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Comprehensive study of the parameters influencing the detection of organotin compounds by a pulsed flame photometric detector in sewage sludge
Journal of Chromatography A, 1188 (2008) 281–285
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Comprehensive study of the parameters influencing the detection of organotin compounds by a pulsed flame photometric detector in sewage sludge ˇ canˇcar a , Martine Potin-Gautier b Tea Zuliani a,b,∗ , Gaetane Lespes b , Radmila Milaˇciˇc a , Janez Sˇ a b
Department of Environmental Sciences, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Laboratoire de Chimie Analytique, LCABIE, UMR CNRS 5034, Universit´e de Pau et des Pays de l’Adour, Avenue de l’Universit´e, BP 1155, 64013 Pau, France
a r t i c l e
i n f o
Article history: Received 3 October 2007 Received in revised form 14 February 2008 Accepted 21 February 2008 Available online 2 March 2008 Keywords: Organotin compounds Sewage sludge Matrix interferences GC-PFPD
a b s t r a c t An investigation of the operating conditions of a pulsed flame photometric detection (PFPD) system for the determination of organotin compounds (OTCs) in sewage sludge is reported. During the analyses, some spectral interferences were observed. For their elimination detector parameters such as gate delay and gate width were investigated. In addition, the applicability of three different internal standards was evaluated. Under optimised analytical conditions (gate delay 3 ms, gate width 2 ms, tripropyltin as internal standard) limits of detection (LOD) were determined. The LOD for butyltins ranged between 8 and 16 ng Sn g−1 , for phenyltins around 8 ng Sn g−1 and for octyltins between 5 and 10 ng Sn g−1 . Since there is no certified reference material (CRM) available for sewage sludge, the accuracy of the analytical procedure was checked by the analysis of CRM PACS-2 (marine sediment) and a spiked sludge sample. Good agreement between determined and certified values was obtained. Sewage sludge from a local wastewater treatment plant was analysed and the results compared with data from the literature. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In recent decades organotin compounds (OTCs) were widely used as biocides, agricultural fertilizers, wood preservatives and plastic stabilizers. Therefore, OTCs are found in numerous environmental compartments such as water, sediments, aquatic organisms, soil and sewage sludge [1]. The first known contamination concerned the aquatic environment because OTCs, especially tributyltin (TBT) and triphenyltins (TPhT), were present in antifouling paints [2,3]. Although there is a lack of data on OTC’s inputs into wastewater [4], the possible sources may be plastic, pesticide, colour and varnish production [5]. During the treatment of wastewater OTCs are mostly removed during the primary treatment (around 80%) [6]. The efficiency of OTCs removal is regulated by their absorption capacities [7]. So, during treatment OTCs are transferred and conserved from wastewater to sewage sludge [8]. The complexity of sewage sludge matrix makes it necessary to ensure the quality of the analytical procedure used. So far, many analytical methods were developed for the determination of OTCs in solid samples. The analytical procedure involves several
∗ Corresponding author at: Department of Environmental Sciences, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Tel.: +386 1 4773900; fax: +386 1 2519385. E-mail address: [email protected] (T. Zuliani). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.02.079
steps including extraction, derivatisation if gas chromatography is then applied, separation and detection. Generally, the analysis is performed using gas chromatography separation followed by element-selective detection. In recent years, the most frequently used detection methods for OTCs determination are the mass spectrometry (MS) [9,10], the inductively coupled plasma mass spectrometry (ICP-MS) [11], the flame photometric detection (FPD) [12,13] and the pulsed flame photometric detection (PFPD) [2,14,15]. PFPD was developed by Amiravet al. [16] and was previously designed for the analysis of sulphur, nitrogen and phosphorus. Later on application of the PFPD was extended to the determination of other elements such as arsenic, selenium, antimony, aluminium, nickel and tin [17]. Recently, an analytical procedure for the determination of OTCs was proposed [14]. The PFPD is based on a discontinuous flame. The pulsed nature of the emitted light enables the observation of time-dependant emission profiles. These profiles are characteristic of the species involved [17]. In Fig. 1 [18] the emission profiles of tin, hydrocarbon (combustion products) and sulphur compounds are presented. Unfortunately, the emission spectra of sulphur and hydrocarbons overlap the tin emission spectrum. This may cause some problems when analysing samples with high carbon and sulphur content. As presented by Bravo et al. [19] and Wasik et al. [20], the sulphur compounds give rise to signal interferences such as unknown peaks and unusual OTCs peak heights. Also, Fernandez-Escobar et al. [21] suggested that large amount of organic substances being
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coated with polydimethyl-siloxane (Quadrex, New Haven, CT, USA). For the separation of OTCs the following temperature programme was applied: at the start the column temperature was held at 80 ◦ C for the first minute, raised to 180 ◦ C at a heating rate of 30 ◦ C min−1 , then to 270 at 10 ◦ C min−1 and held at the final temperature for 7 min. The splitless injector port was kept at a temperature of 290 ◦ C and the temperature of the detector was 350 ◦ C. Nitrogen was used as a carrier gas (2 mL min−1 ). The detector operating conditions have been precisely described elsewhere [14]. For observation of the emission corresponding to the Sn–C molecular bond, a high transmission band filter (320–540 nm; Schott, Mainz, Germany) was used. According to the tin emission profile, signal acquisition was carried out with three different gate settings; (a) gate delay of 2.0 ms and gate width of 3.0 ms; (b) gate delay of 3.0 ms and gate width of 2.0 ms; (c) gate delay of 5.0 ms and gate width of 4.0 ms. Mechanical shaking during the extraction procedure was performed on an elliptical table (KS 2502 basic) from Prolabo (Fontenay Sous Bois, France). Fig. 1. Emission profile of tin and potentially interfering elements [18].
2.2. Standards and reagents extracted from samples may lead to stationary phase overloading and retention time shifting, and consequently data misinterpretation. A decrease in sulphur interferences may be achieved by chemical desulphurisation processes with activated copper [22], AgNO3 -coated silica gel [23], aluminium oxide [23,24], silica gel [25], Florisil [23,26,27], or a combination of these materials [23]. Sulphur interferences may also be decreased by adjusting PFPD parameters such as wavelength and the start (gate delay) and duration (gate width) of the signal detection, according to the profile of the species studied. Variation in the parameters of the detector such as gate delay and gate width strongly influence signal emission, acquisition and processing [28]. In the determination of OTCs with PFPD two wavelengths may be used. One is 390 nm that corresponds to the Sn–C emission, which is 100–1000 times more important than the Sn–H emission at 610 nm [14]. Unfortunately at the wavelength of 390 nm sulphur species also emit [29]. Godoi et al. [30] and Mzoughi et al. [31] studied the possibility of reducing sulphur interferences by recording the OTCs signals at the two wavelengths. The interferences were decreased but also the tin signal was too low for accurate OTCs determination. The aim of our previous work (Zuliani et al.) [15] was the investigation of the influence of different soil matrices on the analytical performance of the headspace solid-phase microextraction (HSSPME) for speciation of OTC by GC-PFPD. The objective of the present work was to optimise the analytical method for the determination of OTC in sewage sludge by GC-PFPD. The possibility of reduction of spectral interferences by changing the detector parameters such as gate delay and gate width was evaluated. Under the optimised parameters three internal standards were tested and the limits of detection (LOD) and quantification (LOQ) were determined. In addition, the concentration of OTCs in a sample of sewage sludge from a local waste water treatment plant was determined. 2. Experimental 2.1. Instrumentation The analyses were carried out on a Varian 3800 gas chromatograph coupled with a pulsed flame photometric detector and a Varian 1079 split/splitless temperature programmable injector (Walnut Creek, CA, USA). The GC was equipped with a 30 m × 0.25 mm DP-5 capillary column (film thickness 0.25 m)
Monobutyltin trichloride (MBT, 95%), monophenyltin trichloride (MPhT, 98%) and diphenyltin dichloride (DPhT, 96%) were purchased from Aldrich (Milwaukee, WI, USA). Dibutyltin dichloride (DBT, 97%), tributyltin chloride (TBT, 96%), triphenyltin chloride (TPhT, 95%) and tripropyltin chloride (TPrT, 98%) were obtained from Merck (Darmstadt, Germany). Monoheptyltin (MHeT, 98%) and diheptyltin (DHeT, 97%) were purchased from LGC Promochem (Teddington, Middlesex, UK). PACS-2 Harbour Sediment was purchased from National Research Council of Canada (Ottawa, Ont., Canada). Organotin standard stock solutions (1000 mg L−1 as Sn) were prepared in methanol. Working standard solutions (10 mg L−1 as Sn) were prepared weekly from stock standard solutions by dilution in Milli-Q water (18.2 M cm−1 ) (Millipore, Bedford, MA, USA). Standard solutions of 100 g L−1 as Sn were daily prepared. All the standards were stored in the dark at 4 ◦ C. Acetic acid, ammonia and nitric acid were obtained from J.T. Baker (Paris, France). Methanol was purchased from Prolabo (Fontenay Sous Bois, France). Sodium tetraethylborate (NaBEt4 ) was purchased from Galab products (Geesthacht, Germany). NaBEt4 was dissolved in Milli-Q water to provide a 2% (m/v) ethylating solution. 2.3. Analytical procedure 2.3.1. Sample preparation The sewage sludge was dried at 50 ◦ C in the dark for 3 days, homogenised and sieved through a 0.2-mm sieve. The sample was then frozen at −20 ◦ C. 2.3.2. Extraction and derivatisation procedure An extraction technique that was previously optimised for sediments [14] was applied for sewage sludge samples. Briefly, 0.5–1 g of air-dried sludge sample was extracted in 20 mL of glacial acetic acid by mechanical stirring (420 rpm) for 16 h. Suspensions were centrifuged at 4000 rpm for 15 min. Aliquots (1–4 mL) of the extract described above, 0.5–1 mL 2% (m/v) solution of NaBEt4 for the derivatisation and 1 mL of isooctane for the extraction of ethylated organotin species were added simultaneously to 20 or 100 mL of sodium acetate–acetic acid buffer (pH 4.8). The mixture was shaken for 30 min. OTCs were determined in the organic phase by GC-PFPD. The results obtained are compared in Table 1. A two-tailed Student’s
T. Zuliani et al. / J. Chromatogr. A 1188 (2008) 281–285 Table 1 Comparison of the results of sewage sludge analysis using 20 or 100 mL of acetic buffer OTC
MBT DBT TBT MOcT DOcT TOcT
Concentrations 100 mL of buffer
20 mL of buffer
129 ± 48 201 ± 11 30 ± 5 99 ± 30 17 ± 3 nda
179 ± 10 192 ± 4 41 ± 3 66 ± 10 25 ± 5 nd
Concentrations are expressed in ng Sn g−1 (dry mass basis). a Not detected.
Fig. 2. Chromatogram of water sample spiked with 50 ng Sn g−1 of OTC standard. Detection parameters: gate delay 3 ms and gate width 2 ms.
t-test using a 5% significance level showed no difference between the two analytical protocols. Consequently, for further analyses the smaller quantity of acetic buffer was used. The same procedure, with the exception that no sample was added, was applied to determine blanks. All the analyses were made in triplicate. 2.3.3. Quantification Quantification of OTCs was performed by applying the standard addition calibration method using 100 ng Sn g−1 of TPrT as internal standard. 3. Results and discussion 3.1. Selectivity of the PFPD detector during sewage sludge analyses During analysis of the sewage sludge matrix interferences were observed. These interferences presumably originated from sulphur compounds that co-elute with some organotin species. The sulphur and tin signals may be distinguished already by their peak shapes. The sulphur peak has a Gaussian shape and is narrow and high because the PFPD is very sensitive toward sulphur compounds. On the contrary, the tin peak displays tailing [17]. The peak tailing of OTCs does not originate from separation problems but from the combustion chemistry of tin compounds and SnO2 deposition in the detector [2,32]. For the determination of OTCs the Varian PFPD manual [18] suggests greater signals are observed at longer measurement times but in the literature no gate setting longer than 8 ms is reported [19,32]. From Fig. 1 it can be seen that sulphur interferences especially may be reduced if the gate width is as narrow as possible.
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The reduction of spectral interferences by gate settings was studied. The chromatogram in Fig. 2 was obtained for an ethylated water standard solution of butyl-, phenyl- and octyltins. In Fig. 3 are presented the chromatograms of a sludge extract recorded at different gate settings. For each setting, the same sewage sludge extract in isooctane was injected five times. The three chromatograms are presented on the same scale in order to show clearly the difference between the responses. As is evident from Fig. 3, there are significant differences between the chromatograms. When the tin signal was recorded at a gate delay of 5 ms and gate width of 4 ms (Fig. 3a), the sensitivity of OTCs was low. At the retention time of TPrT (7.17 min) there is no peak but the nearby peak (retention time 7.3 min) is more likely to be a sulphur than a tin signal. This supposition comes mainly from the shape of the peak. Though, already in 1999, an unknown compound eluting at the retention time of TPrT was reported in extracts obtained from BCR freshwater sediment [20]. The MOcT peak is split and cannot be accurately integrated. However, when a gate delay of 2 ms and gate width of 3 ms (Fig. 3b) was applied the signal increased but degradation of the baseline could be observed. The noise arose from the emission of combustion products which overlaps the tin emission as described earlier (Fig. 1). In the previous chromatogram (Fig. 3a) the peak that was presumed to be TPrT was found to be an unidentified interfering peak. In the chromatogram in Fig. 3b, the TPrT peak is separated from the interfering peak but its integration is still hindered. Also the resolution of the MOcT peak is improved. In continuation of the analyses, the gate delay and gate width were adjusted to 3 and 2 ms, respectively (Fig. 3c). Under these detector conditions good separation of all OTCs present in the sludge was obtained. The interfering peak near TPrT is separated sufficiently (resolution factor Rs = 1.4) to permit its integration. Also, the noise of the baseline was decreased. 3.2. Applicability of different internal standards For reliable quantification of OTCs present in the sample, an appropriate internal standard is required. Three main criteria must be considered for an appropriate choice of internal standard; the selected compound should not be present in the sample, the compound should not interfere with the analytes and the compound should have the same degree of substitution [33]. So far, several internal standards have been used for the determination of butyl- and phenyltin compounds; dimethyltin [34], tetrabutyltin [35], pentyltins [8,36], triethyltin, and tripropyltin which is the most frequently used [33]. ISO standard 17353:2004, determination of selected OTCs in water [37], clearly states that internal standards should be used with regard to the degree of substitution of the OTCs analysed in order to prevent any bias due to possible dependence between the ethylation recovery and the degree of substitution. In the present study, in order to fulfil the requirements of ISO standard 17353:2004 and the requirements for the internal standards previously described, the quantification of OTCs in sewage sludge was done using three internal standards. MHeT was used to quantify mono-, DHeT di-substituted OTCs and TPrT for the quantification of all OTCs present in the sewage sludge sample. The results are presented in Table 2. As can be seen from the table, the results showed a good agreement between the values obtained with all three internal standards. A two-tailed Student’s t-test using a 5% significance level confirmed that there is no significant difference between the values determined when TPrT, rather than MHeT or DHeT, was employed as the internal standard. This means that TPrT may be used as an internal standard for all OTCs, whatever their degree of substitution. On the basis of the presented results, TPrT was applied as the only internal standard in further analyses.
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Fig. 3. Chromatograms of a sewage sludge sample obtained at different gate settings of the PFPD detector. (a) Gate delay 5 ms and gate width 4 ms; (b) gate delay 2 ms and gate width 3 ms; (c) gate delay 3 ms and gate width 2 ms; *impurities that do not disturb OTCs detection. Table 2 Results of the analysis of sewage sludge using different internal standards Concentrations (ng Sn g−1 )
Internal standard
MHeT DHeT TPrT
MBT
DBT
TBT
MOcT
DOcT
83 ± 10 – 102 ± 15
– 184 ± 5 181 ± 8
– – 16 ± 2
100 ± 10 – 123 ± 16
– 19 ± 2 14 ± 1
Concentrations are expressed in ng Sn g−1 (dry mass basis). (–) Not quantified.
3.3. Analytical performance The limit of detection and limit of quantification were determined by the analysis of sewage sludge that did not contain OTCs. The basis for calculation of the LOD and LOQ is the standard deviation of the calibration curve obtained by standard addition. The principle is precisely described in the work of Mocak et al. [38]. The results for LOD and LOQ are presented in Table 3.
Table 3 Limits of detection (LOD) and quantification (LOQ) for sewage sludge sample OTC
LOD (ng Sn g−1 )
LOQ (ng Sn g−1 )
MBT DBT TBT
16.3 10.0 8
37.1 22.7 18
MPhT DPhT TPhT
8.0 6.8 8.1
18.2 15.4 18.4
MOcT DOcT TOcT
9.1 5.1 10.2
20.4 11.4 22.5
Concentrations are expressed in ng Sn g−1 (dry mass basis).
The accuracy and repeatability (as relative standard deviation, RSD) were checked by the analysis of the certified reference material PACS-2, marine sediment. Because PACS-2 is certified only for butyltin compounds, the certified sediment material was additionally spiked with TPhT and TOcT. Generally, a reference material used for the validation of a method should have a matrix close to that of the sample analysed. There is no certified reference material for OTCs in sewage sludge presently available on the market. Therefore, a sample of sewage sludge spiked with TBT, TPhT and TOcT was analysed. Tri-substituted organotin spikes were applied because they are the most toxic of the OTC species. The results are presented in Table 4. Generally, good agreement between certified or spiked and determined values was obtained, confirmed by a two-tailed Student’s t-test using a 5% significance level. The repeatability for butyltin compounds was between 5 and 20%. The concentrations of OTCs in the sewage sludge sample from Slovenia were compared with the results reported from Switzerland and France. The results are presented in Table 5. Data from the Swiss wastewater treatment plant showed considerable organotin residues in the sludge. The concentration of butyltin is much higher than in sewage sludge from Slovenia and France. On the other hand, octyltins were not detected in Swiss sludge. These results show that quite important quantities of OTCs
T. Zuliani et al. / J. Chromatogr. A 1188 (2008) 281–285
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Table 4 Analysis of PACS-2, which was additionally spiked with TPhT and TOcT, and sewage sludge, spiked with TBT, TPhT and TOcT, by GC-PFPD OTC
Certified or spikeda values (ng Sn g−1 ) c
PACS-2
MBT DBT TBT TPhT TOcT
600 1080 ± 150 980 ± 130 200 ± 10 300 ± 10
Sewage sludge
TBT TPhT TOcT
50 ± 2 50 ± 2 50 ± 2
a b c
Determined valuesb (ng Sn g−1 ) 688 1060 880 200 305
± ± ± ± ±
85 45 26 15 15
51 ± 5 37 ± 7 55 ± 9
Repeatability (%)b
Recovery (%)b
10 5 7 8 5
115 98 90 100 102
10 19 16
101 ± 11 84 ± 6 111 ± 18
± ± ± ± ±
20 6 4 11 7
Concentrations of spike. Average of five independent determinations. Indicative value.
Table 5 Comparison of OTC contents in sewage sludge from different locations
References
OTC
Slovenia (1)
Switzerland (2)
France (3)
MBT DBT TBT MOcT DOcT TOcT
179 ± 10 192 ± 4 41 ± 3 66 ± 10 25 ± 5 nda
500 1500 1100 nd nd nd
239 81 54 5.7 19.0 12.4
± ± ± ± ± ±
2 8 5 0.8 0.5 0.2
(1) Present work; (2) Fent [5]; (3) Montigny et al. [33]. Concentrations are expressed in ng Sn g−1 (dry mass basis). a Not detected.
are introduced into the environment via the disposal of sewage sludge. 4. Conclusions In the present work the influences of the PFPD parameters such as gate delay and gate width were investigated in order to minimise spectral interferences arising from the sewage sludge matrix during analysis of OTCs. Under a gate delay of 3 ms and a gate width 2 ms the interferences were reduced. This enabled selective and accurate determination of OTCs in sewage sludge. It was experimentally proven that the use of internal standards with the same degree of substitution as the quantified OTCs is not required. TPrT was confirmed as an appropriate internal standard for the quantification of OTCs in the samples analysed. Under the optimised analytical conditions, sewage sludge from a wastewater treatment plant in Slovenia was analysed and the data compared to those reported from Switzerland and France. Butyltins were present in sewage sludge from all three countries, while octyltins appeared in Slovenian and French sludge. Since OTCs are present in sewage sludge it is important to investigate their fate in the environment by accurate and reliable analytical procedures. Acknowledgements The authors would like to acknowledge the financial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia within the research programme P10143, French Ministry of Education and PROTEUS bilateral project: “Organotin compounds in the Slovenian and French environment”.
[1] K. Fent, CRC Crit. Rev. Toxicol. 26 (1996) 1. [2] J.A. Jacobsen, F. Stuer-Lauridsen, G. Pritzl, Appl. Organomet. Chem. 11 (1997) 737. [3] C. Alzieu, Ecotoxicology 9 (2000) 71. [4] K. Fent, M.D. Muller, Environ. Sci. Technol. 25 (1991) 489. [5] K. Fent, Sci. Total Environ. 185 (1996) 151. [6] N. Voulvoulis, M.D. Scrimshaw, J.N. Lester, Environ. Technol. 25 (2004) 733. [7] O.F.X. Donard, Ph. Quevauviller, A. Bruchet, Water Res. 27 (1993) 1085. [8] K. Fent, J. Hunn, D. Renggli, H. Siegrist, Mar. Environ. Res. 32 (1991) 223. [9] M.D. Muller, Fresen. J. Anal. Chem. 317 (1984) 32. [10] J.A. Stab, U.A.Th. Brinkman, W.P. Cofino, Appl. Organomet. Chem. 8 (1994) 77. [11] S. Aguerre, G. Lespes, V. Desauziers, M. Potin-Gautier, J. Anal. Atom. Spectrom. 16 (2001) 263. [12] C. Carlier-Pinasseau, G. Lespes, M. Astruc, Environ. Technol. 18 (1997) 1179. [13] C. Carlier-Pinasseau, G. Lespes, M. Astruc, Talanta 44 (1997) 1163. [14] C. Bancon-Montigny, G. Lespes, M. Potin-Gautier, J. Chromatogr. A 896 (2000) 149. ˇ canˇcar, M. Potin-Gautier, J. Chromatogr. A [15] T. Zuliani, G. Lespes, R. MIlaˇciˇc, J. Sˇ 1132 (2006) 234. [16] A. Amirav, H. Jing, Anal. Chem. 67 (1995) 3305. [17] H. Jing, A. Amirav, J. Chromatogr. A 805 (1998) 177. [18] A. Amirav, H. Jing, J.J. Robinson, in: N.A. Kirshen (Ed.), Pulsed Flame Photometric Detector for Gas Chromatography, Varian Chromatography Systems, 1998. [19] M. Bravo, G. Lespes, I. De Gregori, H. Pinochet, M. Potin-Gautier, J. Chromatogr. A 1046 (2004) 217. [20] A. Wasik, B. Radke, J. BolaŁek, J. Namie´snik, Chemisphere 68 (2007) 1. [21] I. Fernandez-Escobar, M. Gibert, A. Messeguer, J.M. Bayona, Anal. Chem. 70 (1998) 3703. [22] M. Abalos, J.M. Bayona, R. Comopano, M. Granados, C. Leal, M.D. Prat, J. Chromatogr. A 788 (1997) 1. [23] P. Schubert, I. Fernandez-Escobar, E. Rosenberg, J.M. Bayona, J. Chromatogr. A 810 (1998) 245. [24] W.M.R. Dirkx, M.B. de la Calle, M. Ceulemans, F.C. Adams, J. Chromatogr. A 683 (1994) 51. [25] Y.K. Chau, F. Yang, R.J. Maguire, Anal. Chim. Acta 320 (1996) 165. [26] R. James Meguire, Environ. Sci. Technol. 18 (1984) 291. [27] H. Harino, M. Fukushima, Anal. Chim. Acta 264 (1992) 91. [28] J.P. La Harle, B. Bellier, J. Chromatogr. A 1087 (2005) 124. [29] W.A. Aue, C.G. Flinn, J. Chromatogr. 142 (1977) 145. [30] A.F.L. Godoi, R.C. Montone, M. Santiago-Silva, J. Chromatogr. A 985 (2003) 205. [31] N. Mzoughi, G. Lespes, M. Bravo, M. Dachraoui, M. Potin-Gautier, Sci. Total Environ. 349 (2005) 211. [32] A.F.L. Godoi, R. Favoreto, M. Santiago-Silva, Chromatographyhia 58 (2003) 97. [33] C. Montigny, G. Lespes, M. Potin-Gautier, J. Chromatogr. A 819 (1998) 221. [34] I. Martin-Landa, F. Pablos, L.L. Marr, Appl. Organomet. Chem. 5 (1991) 399. [35] M.D. Muller, Anal. Chem. 59 (1987) 617. [36] K. Fent, Mar. Environ. Res. 28 (1989) 477. [37] Water Quality, Determination of Selected Organotin Compounds—Gas Chromatographic Method, First edition, International Organization for Standardization, Geneva, 2004 (ISO 17353:2004). [38] J. Mocak, A.M. Bond, S. Mitchell, G. Scollary, Pure Appl. Chem. 69 (1997) 297.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Comprehensive study of the parameters influencing the detection of organotin compounds by a pulsed flame photometric detector in sewage sludge ˇ canˇcar a , Martine Potin-Gautier b Tea Zuliani a,b,∗ , Gaetane Lespes b , Radmila Milaˇciˇc a , Janez Sˇ a b
Department of Environmental Sciences, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Laboratoire de Chimie Analytique, LCABIE, UMR CNRS 5034, Universit´e de Pau et des Pays de l’Adour, Avenue de l’Universit´e, BP 1155, 64013 Pau, France
a r t i c l e
i n f o
Article history: Received 3 October 2007 Received in revised form 14 February 2008 Accepted 21 February 2008 Available online 2 March 2008 Keywords: Organotin compounds Sewage sludge Matrix interferences GC-PFPD
a b s t r a c t An investigation of the operating conditions of a pulsed flame photometric detection (PFPD) system for the determination of organotin compounds (OTCs) in sewage sludge is reported. During the analyses, some spectral interferences were observed. For their elimination detector parameters such as gate delay and gate width were investigated. In addition, the applicability of three different internal standards was evaluated. Under optimised analytical conditions (gate delay 3 ms, gate width 2 ms, tripropyltin as internal standard) limits of detection (LOD) were determined. The LOD for butyltins ranged between 8 and 16 ng Sn g−1 , for phenyltins around 8 ng Sn g−1 and for octyltins between 5 and 10 ng Sn g−1 . Since there is no certified reference material (CRM) available for sewage sludge, the accuracy of the analytical procedure was checked by the analysis of CRM PACS-2 (marine sediment) and a spiked sludge sample. Good agreement between determined and certified values was obtained. Sewage sludge from a local wastewater treatment plant was analysed and the results compared with data from the literature. © 2008 Elsevier B.V. All rights reserved.
1. Introduction In recent decades organotin compounds (OTCs) were widely used as biocides, agricultural fertilizers, wood preservatives and plastic stabilizers. Therefore, OTCs are found in numerous environmental compartments such as water, sediments, aquatic organisms, soil and sewage sludge [1]. The first known contamination concerned the aquatic environment because OTCs, especially tributyltin (TBT) and triphenyltins (TPhT), were present in antifouling paints [2,3]. Although there is a lack of data on OTC’s inputs into wastewater [4], the possible sources may be plastic, pesticide, colour and varnish production [5]. During the treatment of wastewater OTCs are mostly removed during the primary treatment (around 80%) [6]. The efficiency of OTCs removal is regulated by their absorption capacities [7]. So, during treatment OTCs are transferred and conserved from wastewater to sewage sludge [8]. The complexity of sewage sludge matrix makes it necessary to ensure the quality of the analytical procedure used. So far, many analytical methods were developed for the determination of OTCs in solid samples. The analytical procedure involves several
∗ Corresponding author at: Department of Environmental Sciences, Joˇzef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Tel.: +386 1 4773900; fax: +386 1 2519385. E-mail address: [email protected] (T. Zuliani). 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.02.079
steps including extraction, derivatisation if gas chromatography is then applied, separation and detection. Generally, the analysis is performed using gas chromatography separation followed by element-selective detection. In recent years, the most frequently used detection methods for OTCs determination are the mass spectrometry (MS) [9,10], the inductively coupled plasma mass spectrometry (ICP-MS) [11], the flame photometric detection (FPD) [12,13] and the pulsed flame photometric detection (PFPD) [2,14,15]. PFPD was developed by Amiravet al. [16] and was previously designed for the analysis of sulphur, nitrogen and phosphorus. Later on application of the PFPD was extended to the determination of other elements such as arsenic, selenium, antimony, aluminium, nickel and tin [17]. Recently, an analytical procedure for the determination of OTCs was proposed [14]. The PFPD is based on a discontinuous flame. The pulsed nature of the emitted light enables the observation of time-dependant emission profiles. These profiles are characteristic of the species involved [17]. In Fig. 1 [18] the emission profiles of tin, hydrocarbon (combustion products) and sulphur compounds are presented. Unfortunately, the emission spectra of sulphur and hydrocarbons overlap the tin emission spectrum. This may cause some problems when analysing samples with high carbon and sulphur content. As presented by Bravo et al. [19] and Wasik et al. [20], the sulphur compounds give rise to signal interferences such as unknown peaks and unusual OTCs peak heights. Also, Fernandez-Escobar et al. [21] suggested that large amount of organic substances being
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coated with polydimethyl-siloxane (Quadrex, New Haven, CT, USA). For the separation of OTCs the following temperature programme was applied: at the start the column temperature was held at 80 ◦ C for the first minute, raised to 180 ◦ C at a heating rate of 30 ◦ C min−1 , then to 270 at 10 ◦ C min−1 and held at the final temperature for 7 min. The splitless injector port was kept at a temperature of 290 ◦ C and the temperature of the detector was 350 ◦ C. Nitrogen was used as a carrier gas (2 mL min−1 ). The detector operating conditions have been precisely described elsewhere [14]. For observation of the emission corresponding to the Sn–C molecular bond, a high transmission band filter (320–540 nm; Schott, Mainz, Germany) was used. According to the tin emission profile, signal acquisition was carried out with three different gate settings; (a) gate delay of 2.0 ms and gate width of 3.0 ms; (b) gate delay of 3.0 ms and gate width of 2.0 ms; (c) gate delay of 5.0 ms and gate width of 4.0 ms. Mechanical shaking during the extraction procedure was performed on an elliptical table (KS 2502 basic) from Prolabo (Fontenay Sous Bois, France). Fig. 1. Emission profile of tin and potentially interfering elements [18].
2.2. Standards and reagents extracted from samples may lead to stationary phase overloading and retention time shifting, and consequently data misinterpretation. A decrease in sulphur interferences may be achieved by chemical desulphurisation processes with activated copper [22], AgNO3 -coated silica gel [23], aluminium oxide [23,24], silica gel [25], Florisil [23,26,27], or a combination of these materials [23]. Sulphur interferences may also be decreased by adjusting PFPD parameters such as wavelength and the start (gate delay) and duration (gate width) of the signal detection, according to the profile of the species studied. Variation in the parameters of the detector such as gate delay and gate width strongly influence signal emission, acquisition and processing [28]. In the determination of OTCs with PFPD two wavelengths may be used. One is 390 nm that corresponds to the Sn–C emission, which is 100–1000 times more important than the Sn–H emission at 610 nm [14]. Unfortunately at the wavelength of 390 nm sulphur species also emit [29]. Godoi et al. [30] and Mzoughi et al. [31] studied the possibility of reducing sulphur interferences by recording the OTCs signals at the two wavelengths. The interferences were decreased but also the tin signal was too low for accurate OTCs determination. The aim of our previous work (Zuliani et al.) [15] was the investigation of the influence of different soil matrices on the analytical performance of the headspace solid-phase microextraction (HSSPME) for speciation of OTC by GC-PFPD. The objective of the present work was to optimise the analytical method for the determination of OTC in sewage sludge by GC-PFPD. The possibility of reduction of spectral interferences by changing the detector parameters such as gate delay and gate width was evaluated. Under the optimised parameters three internal standards were tested and the limits of detection (LOD) and quantification (LOQ) were determined. In addition, the concentration of OTCs in a sample of sewage sludge from a local waste water treatment plant was determined. 2. Experimental 2.1. Instrumentation The analyses were carried out on a Varian 3800 gas chromatograph coupled with a pulsed flame photometric detector and a Varian 1079 split/splitless temperature programmable injector (Walnut Creek, CA, USA). The GC was equipped with a 30 m × 0.25 mm DP-5 capillary column (film thickness 0.25 m)
Monobutyltin trichloride (MBT, 95%), monophenyltin trichloride (MPhT, 98%) and diphenyltin dichloride (DPhT, 96%) were purchased from Aldrich (Milwaukee, WI, USA). Dibutyltin dichloride (DBT, 97%), tributyltin chloride (TBT, 96%), triphenyltin chloride (TPhT, 95%) and tripropyltin chloride (TPrT, 98%) were obtained from Merck (Darmstadt, Germany). Monoheptyltin (MHeT, 98%) and diheptyltin (DHeT, 97%) were purchased from LGC Promochem (Teddington, Middlesex, UK). PACS-2 Harbour Sediment was purchased from National Research Council of Canada (Ottawa, Ont., Canada). Organotin standard stock solutions (1000 mg L−1 as Sn) were prepared in methanol. Working standard solutions (10 mg L−1 as Sn) were prepared weekly from stock standard solutions by dilution in Milli-Q water (18.2 M cm−1 ) (Millipore, Bedford, MA, USA). Standard solutions of 100 g L−1 as Sn were daily prepared. All the standards were stored in the dark at 4 ◦ C. Acetic acid, ammonia and nitric acid were obtained from J.T. Baker (Paris, France). Methanol was purchased from Prolabo (Fontenay Sous Bois, France). Sodium tetraethylborate (NaBEt4 ) was purchased from Galab products (Geesthacht, Germany). NaBEt4 was dissolved in Milli-Q water to provide a 2% (m/v) ethylating solution. 2.3. Analytical procedure 2.3.1. Sample preparation The sewage sludge was dried at 50 ◦ C in the dark for 3 days, homogenised and sieved through a 0.2-mm sieve. The sample was then frozen at −20 ◦ C. 2.3.2. Extraction and derivatisation procedure An extraction technique that was previously optimised for sediments [14] was applied for sewage sludge samples. Briefly, 0.5–1 g of air-dried sludge sample was extracted in 20 mL of glacial acetic acid by mechanical stirring (420 rpm) for 16 h. Suspensions were centrifuged at 4000 rpm for 15 min. Aliquots (1–4 mL) of the extract described above, 0.5–1 mL 2% (m/v) solution of NaBEt4 for the derivatisation and 1 mL of isooctane for the extraction of ethylated organotin species were added simultaneously to 20 or 100 mL of sodium acetate–acetic acid buffer (pH 4.8). The mixture was shaken for 30 min. OTCs were determined in the organic phase by GC-PFPD. The results obtained are compared in Table 1. A two-tailed Student’s
T. Zuliani et al. / J. Chromatogr. A 1188 (2008) 281–285 Table 1 Comparison of the results of sewage sludge analysis using 20 or 100 mL of acetic buffer OTC
MBT DBT TBT MOcT DOcT TOcT
Concentrations 100 mL of buffer
20 mL of buffer
129 ± 48 201 ± 11 30 ± 5 99 ± 30 17 ± 3 nda
179 ± 10 192 ± 4 41 ± 3 66 ± 10 25 ± 5 nd
Concentrations are expressed in ng Sn g−1 (dry mass basis). a Not detected.
Fig. 2. Chromatogram of water sample spiked with 50 ng Sn g−1 of OTC standard. Detection parameters: gate delay 3 ms and gate width 2 ms.
t-test using a 5% significance level showed no difference between the two analytical protocols. Consequently, for further analyses the smaller quantity of acetic buffer was used. The same procedure, with the exception that no sample was added, was applied to determine blanks. All the analyses were made in triplicate. 2.3.3. Quantification Quantification of OTCs was performed by applying the standard addition calibration method using 100 ng Sn g−1 of TPrT as internal standard. 3. Results and discussion 3.1. Selectivity of the PFPD detector during sewage sludge analyses During analysis of the sewage sludge matrix interferences were observed. These interferences presumably originated from sulphur compounds that co-elute with some organotin species. The sulphur and tin signals may be distinguished already by their peak shapes. The sulphur peak has a Gaussian shape and is narrow and high because the PFPD is very sensitive toward sulphur compounds. On the contrary, the tin peak displays tailing [17]. The peak tailing of OTCs does not originate from separation problems but from the combustion chemistry of tin compounds and SnO2 deposition in the detector [2,32]. For the determination of OTCs the Varian PFPD manual [18] suggests greater signals are observed at longer measurement times but in the literature no gate setting longer than 8 ms is reported [19,32]. From Fig. 1 it can be seen that sulphur interferences especially may be reduced if the gate width is as narrow as possible.
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The reduction of spectral interferences by gate settings was studied. The chromatogram in Fig. 2 was obtained for an ethylated water standard solution of butyl-, phenyl- and octyltins. In Fig. 3 are presented the chromatograms of a sludge extract recorded at different gate settings. For each setting, the same sewage sludge extract in isooctane was injected five times. The three chromatograms are presented on the same scale in order to show clearly the difference between the responses. As is evident from Fig. 3, there are significant differences between the chromatograms. When the tin signal was recorded at a gate delay of 5 ms and gate width of 4 ms (Fig. 3a), the sensitivity of OTCs was low. At the retention time of TPrT (7.17 min) there is no peak but the nearby peak (retention time 7.3 min) is more likely to be a sulphur than a tin signal. This supposition comes mainly from the shape of the peak. Though, already in 1999, an unknown compound eluting at the retention time of TPrT was reported in extracts obtained from BCR freshwater sediment [20]. The MOcT peak is split and cannot be accurately integrated. However, when a gate delay of 2 ms and gate width of 3 ms (Fig. 3b) was applied the signal increased but degradation of the baseline could be observed. The noise arose from the emission of combustion products which overlaps the tin emission as described earlier (Fig. 1). In the previous chromatogram (Fig. 3a) the peak that was presumed to be TPrT was found to be an unidentified interfering peak. In the chromatogram in Fig. 3b, the TPrT peak is separated from the interfering peak but its integration is still hindered. Also the resolution of the MOcT peak is improved. In continuation of the analyses, the gate delay and gate width were adjusted to 3 and 2 ms, respectively (Fig. 3c). Under these detector conditions good separation of all OTCs present in the sludge was obtained. The interfering peak near TPrT is separated sufficiently (resolution factor Rs = 1.4) to permit its integration. Also, the noise of the baseline was decreased. 3.2. Applicability of different internal standards For reliable quantification of OTCs present in the sample, an appropriate internal standard is required. Three main criteria must be considered for an appropriate choice of internal standard; the selected compound should not be present in the sample, the compound should not interfere with the analytes and the compound should have the same degree of substitution [33]. So far, several internal standards have been used for the determination of butyl- and phenyltin compounds; dimethyltin [34], tetrabutyltin [35], pentyltins [8,36], triethyltin, and tripropyltin which is the most frequently used [33]. ISO standard 17353:2004, determination of selected OTCs in water [37], clearly states that internal standards should be used with regard to the degree of substitution of the OTCs analysed in order to prevent any bias due to possible dependence between the ethylation recovery and the degree of substitution. In the present study, in order to fulfil the requirements of ISO standard 17353:2004 and the requirements for the internal standards previously described, the quantification of OTCs in sewage sludge was done using three internal standards. MHeT was used to quantify mono-, DHeT di-substituted OTCs and TPrT for the quantification of all OTCs present in the sewage sludge sample. The results are presented in Table 2. As can be seen from the table, the results showed a good agreement between the values obtained with all three internal standards. A two-tailed Student’s t-test using a 5% significance level confirmed that there is no significant difference between the values determined when TPrT, rather than MHeT or DHeT, was employed as the internal standard. This means that TPrT may be used as an internal standard for all OTCs, whatever their degree of substitution. On the basis of the presented results, TPrT was applied as the only internal standard in further analyses.
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Fig. 3. Chromatograms of a sewage sludge sample obtained at different gate settings of the PFPD detector. (a) Gate delay 5 ms and gate width 4 ms; (b) gate delay 2 ms and gate width 3 ms; (c) gate delay 3 ms and gate width 2 ms; *impurities that do not disturb OTCs detection. Table 2 Results of the analysis of sewage sludge using different internal standards Concentrations (ng Sn g−1 )
Internal standard
MHeT DHeT TPrT
MBT
DBT
TBT
MOcT
DOcT
83 ± 10 – 102 ± 15
– 184 ± 5 181 ± 8
– – 16 ± 2
100 ± 10 – 123 ± 16
– 19 ± 2 14 ± 1
Concentrations are expressed in ng Sn g−1 (dry mass basis). (–) Not quantified.
3.3. Analytical performance The limit of detection and limit of quantification were determined by the analysis of sewage sludge that did not contain OTCs. The basis for calculation of the LOD and LOQ is the standard deviation of the calibration curve obtained by standard addition. The principle is precisely described in the work of Mocak et al. [38]. The results for LOD and LOQ are presented in Table 3.
Table 3 Limits of detection (LOD) and quantification (LOQ) for sewage sludge sample OTC
LOD (ng Sn g−1 )
LOQ (ng Sn g−1 )
MBT DBT TBT
16.3 10.0 8
37.1 22.7 18
MPhT DPhT TPhT
8.0 6.8 8.1
18.2 15.4 18.4
MOcT DOcT TOcT
9.1 5.1 10.2
20.4 11.4 22.5
Concentrations are expressed in ng Sn g−1 (dry mass basis).
The accuracy and repeatability (as relative standard deviation, RSD) were checked by the analysis of the certified reference material PACS-2, marine sediment. Because PACS-2 is certified only for butyltin compounds, the certified sediment material was additionally spiked with TPhT and TOcT. Generally, a reference material used for the validation of a method should have a matrix close to that of the sample analysed. There is no certified reference material for OTCs in sewage sludge presently available on the market. Therefore, a sample of sewage sludge spiked with TBT, TPhT and TOcT was analysed. Tri-substituted organotin spikes were applied because they are the most toxic of the OTC species. The results are presented in Table 4. Generally, good agreement between certified or spiked and determined values was obtained, confirmed by a two-tailed Student’s t-test using a 5% significance level. The repeatability for butyltin compounds was between 5 and 20%. The concentrations of OTCs in the sewage sludge sample from Slovenia were compared with the results reported from Switzerland and France. The results are presented in Table 5. Data from the Swiss wastewater treatment plant showed considerable organotin residues in the sludge. The concentration of butyltin is much higher than in sewage sludge from Slovenia and France. On the other hand, octyltins were not detected in Swiss sludge. These results show that quite important quantities of OTCs
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Table 4 Analysis of PACS-2, which was additionally spiked with TPhT and TOcT, and sewage sludge, spiked with TBT, TPhT and TOcT, by GC-PFPD OTC
Certified or spikeda values (ng Sn g−1 ) c
PACS-2
MBT DBT TBT TPhT TOcT
600 1080 ± 150 980 ± 130 200 ± 10 300 ± 10
Sewage sludge
TBT TPhT TOcT
50 ± 2 50 ± 2 50 ± 2
a b c
Determined valuesb (ng Sn g−1 ) 688 1060 880 200 305
± ± ± ± ±
85 45 26 15 15
51 ± 5 37 ± 7 55 ± 9
Repeatability (%)b
Recovery (%)b
10 5 7 8 5
115 98 90 100 102
10 19 16
101 ± 11 84 ± 6 111 ± 18
± ± ± ± ±
20 6 4 11 7
Concentrations of spike. Average of five independent determinations. Indicative value.
Table 5 Comparison of OTC contents in sewage sludge from different locations
References
OTC
Slovenia (1)
Switzerland (2)
France (3)
MBT DBT TBT MOcT DOcT TOcT
179 ± 10 192 ± 4 41 ± 3 66 ± 10 25 ± 5 nda
500 1500 1100 nd nd nd
239 81 54 5.7 19.0 12.4
± ± ± ± ± ±
2 8 5 0.8 0.5 0.2
(1) Present work; (2) Fent [5]; (3) Montigny et al. [33]. Concentrations are expressed in ng Sn g−1 (dry mass basis). a Not detected.
are introduced into the environment via the disposal of sewage sludge. 4. Conclusions In the present work the influences of the PFPD parameters such as gate delay and gate width were investigated in order to minimise spectral interferences arising from the sewage sludge matrix during analysis of OTCs. Under a gate delay of 3 ms and a gate width 2 ms the interferences were reduced. This enabled selective and accurate determination of OTCs in sewage sludge. It was experimentally proven that the use of internal standards with the same degree of substitution as the quantified OTCs is not required. TPrT was confirmed as an appropriate internal standard for the quantification of OTCs in the samples analysed. Under the optimised analytical conditions, sewage sludge from a wastewater treatment plant in Slovenia was analysed and the data compared to those reported from Switzerland and France. Butyltins were present in sewage sludge from all three countries, while octyltins appeared in Slovenian and French sludge. Since OTCs are present in sewage sludge it is important to investigate their fate in the environment by accurate and reliable analytical procedures. Acknowledgements The authors would like to acknowledge the financial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia within the research programme P10143, French Ministry of Education and PROTEUS bilateral project: “Organotin compounds in the Slovenian and French environment”.
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