. The samples were spiked with BT (addition level 5.7 g L−1 ) and 2-MBT (addition level 29.2 g L−1 ). To eliminate instrument variability, precision for the analysis of BT was determined by the relative standard deviation (RSD) of the ratio of the peak areas of m/z 135 to m/z 149. The RSD was 9.8%, indicating a good inter-day repeatability of the method. A laboratory blank was taken into account when calculating the limit of detection (LOD) according to DIN 32645 [18] (p: 0.05, N = 1, N = 4). With a t-value of 2.353, the LOD was 0.256 g L−1 . The concentration of BT in untreated wastewater was determined using the standard addition procedure. 18 mL-aliquots of an untreated wastewater sample were spiked with BT (addition levels: 5.7 g L−1 and 11.3 g L−1 ). One non-spiked sample was also analysed. The resulting calibration curve is shown in Fig. 4 (the y-axis is labelled with the ratio of m/z 135 to m/z 149). The value of R2 of the calibration curve was 0.999, demonstrating a very good linearity in the studied concentration range. The BT concentration in untreated wastewater was determined to be 1.04 g L−1 . This was rather similar to the mean concentration of 0.85 g L−1 determined in untreated wastewater collected from a municipal WWTP in Berlin [5]. 3.5. Comparison of SBSE to other extraction methods
3.2. Optimisation of extraction conditions
3.3. Matrix effects SBSE can be significantly affected by the composition of the sample matrix. Thus, matrix effects were studied by extracting
A relatively high LOD of 2.5 g L−1 was achieved in the analysis of BT in industrial wastewater after direct injection of 100 L sample volume using LC/ESI-MS/MS [19]. Lower LODs were achieved by applying different extraction methods. The limit of quantification (LOQ) for the analysis of BT in untreated wastewater by SPE/LC–MS was 0.420 g L−1 (LOQ: signal/noise >10) when using a sample volume of 50 mL [6]. A LOD value of 10 ng L−1 was obtained
BT 8.E+05
Abundance
The effects of ionic strength and extraction time on the response of BT were studied and optimised by analysing fortified ultra pure water samples (addition levels: 11.3 g L−1 for BT and 29.2 g L−1 for 2-MBT). The results are shown in Fig. 2. First, the influence of the extraction time was studied by performing SBSE for 60, 240 and 360 min. NaCl concentration was 30% (w/w). Increasing the extraction time from 60 to 240 min resulted in a BT response that was approximately four times higher. The maximum response was observed for an extraction time of 240 min, indicating that equilibrium was reached between the aqueous phase and the PA coating. Secondly, the influence of ionic strength was studied. The extraction time was 240 min. SBSE was performed without NaCl addition (0%, w/w) and with the addition of NaCl (30%, w/w). An increase in the BT response by a factor of approximately three was observed when NaCl was added. A NaCl concentration of 30% and an extraction time of 240 min were selected as optimum parameters for all further analyses.
2-MBT
4.E+05
0.E+00 Ultra pure water
River water
Untreated wastewater
Fig. 3. Influence of sample matrix on the responses of BT and 2-MBT.
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0.6
Ratio m/z 135 to 149
A much lower sample volume was required to reach a similar LOD as methods reported previously. A good precision with an RSD of 9.8% indicated the method’s good inter-day repeatability. Compared to analytical techniques previously reported, the polarphase SBSE/TDS–GC–MS method saves analytical time and costs because no filtering and clean-up steps or solvents were necessary. SBSE/TDS–GC–MS can also have a great potential for the extraction of polar substances with similar physico-chemical properties such as BT from untreated wastewater.
y = 0.0548x + 0.0567 R2 = 0.9999
0.4
0.2
Acknowledgements
0 0
5
10
Concentration [µg L-1] Fig. 4. Calibration curves resulting from standard addition of BT and 2-MBT to untreated waste water.
for the analysis of BT in raw wastewater by SPE coupled to twodimensional GC and time-of-flight MS when a sample volume of 500 mL was used [7]. Compared to other sample preparation techniques reported previously for the analysis of BT in untreated wastewater, the advantage of the SBSE/TDS–GC–MS method presented in this study is a low LOD, although only a few mL of the sample were used. Thus, transport costs are significantly reduced. BT can be volatilised during extraction procedures [6], resulting in low recoveries for LLE and SPE. Thus, one main advantage of SBSE is that losses via volatilisation during extraction are omitted because extraction is performed in sealed vials. Prior LLE and SPE wastewater samples were filtered [5–7,9–19]. A clean-up step is usually applied prior to GC analysis [10]. For the SBSE/TDS–GC–MS method presented in this study, neither filtering nor clean-up was necessary. SBSE is a solvent-free technique that clearly performs better than all previously used extractions techniques for the analysis of BT in wastewater in terms of easy use, sample throughput and analytical costs. 4. Conclusions A solvent-free extraction method based on polar-phase SBSE followed by TDS–GC–MS using a novel PA Twister® has been applied for the first time to analyse BT in untreated wastewater.
The authors are grateful to Dr. Eike Kleine-Benne from the company Gerstel for kindly providing the novel PA Twister® and to Melanie Wissing for her help with the analysis. The financial support granted by the University of Osnabrueck is gratefully acknowledged. References [1] B.G. Brownlee, J.H. Carey, G.A. MacInnis, I.T. Pellizzari, Environ. Toxicol. Chem. 11 (1992) 1153. [2] H.W. Engels, H.J. Weidenhaupt, M. Abele, M. Pieroth, W. Hofmann, in: B. Elvers, S. Hawkins, W. Russey, G. Schulz (Eds.), Ullmann’s Encyclopedia of Industrial Chemistry, vol. AZ3, 5th ed., VCH, Weinheim, 1993, p. 365. [3] T. Reemtsma, O. Fiehn, G. Kalnowski, M. Jekel, Environ. Sci. Technol. 29 (1995) 478. [4] A. Wik, G. Dave, Environ. Pollut. (2009) 1. [5] A. Kloepfer, M. Jekel, T. Reemtsma, Environ. Sci. Technol. 39 (2005) 3792. [6] A. Kloepfer, M. Jekel, T. Reemtsma, J. Chromatogr. A 1058 (2004) 81. [7] E. Jover, V. Matamoros, J.M. Bayona, J. Chromatogr. A 1216 (2009) 4013. [8] M. Matamoros, E. Jover, J.M. Bayonea, Water Sci. Technol. 61 (2010) 191. [9] O. Fiehn, T. Reemtsma, M. Jekel, Anal. Chim. Acta 295 (1994) 297. [10] H.G. Ni, F.H. Lu, X.L. Luo, H.Y. Tian, E.Y. Zeng, Environ. Sci. Technol. 42 (2008) 1892. [11] J. Van Leerdam, A.C. Hogenbooma, M.M.E. van der Kooia, P. de Voogta, Int. J. Mass Spectrom. 282 (2009) 99. [12] V. Bellavia, M. Natangelo, R. Fanelli, D. Rotilio, J. Agric. Food Chem. 48 (2000) 1239. [13] C.L. Arthur, K. Pratt, S. Motlagh, J. Pawliszyn, R.L.J. Belardi, High Resolut. Chromatogr. 15 (1992) 741. [14] J. Beltran, F.J. Lopez, O. Cepria, F. Hernandez, J. Chromatogr. A 808 (1998) 257. [15] E. Baltussen, P. Sandra, F. David, C. Cramers, J. Microcolumn Sep. 11 (1999) 737. [16] A. Hoffmann, R. Bremer, P. Sandra, F. David, Gerstel Aktuell. 24 (2000) 4. [17] R. Rodil, J. von Sonntag, L. Montero, P. Popp, M.R. Buchmeiser, J. Chromatogr. A 1138 (2007) 1. [18] DIN (Deutsches Institut für Normung e.V.—German Institute for Standardisation) Detection limit, reporting limit and quantification limit (in German). DIN 32645, Beuth, Berlin, Germany, 1994. [19] T. Reemtsma, Rapid Commun. Mass Spectrom. 14 (2000) 1612.