DEVELOPMENT OF A SEMI-QUANTITATIVE DETECTION METHOD FOR POLYCHLORINATED ALKANES

Transcription

1 DEVELOPMENT OF A SEMI-QUANTITATIVE DETECTION METHOD FOR POLYCHLORINATED ALKANES Ike van der Veen Universiteit van Amsterdam Vrije Universiteit Amsterdam Nieuwe achtergracht 166 De Boelelaan WW Amsterdam 1081 HV Amsterdam Netherlands Institute of Fishery Research Institute of Environmental Studies Wageningen University and Research Centre Vrije universiteit Amsterdam Haringkade 1 De Boelelaan CP IJmuiden 1081 HV Amsterdam Supervision: Dr P.E.G. Leonards Dr. J.J. Vreuls Prof. dr. H. Irth

2 CONTENTS Summary Chapter 1. General introduction Preface Scope of the study 2 Chapter 2. Separation and detection method Comprehensive two-dimensional gas chromatography Advantages of GCxGC Principles of GCxGC Modulator Dual CO2 jet modulator Wrap-around GCxGC separation of PCAs (State-of-the-art) Time-of-flight mass spectrometry Time-of-flight mass spectrometer Principle of the time-of-flight mass spectrometer Reflectrons Chemical Ionization Positive or negative chemical ionization TOF analysis of PCAs (State-of-the-art) 13 Chapter 3. Optimization of GCxGC-TOF MS for PCA analysis Introduction Objective Experimental Optimization of the separation of PCAs Optimization of the modulator Mass spectra PCAs Ionization gas Results & Discussion Optimization of the separation of PCAs Optimization of the modulator Mass spectra PCAs Ionization gas for PCA analysis Conclusions 27 Chapter 4. Analysis of short, medium and long chain PCAs Introduction Objective Experimental Analysis of mixtures of PCAs Measurement of individual PCAs Results & Discussion 30 2

3 4.3.1 Analysis of mixtures of PCAs Chlorine content of C 10 -C 13 mixtures Chlorine content of C 14 -C 17 mixtures Comparing mixtures of different chain lengths Measurement of individual PCAs Conclusions 33 Chapter 5. Semi-quantitative measurement of PCAs Introduction Objective Experimental Results & Discussion Conclusions 35 Chapter 6. Clean up method for PCA analysis Introduction Experimental Gel Permeation Chromatography Sulphuric acid Acidic silica Silica gel Results & Discussion Gel Permeation Chromatography Sulphuric acid Acidic silica Silica gel Conclusions 44 Chapter 7. Biomonitoring Introduction Experimental Pilot study Samples Results & Discussion Pilot study Samples Conclusions 50 Chapter 8. Conclusions and recommendations 51 Acknowledgements 53 References 55 List of abbreviations 58 3

4 Appendix 1 59 Appendix 2 62 Appendix

5 SUMMARY The scope of the study was to develop an analytical method for the determination of polychlorinated alkanes (PCAs) in environmental samples. A first attempt to develop a semi quantitative method by measuring intensities of several individual PCA standards using comprehensive multidimensional gas chromatography (GC) combined with electron capture negative chemical ionisation time-of-flight mass spectrometry (ECNI-TOF-MS) was made. The electronegative chemical ionization mode for analysis of polychlorinated alkanes compared to positive chemical ionization or electron impact is discussed. The separation of PCA groups by GCxGC was improved by using a longer column in the second dimension. The use of ammonia as ionization gas in the mass spectrometer rather than 100 % methane improved the stability of the signal and cleanness of the source. For the sample treatment of environmental samples various clean up techniques were compared. Acidic silica clean up in combination with silica fractionation is a promising method for cleaning environmental samples for the analysis of short chain PCAs. However, the method should be further validated for medium and long chain PCAs. Silica gel is a good clean up and fractionation method for PCAs from other chlorine containing compounds like PCBs that can interfere with the analysis (same m/z value as PCAs). The PCAs elute in the second fraction and PCBs in the first fraction. Sulphuric acid is not suitable as a clean up method as various PCA groups were lossed. Gel permeation chromatography (GPC) was not sufficient for cleaning extracts for PCA analysis because lipids are coëluting with some PCAs. Two-dimensional chromatograms of mixtures of PCAs with different chain lengths (short, medium and long chain PCAs), and different chlorine contents are compared and showed that the longer the carbon chain, the longer the first dimension retention time. The observed group separation is based on the number of carbon and chlorine atoms in the compounds. A number of individual PCAs were analysed for identification of the groups in the chromatograms of the mixtures. However, the individual PCAs showed too low sensitivity with ECNI. A preliminary study for the set up of a semi-quantitative method for PCA analysis was carried out. The analysis of a number of individual PCAs showed that the individual PCAs had a low response due to the low chlorine content. A semi-quantitative method based on individual PCAs could not be established. Finally, PCAs were analysed in a number of environmental samples with the developed sample treatment method and GCxGC-ECNI-MS system. Although the GCxGC-ECNI-MS system was not stable, PCA groups were detected in an extract of tern egg and in an extract of suspended matter. Analysis of extracts of different types of fish gave poor results, of which no qualitative or semi quantitative conclusions could be made, due to the poor stability and robustness of the TOF instrument. In conclusion, a method is developed for PCA analysis in environmental and biota samples, including a Soxhlet extraction, followed by fractionation with silica and a clean up step with 5

6 acidic silica. Analysis is performed by GCxGC-ECNI-TOF MS. The second dimension separation of PCAs is improved by using a longer second dimension column. However, the TOF mass spectrometer in ENCI mode could be used for qualitative analysis, but is not a suitable instrument for quantitative analysis due to the instability of the detector response. 6

7 1. GENERAL INTRODUCTION 1.1 Preface Polychloroalkanes (PCAs), also known as chloroparaffines, are complex mixtures of chlorinated n-alkanes with carbon chain lengths of 10 to 30 and a chlorination degree between 30% and 70% by mass. Characterization of PCAs is performed by their alkane chain lengths. They are divided in three groups: short chain (C 10 -C 13 ), medium chain (C 14 -C 17 ) and long chain (C >17 ) PCAs. PCA mixtures were synthesized since the 1930s and over 200 commercial products, containing PCAs, have been produced since (Serrone et al., 1987). PCAs are used in several industrial applications like flame-retardants in the rubber industry, as high temperature lubricants and cutting fluids in the metalworking industry and as additives in liquids, in paints and textile. Worldwide over ton PCAs are produced per year (Cooley et al., 2001). PCAs can have a toxic effect on terrestrial and aquatic organism (OSPAR, 2001). Although the acute toxicity is low, PCAs can effect the growth of an organism when they bioconcentrate (WHO, 1996). They are potential carcinogen and under Canada s Environmental Protection Act, PCAs have been classified as priority toxic substances (CEPA, 1993). They have also been placed on the U.S. Environmental Protection Agency Risk Reduction List (EPA, cited in Tomy et al., 1997), and they are included on the list of priority hazardous substances by the European Union (Off.J.Eur.Commiun, 2000 and 2001 cited in Korytár et al., 2006) and since 2004 the short chain PCAs may not be used in concentrations higher than 1 % in metalworking and for liquoring of leather in the European Union (Stejnarova et al., 2005). Knowledge of concentrations of PCAs in the environment is needed to estimate environmental exposure and to understand their fate. Therefore, a sensitive and specific method for quantitative analysis of PCAs is required for their exposure assessment. The short chain PCAs are most likely to be found in the environment due to their high vapor pressure, persistency and high water solubility. These compounds are highly toxic for aquatic organism and on long term they are expected to cause negative effects on the aquatic environment (OSPAR, 2001). The group of PCAs exists of thousands of isomers and due to the complex character of the mixtures of PCAs only a few laboratory in the world are analysing PCAs. No GC technique is available to separate all individual isomers, but comprehensive two-dimensional GC combined with time-of-flight mass spectrometry (GCxGC-TOF MS) seems to be a promising technique to characterize the PCA groups. Limited methods are available for analysing and quantifying PCAs. This is due to several reasons. The first reason is that thousands of isomers do exist and it is 1

8 impossible to quantify each compound separately. A second reason is that no individual reference standards are available, only various types of mixtures. In this study a semi-quantitative analytical method for PCAs in environmental samples by use of GCxGC-ECNI-TOF mass spectrometry is developed. 1.2 Scope of the study The aim of the study is to develop an analytical method for determination of polychlorinated alkanes in environment samples, to separate the isomers and congeners in characteristic groups and to make a first attempt to set up a semi quantitative method. The first goal of the study is to further develop the existing method (Korytár et al., 2006), for the separation of PCAs, using technical mixtures and environmental samples. The second goal of the study is to optimise the extraction and the clean up method for environmental samples. PCAs should be separated from other interfering compounds for quantification. A preliminary quantification method for congener/isomer groups in the GCxGC contour plots is set up. The final goal of the study is to analyse environmental samples. In the second part of the study the focus is on the analysis of samples of fish, shrimps and mussels. Recently, differences in congener PCA patterns between fish from the North Sea and from the Baltic Sea (Reth et al., 2005) and difference in congener pattern between fish types have been found. 2

9 2. SEPARATION AND DETECTION METHOD Comprehensive two-dimensional gas chromatography with electron capture negative ionization (ECNI) time-of-flight mass spectrometry (GCxGC-ECNI- TOF MS) is used to study the composition and characteristics of short-, mediumand long-chain polychlorinated n-alkane (PCA) mixtures. In paragraph 2.1 comprehensive two-dimensional gas chromatography and in paragraph 2.2 timeof-flight mass spectrometry is discussed. 2.1 Comprehensive two-dimensional gas chromatography Conventional one-dimensional GC is a well-known separation technique. However, for separation of complex mixtures the technique is insufficient. It is impossible to separate a very large number of compounds with one-dimensional GC. The group of PCAs consists of thousands of isomers and one-dimensional GC is not sufficient for separation of PCAs. Comprehensive two-dimensional gas chromatography is a technique that is currently being used for a wide range of applications. Ventura et al. (2008) used GCxGC for analysis of unresolved complex mixtures of hydrocarbons extracted from Late Archean sediments, which were too complex to resolve by traditional capillary gas chromatography. Klimankova et al. (2008) used GCxGC coupled to time-of-flight mass spectrometry for profiling volatile compounds released from five Ocimum basilicum L. cultivars. Junge et al. (2007) used GCxGC for the chiral separation of amino acids derivatised with ethyl chloroformate. For PCA analyses comprehensive two-dimensional gas chromatography seems to be a promising method. The increase in peak capacity compared to one-dimensional GC makes it a much better separation technique for very complex samples. Another benefit of twodimensional GC is the structured separation of compounds in groups, resulting in improved identification Advantages of GCxGC Compared to conventional GC, two-dimensional GCxGC shows the advantage that the peak capacity is much higher than in conventional GC. Compounds will be more separated from each other, but also from interfering compounds (e.g. background matrix), which are present in the sample (Dallüge et al., 2003, Korytár, 2006). A second advantage of GCxGC is that due to the refocusing process in the cooling device, the peaks will be narrowed. Due to this refocusing effect and improved separation, sensitivity is improved. 3

10 A third advantage of GCxGC is the structured separation. Chemical related compounds will be ordered in a chromatogram. These structured chromatograms result in improved identification and make it possible to identify unknowns in a chromatogram by their chemical structure (Dallüge et al., 2003) Principles of GCxGC GCxGC is a separation technique based on two different separation mechanisms. Two columns with different stationary phases will be coupled in series. Between the columns a trapping and cooling device is often used, which is called the modulator. A schematic overview of a GCxGC system is shown in Figure 1. Figure 1. Schematic overview of a GCxGC system. I=injector, M=modulator, D=detector, 1st=GC-oven with first-dimension column, 2 nd =GC oven with second-dimension column (Figure taken from Dallüge et al., 2003). The first dimension separation is a conventional GC-separation, with a normal GC-column length in an oven that will be temperature programmed. The onedimensional (1D) chromatogram, which is obtained by the GCxGC separation, shows a chromatogram in which many second dimension chromatograms are displayed. These second dimension chromatograms can be transformed to a twodimensional (2D) chromatogram. This transformation is shown in Figure 2. In the two-dimensional chromatogram the x-as represents the retention time of the separation on the first column and de y-ax represents the retention time on the second column. Software programs are used to transform the 1D chromatogram into a 2D chromatogram. This 2D chromatogram is displayed as a contour plot with different colors representing the different intensities of the signal. The contour plot can be displayed as a 3D plot as well. 4

11 2.1.3 Modulator With GCxGC, compounds that are separated on the first column should be injected on the second column without loosing the separation from the first column. To manage this, separate fractions from the first column should be collected and injected individually on the second column. For this transfer from the first to the second column, a modulator is used. The modulator is a very important part of the GCxGC system and is placed between the first and the second column, see Figure 1. It is used to trap, refocus and inject the fraction from the first column on the second column, and after that to collect, refocus and inject the next fraction of the first column. Figure 2. Generation and visualization of a GCxGC chromatogram (Figure taken from Dallüge et al., 2003). 5

12 The separation on the second column is very fast and takes only a few seconds. The frequency of the switching of the modulator will be so high that the second column separation will be finished before the next fraction is injected on the second column. When the separation is not completed when the next fraction is injected, an effect, which is called wrap-around, will show up (see paragraph ). Different types of modulators are available. The sweeper is one of these and a valve modulator also is used. The last types of modulator are modulators based on a cryogenic principle (Dallüge et al., 2003). The Capillary Flow Technology (CFT) from Agilent is the latest type of modulator. The dual CO 2 jet cryogenic modulator is used in this study and will be explained in more detail Dual CO 2 jet modulator The dual CO 2 jet modulator consists of two CO 2 jets, placed over the first part of the second column. Expanding liquid CO 2 is coming out one of the jets, and after a few seconds (modulation time) the CO 2 spray is switched to the second jet. This process is repeated during the entire run. Figure 3. Schematic overview of a two-jet cryogenic modulator (Figure taken from Dallüge et al., 2003). 6

13 When CO 2 is sprayed from the first jet, the compounds that elute from the first column will be trapped due to the cooling effect of the CO 2. When the cooling is switched to the second jet, the trapped fraction will move on to the second jet and refocus. After switching back to the first jet, the compounds are injected on the second column. The first jet starts to cool and the next fraction out of the first column will be trapped. In this way the modulator takes care of fractionating the compounds of the first column and injecting the fractions separately on the second column (Dallüge et al., 2003). An advantage of this modulator is that there are no moving parts and the jet system is therefore more robust. A schematic overview of the general set-up of the dual CO 2 jet modulator is shown in Figure Wrap- around In GCxGC the separation of compounds on the second column only takes a few seconds. When the modulation time is shorter than the elution time of the compounds, compounds will elute in the next fraction. It is possible that peaks from different fractions coëlute in one modulation. This results in a less good separation and ordered structures will be lost. This effect of compound eluting in the next modulation is called wrap-around and should be avoided as much as possible by working with a modulation time longer than the retention time of the most retained peak in the second dimension (Dallüge et al., 2003) GCxGC separation of PCAs (State-of-the-art) Limited methods are available for analysing and quantifying PCAs. In Figure 4, a chromatogram is shown from a one- dimensional high resolution GC separation of PCA-60 (a technical mixture of short chain PCAs) (Tomy et al., 1997). Figure 4. High resolution GC/ECNI-MS total ion chromatogram (m/z ) of PCA-60 (Figure taken from Tomy et al., 1997). 7

14 Korytár et al. (2005) used GCxGC for analysis of PCAs with a column combination of a 30m x 0.25mm, 0.25 µm DB-1 column (100% methylsiloxane) in the first dimension and a 1.0 m x 0.1 mm x 0.1 µm HT column (65% phenyl-methylpolysiloxane) in the second dimension, because it provides most information in terms of ordered structures, i.e. of group and sub-group separation. In a GCxGC chromatogram of a PCA-mixture, compounds having the same chlorine substitution pattern but different carbon chain length are ordered as more or less parallel horizontal lines. This is an expected result because the compounds within each group have essentially the same polarity because their substitution pattern is the same and they only differ in their boiling points due to the different lengths of the carbon chain (see Figure 5). Figure 5. GC GC _ECD chromatograms of PCA-60. Column combination: 30 m x 0.25 mm x 0.25 m DB-1 X 1.0 m x 0.1 mm x 0.1 µm HT. The four discrete peaks/spots visible are due to the added internal standards. modulator (Figure taken from Korytár et al., 2005). On the other hand, there are obvious differences in polarity between the various groups. Compounds, which have substituents on only one end of the carbon chain, are less polar and thus have shorter second-dimension retention times than the compounds with chlorine substituents distributed over the entire length of the molecule when using the column combination 30m x 0.25mm, 0.25 µm DB-1 column in the first dimension and a 1.0 m x 0.1 mm x 0.1 µm HT column in the second dimension. A disadvantage is that there is some overlap with neighboring bands (Korytár, 2006). 8

15 The main observation is that with increasing length of the carbon skeleton, boiling points become higher and first dimension retention times consequently increase. A disadvantage is that, an extra chlorine atom is just as volatile as an extra carbon-atom (Korytár et al., 2006). So, in the first dimension, compounds with one carbon atom more and a chlorine atom less have the same retention time. 2.2 Time-Of-Flight Mass Spectrometry Different types of mass spectrometers exist: Quadrupole, triple quad, iontrap and time-of-flight mass spectrometers are well known. Often a TOF MS is used, because of the fast scan speed and low limit of detection. A high scan speed (50 Hz or higher) is needed for GCxGC analysis, as the second dimension peak widths are generally below 200 ms. Currently, fast scanning quadrupole MS systems are available. Generally these are able to scan a limited mass range with the obtained scanning speed (Adahchour et al., 2005). In this study a time-offlight analyser, which is described in paragraph 2.2.1, is used for the analysis of PCAs Time-Of-Flight Mass Spectrometer The time-of-flight mass spectrometer is one of the first existing mass spectrometers. The principle of TOF is based on the flight time of ions over a certain distance. Even though the time-of-flight mass spectrometer already existed for decades it was not used very often in the beginning, because it was not possible to measure the time-of-flight of the ions accurately. To be more precise in time measurement, the flight tube should be very long and so the mass spectrometer was a huge instrument. Nowadays it is possible to measure time very precise, even in nanoseconds. Because of this development, the time-of-flight mass spectrometer has a better resolution, is not very large anymore and is used very often for analysis Principle of the Time-Of-Flight Mass Spectrometer Figure 6 shows a schematic overview of the time-of-flight mass spectrometer of Thermo Finnigan (Tempus update), which is used for the analysis of PCAs in our study, as this is a TOF with a high scan speed and the option of using both electron impact and chemical ionization; the latter is the preferred ionization method for PCA analysis. At the time of our study no other TOF systems with these options were available. 9

16 Figure 6. Schematic overview of the tempus time-of-flight mass spectrometer of Thermo Finnigan, which is used for the PCA analysis in this study. The ions in a time-of-flight mass spectrometer are travelling a certain distance through a flight tube due to a difference in potential between the source and the detector. The mass spectrometer measures the time between the injection and the arrival of the ions at the detector and calculates from the travelling time the mass/charge ratio of the ion by the following equation (Hoffmann et al., 2002): m/z = t 2 * (2 V e/ d 2 ) m/z = mass/charge ratio of the ion t = travelling time of the ion V = Potential e = charge of an electron d = distance 10

17 The ions will be travelling faster if the mass is lower and they will be flying slower if the mass of the ions is higher. The time-of-flight mass spectrometer has some advantage compared to other mass spectrometers. The first advantage of the time-of-flight mass spectrometer is that the spectrometer has no upper mass limit, which makes it possible to measure large molecules. Another advantage of the time-of-flight mass spectrometer is the very high sensitivity. All the ions, which have been produced, will be analysed by the mass spectrometer and almost none will be lost. Unfortunately the time-of-flight mass spectrometer has also a disadvantage. It has a poor mass resolution due to a distribution in flight time between ions with the same mass/charge ratio. This distribution in flight time is caused by several parameters, e.g. the length of the ion pulse, the size of the volume where the ions are formed, the variation of the internal kinetic energy of the ions and the electronics for the time measurement. When a longer flight tube is used the mass resolution will increase because the flight time will be longer. A longer flight time results in a better mass resolution (Hoffmann et al., 2002) Reflectrons In the past the flight tube of a time-of-flight mass spectrometer was a very long tube. Nowadays a reflectron is used in the time-of-flight mass spectrometer. The reflectron makes the instrument smaller, but the main advantage of the reflectron is the gain in mass-resolution. The reflectron is correcting for the energy dispersion of the ions leaving the source. In Figure 7 a schematic overview of a time-of-flight mass spectrometer equipped with a reflectron is given. The reflectron is an electronic field that is acting like a mirror for the ions in the flight tube. When the ions enter the field, the flight direction will be reversed and they fly towards the detector. The ions with more kinetic energy are entering the field deeper and so it will take a longer time to reach the detector. Ions with a lower kinetic energy will enter the field less deep and so the flight time will be shortened. Due to this effect the ions with the same mass/charge ratio will arrive at the detector at the same time. 11

18 Figure 7. Schematic overview of a TOF instrument equipped with a reflectron ( ) ions of a given mass with correct kinetic energy; ( ) ions of the same mass but with a kinetic energy that is too low. The latter reach the reflectron later, but come out with the same kinetic energy as before. With properly chosen voltages, path lengths and fields, both kinds of ions reach the detector simultaneously (Figure taken from Hoffmann et al., 2002). Although the use of a reflectron has the big advantage of higher mass resolution, it also does have some disadvantages. Contrary to what was mentioned in paragraph , that the mass spectrometer did not have an upper mass range, when a reflectron is used a mass range limitation does appear. An other disadvantage of the use of a reflectron is the loss in sensitivity Chemical Ionization When the mass spectrometry technique is used, it is often performed with electron impact ionization (EI). On the other hand, for the analysis of PCAs chemical ionization (CI) is used instead of EI, because EI leads to a strong fragmentation of the molecular ion, whereas CI does not give much fragmentation. In spectra obtained with CI most often the molecular mass of the analysed compounds can be found. In CI an ionization gas is used. Ions are formed due to collision of the molecules with molecules of the ionization gas present in the source (Hoffmann et al., 2002). 12

19 Positive or Negative Chemical Ionization Almost all neutral substances are able to yield positive ions, whereas negative ions require the presence of acidic groups or electronegative. PCAs do have the electronegative chlorine atoms in the molecules, which makes negative chemical ionization for the detection and identification of PCAs the preferred method. The ionization gas is introduced into the source. The ionization gas moderates the energy of the electrons to thermal levels and, most organic compounds will then capture electrons to form negative molecular ions (Iowa State university, 2007). Ionization takes place in several general steps. In the first step: R + e - R - R = ionization gas In the second step collision takes place of the reagent ion with the compounds of interest (M), yielding analyte ions: R - + M M - + R In the third step the analytes may fragmentize by one or more pathways. The ionization step can be reached through many types of reactions, proton transfer, charge transfer, electron capture and adduct formation (Kimmel, 2006). In this study of PCAs ionization takes place by the electron capture principle TOF analysis of PCAs (State-of-the-art) In this study of PCAs a Time-off-flight mass spectrometer is used since a high scan speed is needed for GCxGC analysis. No information can be found on PCA analysis performed earlier with Time-off-flight-mass spectrometry except for the study of Korytár et al (2005). He performed analyses on standard solutions and on two dust samples of household origin, collected in central part of Spain and in northeast part of Slovakia. Analysis of the dust samples is performed qualitatively and no quantitative analyses have been performed. Electron capture negative ionization mass spectrometry has been used for PCA analysis (Pribylova et al., 2006; Nicholls et al., 2001; Reth et al., 2005). Molecular ions observed in negative ion chemical ionization mass spectra are usually M - or [M- H] - (Iowa State university, 2007). The appropriate ions for quantification of PCAs in the ECNI mode, which are always present in the mass spectra of PCA-mixtures are in the low m/z region [HCl 2 ] - and [Cl 2 ] -., according to Castells et al. (2004). 13

20 3. OPTIMIZATION OF GCxGC-TOF MS FOR PCA ANALYSIS 3.1 Introduction Korytár et al. (2006) tested in his study several column combinations for the analysis of PCAs with GCxGC. The results showed that the most promising column combination was a 30m x 0.25mm, 0.25 µm DB-1 column (100% methylsiloxane) in the first dimension and a 1.0 m x 0.1 mm x 0.1 µm HT column (65% phenyl-methylpolysiloxane) in the second dimension, because it provides most information in terms of ordered structures, i.e. of group and subgroup separation (see paragraph 2.1.4). In the first dimension, compounds with one carbon atom more and a chlorine atom less have the same retention time. In the second dimension the compounds with longer carbon chains have lower second-dimension retention times (Korytár, 2006). This carbon chain length selectivity helps to create a distinct separation of compounds, which differ by at least three carbons (Korytár, 2006). For a separation of compounds, which differ less than three carbons, optimization needs to be performed on the second dimension separation. Molecular ions observed in negative ion chemical ionization mass spectra are usually M - or [M- H] - (Iowa State university, 2007). The appropriate ions for quantification of PCAs in the NCI mode, which are always present in the mass spectra of PCA-mixtures are in the low m/z region [HCl 2 ] - and [Cl 2 ] -., according to Castells et al. (2004). The ionization gases used in ECNI may vary. To make a good choice which gas should be used the proton affinity and the energy transfer are important. Two examples of ionization gases used in this study are methane (CH 4 ) and ammonia (NH 3 ). In chemical ionization ammonia is the most used ionization gas because of the low energy transfer of ammonia compared to methane (Iowa State university, 2007). It is not possible to use ammonia as ionization gas to analyse alkanes, aromatic, ethers and nitrogen compounds other then amines, because they will almost not be ionised. Polar molecules and those able to form hydrogen bonds and having no or little basic character will form adducts (Hoffmann et al., 2002) Objective The objective in the present study was to optimize the GCxGC-TOF MS system for analysis of PCAs by: Improvement of the separation of PCA-groups. Optimization of the modulator. Determination of the mass spectra of PCAs. Optimization of the ionization gas. 14

21 3.2 Experimental In this study, the analysis of PCAs is performed by comprehensive twodimensional gas chromatography with electron capture negative ionization timeof-flight mass spectrometry. The used GC was a Trace GCxGC (Thermo Finnigan) and the mass spectrometer used was a Tempus Update Time-of-flight mass spectrometer (Thermo Finnigan). Separation at the start of the study was performed on a 30 m x 0.25 mm x 0.25 µm DB-1 column (Agilent J&W, Amstelveen, Netherlands) in the first dimension and on a 1.0 m x 0.1 mm x 0.1 µm HT column (Quadrex, New Haven, CT, USA) in the second dimension. The GCxGC method used in the beginning of this study was the same as described by Korytár et al. (2006). The carrier gas was helium at a flow of 1.2 ml/min. The temperature programme of the GC started at 65 C for 2 min, then the GC oven was heated to 140 C at a rate of 20 C/min, followed by an increase of 3 C/min to 320 C and held at this temperature for 5 min. Injection volume of the samples was 1 µl into a PTV inlet port operated in the constant-temperature splitless mode at 280 C, with the split opened for 2 min. The modulator used in this study was a two-jet cryogenic modulator as described in paragraph The MS source temperature was 160 C and the interface temperature was 160 C. Methane was used as ionization gas at the beginning of this study at a flow of 3.0 ml/min and the temperature of the GC-MS transfer line was set at 320 C. The acquired mass range was Da at a data acquisition rate of 40 Hz. The software used was Xcalibur software (Thermo Electron) for controlling the GCxGC-TOF MS and to acquire data. For 1-dimensional chromatograms XCalibur was used, for GCxGC data analysis and visualization in 2-dimensional chromatograms, raw data files were imported into HyperChrom Software (Thermo Electron) Optimization of the separation of PCAs In this experiment the differences in separation of PCAs between a 1.0 m x 0.1 mm x 0.1 µm HT column, and a 3.0 m x 0.1 mm x 0.1 µm HT column in the second dimension was studied. As first dimension column a DB-1 was used. The modulation time is increased to 12 seconds, to avoid wrap around since the second dimension retention times are longer. With the used instrument, it was not possible to use a modulation time higher than 12 seconds. 15

22 3.2.2 Optimization of the modulator In this study a dual CO 2 jet cryogenic modulator is used, as discussed in paragraph The alignment and the performance of the CO2 jets were studied Mass spectra PCAs Mass spectra of the different PCA groups of a PCA-60 (145 µg/ml in iso-octane) mixture were determined with the GCxGC-TOF MS system in ECNI-mode. The appropriate m/z range for extracted ion chromatograms for PCA analysis is determined by determining the most abundant m/z range from the mass spectra of the PCA-60 mixture Ionization gas A comparison is made between the use of methane and the use of ammonia as ionization gas for negative chemical ionization. The signal intensity, the background noise, the stability and the mass spectra are investigated. TOF-MS settings are as described in paragraph Results & Discussion Optimization of the separation of PCAs Figure 8 shows the separation of PCA-60 (145 µg/ml), a technical mixture of short chain PCAs, with a 30m x 0.25mm, 0.25 µm DB-1 column in the first dimension and a 1.0 m x 0.1 mm x 0.1 µm HT column in the second dimension. Figure 9 shows the separation of PCA-60 (145 µg/ml), with a 3.0 m x 0.1 mm x 0.1 µm HT in the second dimension. 16

23 Figure 8. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a PCA-60 solution (145 µg/ml), obtained with a 30 m x 0.25 mmx 0.25 µm DB-1 column in the first dimension and a 1.0 m x 0.1 mm x 0.1 µm HT column in the second dimension. The numbers indicate number of (carbon + chlorine) atoms of the compounds present in the bands. 15: C 10 Cl 5 -C 12 Cl 3 ; 16: C 10 Cl 6 -C 13 Cl 3 ; 17: C 10 Cl 7 -C 13 Cl 4 ; 18: C 10 Cl 8 - C 13 Cl 5 ; 19: C 10 Cl 9 -C 13 Cl 6 ; 20: C 10 Cl 10 -C 13 Cl 7 ; 21: C 10 Cl 11 -C 13 Cl 8. Comparing the results of both columns, the longer column gives clearly more separation. The group separation, which is observed, is based on the number of atoms (carbon and chlorine) in the compounds, which is shown in Figure 8 and Figure 9. The group bands are longer in the second dimension and due to that they are smaller in the first dimension. The groups are all more separated from each other. On top of the bands, smaller groups can be observed. These groups are more separated by the 3 meter column in the second dimension. Why these groups are separated is not jet known. One possibility is that there is a lack of some compounds in the PCA-60 solution that would fill up the space between the larger en the smaller group on top of it. An other possibility is that the separation is due to structural difference. In the later case, it is useful to have the smaller group completely separated from the larger group. Identification of the 17

24 small groups should be performed by NMR to draw any conclusion about the structure of the compounds in the smaller groups and if the structural difference is the reason for the separation. Figure 9. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a PCA-60 solution (145 µg/ml), obtained with a 30 m x 0.25 mmx 0.25 µm DB-1 column in the first dimension ande3 a 3.0 m x 0.1 mm x 0.1 µm HT column in the second dimension. The numbers indicate number of (carbon + chlorine) atoms of the compounds present in the bands (see figure 8) Optimization of the modulator In this study a dual CO 2 jet cryogenic modulator is used, as discussed in paragraph The critical factor with this type of cooling device is the positioning of the CO 2 jets over the column. When the sprays out of the CO 2 jets were inline with the column, the cooling could be too much, so that the compounds from the first column are trapped too long on the second column, resulting in chromatograms like Figure 10. When the sprays of the CO 2 jets were too less inline with the column, the cooling would not be enough and the 18

25 compounds are not trapped enough for injection on the second column, resulting in chromatograms like Figure 10. Figure 10. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a PCA-60 solution with the cooling device of the modulator cooling to much or to less. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. The temperature of the CO 2 cylinder is an important aspect for a proper working of the modulator. It had to be on room temperature before the CO 2 gas could be used. When the temperature of the cylinder was to low, the gas pressure of the CO 2 in the bottle would be to low, and no gas, or a small amount of gas would come out of the CO 2 jets of the modulator. Compounds eluting from the first column will than not be trapped enough for injection on the second column, which results in the same type of chromatogram as shown in Figure 10. When using CO 2 the pressure of the CO 2 in the bottle will decrease. Due to this, in the beginning of a sample sequence, the cooling device can work properly, resulting in good chromatograms, while after a number of samples the cooling will not be sufficient anymore. 19

26 The CO 2 jets used have several holes, placed in line, out of which the CO2 is spraying. The holes are very narrow and can easily be clocked. When some of the holes in the middle are clocked the compounds from the first column are trapped in two parts, resulting in a chromatogram as Figure 11. Figure 11. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a PCA-60 solution with some of the holes of the jet clocked. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT Mass spectra PCAs Figure 12 shows the spectrum (Rt minutes) of a PCA-60 solution obtained in this study with GCxGC-ECNI-TOF MS. As can be seen in Figure 12, the most abundant ions are at m/z 70, which correspond to [Cl 2 ] -. Other clusters that can be seen in Figure 12 are m/z , m/z and m/z corresponding to [M+ Cl] - of C 8 Cl 5 H 13, C 9 Cl 5 H 15, and C 13 Cl 4 H 24 and the clusters m/z , m/z , m/z and m/z , corresponding to [M-CL] - of C 11 Cl 7 H 17, C 12 Cl 7 H 19, C 11 Cl 8 H 16 and C 12 Cl 8 H

27 The extracted ion chromatograms for PCAs shown in the rest of this study are m/z PCA_60_145µGMLBDE127CB40_ # RT: AV: NL: 3.15E3 T: {0,0} - c CI Full [ ] Relative Abundance m/z Figure 12. GCxGC-ECNI-TOF MS mass spectrum of a PCA-60 solution. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 1.0 m x 0.1 mm x 0.1 µm HT Ionization gas for PCA analysis In the beginning of the study of PCA analysis, methane has been used in the negative chemical ionization mode. The TOF MS instrument from Thermo Finnigan had some disadvantages in negative chemical ionization mode. A disadvantage of the instrument is that it has no automatic tuning for chemical ionization. To get the instrument tuned well is very time consuming. With the use of methane, only a few injections could be performed before the signal decreased dramatically. Figure 13 shows a chromatogram (m/z 68-72) of PCA-60, with the use of methane as ionization gas, injected at a time the instrument performed well. Figure 14 shows a chromatogram (m/z 68-72) of the injection of the same PCA- 21

28 RT: Intensity Time (min) NL: 5.00E5 m/z= F: MS PCA60_29 0_µgml9 Figure 13. GCxGC-ECNI-TOF MS 1-dimensional chromatogram (m/z 68-72) of a PCA-60 solution, obtained when the instrument was performing well, using methane as ionization gas. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 1.0 m x 0.1 mm x 0.1 µm HT. RT: Intensity Time (min) NL: 5.00E5 m/z= F: MS PCA60_29 0_µgml10 Figure 14. GCxGC-ECNI-TOF MS 1-dimensional chromatogram (m/z 68-72) of a PCA-60 solution, obtained directly after the run of the chromatogram of Figure 13. Methane was used as ionization gas. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 1.0 m x 0.1 mm x 0.1 µm HT.

29 60 solution, directly obtained after the run of the injection of Figure 13. Both chromatograms are shown on the same absolute scale. It can be seen that the signal decreased dramatically. The ion source than had to be cleaned and tuned again before the work could be continued. In this study a lot off time was spend on cleaning the ion source, proper tuning of the MS and, varying gas flows of methane to optimise the signal. The use of ammonia as ionization gas resulted in a more stable signal. The ion source didn t need so much cleaning anymore and more samples could be run in a row. Amendola et al. (2002) showed that the use of pure ammonia leads to a marked reduction of the background noise, with parallel improvement of the overall sensitivity. This is also observed in the present study. Tuning the instrument with the calibration gas (heptacosafluorotributlamine) with methane as ionization gas gave an overall signal between 3000 and 4000, where a signal of could be obtained by using ammonium as ionization gas, so the sensitivity increased by at least a factor 2 by using ammonia instead of methane, which is a big advantage of using ammonia. Amendola et al. (2002) found for pesticides significant differences in the mass spectra by changing the ionization gas. Also the chemical facility Iowa State University (2007) found that the ionization gas influences the amount of fragmentation and the prominence of the protonated molecular ion detected. In the study of PCAs no differences in the mass fragmentation of PCA-60 can be found between methane and ammonia as shown in Figure 15 and Figure 16. Figure 15 and Figure 16 show the spectra of PCA-60 with ionization gas methane and ammonia at retention time (Rt) min. As can be seen in Figure 15A and in Figure 16A comparable mass spectra were found between methane and ammonia ionization. In Figure 15B and in Figure 16B m/z is displayed. These spectra also look similar. Most abundant peaks with methane and ammonia are: m/z 311/313 from [M-HCl].- of C 10 H 16 Cl6; m/z 325/327 from [M-HCl].- of C 11 H 18 Cl 6 ; m/z 339/341 from [M-HCl].- of C 12 H 20 Cl 6 ; m/z 361/363 from [M].- of C 11 H 18 Cl 6 and from [M-HCl].- of C 11 H 17 Cl 7 ; m/z 375/377 from [M- Cl] - of C 12 H 19 Cl 7 ; m/z 395/397 from [M-Cl] - of C 11 H 16 C 8 and m/z 409/411 from [M-Cl] - of C 12 H 18 Cl 8. In conclusion, in the study of PCAs no differences in the mass fragmentation of PCA-60 can be found between methane and ammonia as ionization gas. 23

30 A 112 PCA_60_290µgml_ # RT: AV: NL: 4.88E3 T: {0,0} - c CI Full [ ] Relative Abundance m/z B 112 PCA_60_290µgml_ # RT: AV: NL: 1.95E2 T: {0,0} - c CI Full [ ] Relative Abundance m/z Figure 15. GCxGC-ECNI-TOF MS spectra of a PCA-60 solution by use of methane as ionization gas. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. A) m/z B) m/z

31 A PCA_60_145µGMLBDE127CB40_ # RT: AV: NL: 3.44E3 T: {0,0} - c CI Full [ ] Relative Abundance m/z B PCA_60_145µGMLBDE127CB40_ # RT: AV: NL: 1.71E2 T: {0,0} - c CI Full [ ] Rel60 ativ e 55 Ab und50 anc e m/z Figure 16. GCxGC-ECNI-TOF MS spectra of a PCA-60 solution by use of ammonia as ionization gas. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. A) m/z B) m/z

32 Figure 17 shows the GCxGC contour plot (m/z 68-72) of the mixture of PCA-60 (290 µg/ml) by use of methane as ionization gas and Figure 18 shows the GCxGC contour plot of the mixture of PCA-60 (145 µg/ml) with ammonia as ionization gas. Comparing the plot of methane as ionization gas and the plot of ammonia as ionization gas, the higher sensitivity with ammonia is observed. Figure 17. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a PCA-60 solution (290 µg/ml) by use of methane as ionization gas. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 26

33 Figure 18. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a PCA-60 solution (145 µg/ml) by use of ammonia as ionization gas. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 3.4 Conclusions For the analysis of PCA s with GCxGC-ENCI-TOF-MS the column combination with a 30m x 0.25mm, 0.25 µm DB-1 column in the first dimension and a 3.0 m x 0.1 mm x 0.1 µm HT column in the second dimension showed a improved separation compared to the column combination with a 30m x 0.25mm, 0.25 µm DB-1 column in the first dimension and a 1.0 m x 0.1 mm x 0.1 µm HT column in the second dimension. In the study for PCA analysis a dual CO2 jet cryogenic modulator is used. For this device the following aspects are very critical: the positioning of the jets over the column, a constant temperature of the CO2 bottle and to avoid clocking of the jets. In the TOF MS spectra of a PCA mixture the most abbunded peaks are m/z 68-27

34 72. Because the most abbunded peaks in the TOF MS spectra of a PCA mixture are in the range m/z 68-72, the extracted mass range for the chromatograms of the PCA analysis in this study is m/z In ENCI mass spectrometry ammonia should be used as ionization gas instead of methane. A more stable and intense signal is obtained and the ion source remains cleaner when using ammonia as ionization gas and for the analysis of PCAs. The mass spectra obtained with ammonia and obtained with methane as ionization gas are comparable. 28

35 4. ANALYSIS OF SHORT, MEDIUM AND LONG CHAIN PCAS 4.1 Introduction PCAs can be characterized by their alkane-chain lengths and they can be divided in three groups: short chain (C 10 -C 13 ), medium chain (C 14 -C 17 ) and long chain (C >17 ) PCAs. PCA-60 is a mixture of short-chain PCAs. Compounds having the same chlorine substitution pattern but different carbon chain length are ordered as more or less parallel horizontal lines (Korytár, 2006). Separation in the first dimension is based on boiling point. Compounds within each group have essentially the same polarity because their substitution pattern is the same and they only differ in boiling points due to the different lengths of the carbon chain. On the other hand, there are obvious differences in polarity between the various groups. Compounds which have substituents on only one end of the carbon chain are less polar and, thus, have a shorter second-dimension retention time than the compounds with chlorine substituents distributed over the length of the molecule (Korytár, 2006) Objective The first objective in the present study was to analyse short, medium and long chain PCAs mixtures with GCxGC-TOF MS with a 3.0 meter column in the second dimension. The second objective was to analyse a number of individual PCAs, firstly to determine their retention time and to study what the influence of carbon chain length and chlorine content and chlorine substituents on the first and second retention times are. 4.2 Experimental In this study, the analysis of PCAs is performed by comprehensive twodimensional gas chromatography with electron capture negative ionization timeof-flight mass spectrometry as described in paragraph 3.2. The column used in the second dimension is a 3.0 m x 0.1 mm x 0.1 µm HT column. Ammonia was used as ionization gas Analysis of mixtures of PCAs Several mixtures of short, medium and long chain PCA s were analysed to investigate the retention times in relation to chlorine content and carbon length of the PCAs. 29

36 The following mixtures of PCAs were studied: C 10 -C 13 with 51.5 % chlorine (i.e. short chain) (100 µg/ml in iso-octane) C 10 -C 13 with 55.5 % chlorine (i.e. short chain) (100 µg/ml in iso-octane) C 10 -C 13 with 63 % chlorine (i.e. short chain) (100 µg/ml in iso-octane) C 14 -C 17 with 42 % chlorine (i.e. medium chain) (100 µg/ml in iso-octane) C 14 -C 17 with 57 % chlorine (i.e. medium chain) (100 µg/ml in iso-octane) C 18 -C 20 with 30 % chlorine (i.e. long chain) (100 µg/ml in iso-octane) Measurement of individual PCAs In Table 1 the individual PCAs (10 µg/ml), analysed by GCxGC-ECNI-TOF MS, are given. Table 1: PCAs, which are measured individually by GCxGC-ECNI-TOF MS. Name abbreviation Formula 1,1,1,3- tetrachlorooctane TCO C 8 H 14 Cl 4 1,2- dichlorotetradecane DCTD C 14 H 28 Cl 2 1,2- dichlorotridecane DCTrD C 13 H 26 Cl 2 1,2- dichlorododecane DCDD 2 C 12 H 24 Cl 2 1,12- dichlorododecane DCDD 12 C 12 H 24 Cl 2 1,2- dichloroundecane DCUD C 11 H 22 Cl 2 1,2- dichlorodecane DCD C 10 H 20 Cl 2 1,1,1,3- tetrachlorotetradecane TCTD C 14 H 26 Cl 4 1,1,1,3- tetrachlorotridecane TCTrD C 13 H 24 Cl 4 1,1,1,3- tetrachlorododecane TCDD C 12 H 22 Cl 4 1,1,1,3- tetrachloroundecane TCUD C 11 H 20 Cl 4 1,1,1,3- tetrachlorodecane TCD C 10 H 18 Cl 4 1,1,1,3- tetrachlorononane TCN C 9 H 16 Cl Results & Discussion Analysis of mixtures of PCAs GCxGC-ECNI-TOF MS chromatograms (m/z 68-72) of each mixture are shown in appendix 2. As expected PCAs of the mixture of C 18 -C 20 (appendix 2f) do 30

37 have a much longer retention time in the first dimension than those in the mixtures of C 14 -C 17 (appendix 2d and 2e), which have a longer retention time than the PCAs in the mixtures of C 10 -C 13 (appendix 2a, 2b and 2c). This is due to the longer carbon chain of the PCAs in the mixtures of C 18 -C 20 compared to those in the mixture of C 14 -C 17 and of the longer carbon chain length of the PCAs in the mixture of C 14 -C 17 compared to those in the mixtures of C 10 -C 13. When the carbon chain is longer, the boiling point will be higher and so the retention time in the first dimension will be longer Chlorine content of C 10-C 13 mixtures Comparing the chromatograms (m/z 68-72) of the three mixtures C 10-C 13 (appendix 2a, 2b and 2c) more PCA groups can be found at shorter retention time in the mixture with 51,5 % chlorine and more PCA groups can be found at higher retention time in the mixture with 63 % chlorine. According to Korytár et al. (2006), this was due to the fact that more isomers are present in the mixture by higher chlorine contents than by lower chlorine contents. To prove this, chromatograms of three different m/z, from [M].- of C 10 H 18 Cl 4, from [M-Cl] - of C 12 H 20 Cl 6 and 375 from [M].- of C 12 H 20 Cl 6, were extracted. For these m/z values in all three chromatograms the peaks were found at the exact same retention time in the first dimension. However, for m/z 339 and m/z 375 a number of other peaks appear in the chromatogram of the C 10-C % mixture, which do not appear in the chromatograms of the other two mixtures. This can be due to the higher chlorine content. The mixture of 63% chlorine contains more isomers of C 12 H 20 Cl 6, than the other two mixtures Chlorine content of C 14-C 17 mixtures. Comparing the chromatograms (m/z 68-72) of the two mixtures C 14-C 17 (appendix 2d and 2e) the same observation is found as for the mixtures of C 10- C 13 (appendix 2a, 2b and 2c). The mixture with the lower chlorine content shows more PCA groups at lower first dimension retention time and the mixture with the higher chlorine content shows more PCA groups at higher first dimension retention time. This is probable due to the fact that more isomers are present in the mixture by higher chlorine contents than by lower chlorine contents Comparing mixtures of different chain lengths When comparing the C 10-C % mixture (appendix 2b) with the C 14-C 17 57% mixture (appendix 2e), the first dimension retention time is longer for the 31

38 C 14-C 17 mixture. This is due to the carbon chain length, which gives the compound a higher boiling point and thus a higher first dimension retention time. When comparing the C 14 -C % mixture with the C 18 -C % mixture (appendix 2f), the first dimension retention time of C 18-C 20 is longer than that of C 14-C 17 as was expected. In the chromatograms (m/z 68-72) of the individual mixtures and of the mix of mixtures, several groups can be observed. Korytár et al. (2006) already identified the groups of PCAs. The groups are more separated from each other and longer in the second dimension in the present study due to the use of a 3.0 m x 0.1 mm x 0.1 µm HT column instead of a 1.0 m x 0.1 mm x 0.1 µm HT column in the second dimension. By using a longer column in the second dimension, the groups are more separated from each other, but the order of appearance will not be changed. A chromatogram (m/z 68-72) of a mixture of all the tested mixtures mentioned above is shown in appendix 3. The individual mixtures do overlap in this chromatogram, but the PCA groups are still separated as can be observed in the chromatogram Measurement of individual PCAs Although concentrations of 10 µg/ml of the individual PCAs were analysed, no peaks at any mass were found in the chromatograms. The ion abundances are proportional to the number of chlorine atoms on the alkane skeleton, but molecules with less than 5 chlorine atoms are not readily ionised in ECNI mode (Moore et al., 2004). All the measured compounds do have less than 5 chlorine atoms, so it is possible that no masses were observed due to the lack of ionization. Because no peaks were found, no calculations can be performed on intensities of the signals. Other individual PCAs with a higher chlorine content should be measured to determine their retention time to study what the influence of carbon chain length and chlorine content and chlorine substituents on the first and second retention times are, and to study the relationship between response factors and chlorine contents and carbon chain lengths. Standards of individual PCAs, containing 6 or 8 chlorine atoms in the molecule, are now commercially available. Analysis of these individual standards is not performed due to a lack of time. 32

39 4.4 Conclusions PCAs with longer carbon chains have longer retention times in the first dimension. The use of a 3.0 m x 0.1 mm x 0.1 µm HT column instead of a 1.0 m x 0.1 mm x 0.1 µm HT column in the second dimension resulted in a improved separation of PCA groups in the chromatograms of the mixtures of PCAs in the second dimension and in a longer second dimension retention time. No calculations could be performed on intensities of the signals of individual PCAs, due to a low response, which was a result of the low chlorine content of the studied PCAs. 33

40 5. SEMI-QUANTITATIVE MEASUREMENT OF PCAS 5.1 Introduction Because of the toxicity of PCAs and the expected carcinogenity to humans, it is important to be able to quantify PCAs in environmental samples. Due to the fact that thousands of isomers, enantiomers and diastereomers do exist in the environment it is not possible to quantify each compound separately. Until now PCAs have been quantified and calculated by the total amount of PCAs in a sample compared to the total amount of PCAs in a standard of a mixture of PCAs (Tomy et al., 1997; Reth et al., 2005). Quantification of PCAs would be more easy if the mass spectrometer signal intensities do not vary by carbon chain length differences and do not vary by different chlorine content or substitution. If the intensities of the signals are equal, the area of all peaks of a group could be integrated as a whole and calculated with a standard of a mixture of any type of PCA when the overall concentration of the mixture is known. However, this ideal situation is not the case in practice. Moore et al. (2004) already found that the ion abundances are proportional to the number of chlorine atoms on the alkane skeleton. When intensities of the mass spectrometer signal do vary with carbon chain length and also vary with chlorine content and substitution, it is important to find the relationship between these parameters in order to make a quantitative method based on the group separation. An other possibility is that all isomers of one carbon chain length and chlorine content would have more or less the same sensitivity. In this case it would be important to have standards of mixtures available of known concentration, only from one group. Quantification can then be performed by summarizing all off the peak areas of one group. One drawback of this method is that groups in a chromatogram not simply contain one carbon length or an equal amount of chlorine. Quantification on m/z is not possible and, it would be necessary to quantify on the [M-Cl] - and [M-HCl].-, which are less sensitive ions Objective The objective in the present study was to developed a semi quantitative analysis method for PCAs by GCxGC-ECNI-TOF MS. The relationship between the abundance of the signal of individual PCAs compared to chlorine contents and carbon chain length was investigated. 34

41 5.2 Experimental To set up a semi-quantitative method for PCA analysis, the intensity of the signal of individual compounds is investigated, compared to chlorine contents and carbon chain length. Individual PCA standards from Table 1 were analysed in duplo at a concentration of 7.5 µg/ml in iso-octane and in duplo at a concentration of 10 µg/ml in iso-octane, with comprehensive two- dimensional gas chromatography with electron capture negative ionization time-of-flight mass spectrometry as described in paragraph 3.2. The column used in the second dimension is a 3.0 m x 0.1 mm x 0.1 µm HT column. Ammonia was used as ionization gas. As internal injection standard CB 112 was used with a concentration of 340 ng/ml. 5.3 Results & Discussion In the chromatograms of the individually analysed PCAs, CB 112 was found with m/z at a retention time of 35.7 minutes. In all chromatograms except those of DCTRD and of TCO a small peak was found. The areas of CB 112 are too low to be quantified. Unfortunately no other peaks than CB 112 were found in the chromatograms, even though concentrations of 10 µg/ml of the individual PCAs were injected, as discussed in paragraph 4.3.2, except in the chromatograms of TCO. At Rt 17.6 minutes a peak is found with m/z 223 and m/z These masses, however, cannot be explained to be a fragment of TCO. 5.4 Conclusions Because no peaks were found in most of the chromatograms of the individual PCAs analysed, no information can be obtained about the relationship of sensitivity and carbon chain length and chlorine content and substitution. 35

42 6. CLEAN UP METHOD FOR PCA ANALYSIS 6.1 Introduction For the extraction of PCAs from biota samples a Soxhlet extraction with hexane/ acetone (3:1 v/v) is used. With this type of extraction lipids are also co-extracted, which have to be removed before extracts can be analysed with GCXGC TOF MS. Another problem is that with the PCA analysis using ECNI-TOF MS a number of other chlorinated compounds (e.g. PCBs) with similar m/z values can interfere. The objective of this study was to test different types of clean up methods to remove lipids, for the analysis of short chain PCAs; Gel Permeation Chromatography (GPC), sulphuric acid and acidic silica gel. The second objective was to test a silica gel fractionation for removing other chlorinated compounds then PCAs to avoid interference of these compounds by analysis of PCAs by ENCI-TOF. 6.2 Experimental To remove lipids from the extracts different types of clean up methods were tested for the analysis of short chain PCAs; Gel Permeation Chromatography (GPC), silica gel, sulphuric acid and acidic silica gel. Analysis are performed by comprehensive two- dimensional gas chromatography with electron capture negative ionization time-of-flight mass spectrometry as described in paragraph 3.2. The column used in the second dimension is a 3.0 m x 0.1 mm x 0.1 µm HT column. Ammonia was used as ionization gas Gel Permeation Chromatography For the clean up with GPC a PL gel 600 mm X 20 mm column is used, with a mobile phase of dichloromethane (10 ml/min). Two ml of an extract in dichloromethane is injected using a Gilson autosampler. The GPC clean up is tested with 2 ml of a PCA-60 solution (265 µg/ml). CB 112 and BDE 127 are added as internal standards. Fourteen fractions have been collected after separation on the GPC column to determine the elution pattern of the PCAs. In Table 2 the collection time of each fraction is given. The fractions 4 and 5 where analysed on the GCxGC-TOF mass spectrometer to investigate if PCAs are present in the lipid fraction, which is between 14 and 17.5 minutes. 36

43 Table 2: Collected fractions with GPC. Fraction no. Time (min) Fraction no. Time (min) Sulphuric acid For removing lipids from extracts of biota, concentrated sulphuric acid can be used. To test if sulphuric acid can be used for cleaning extracts for PCA analysis, PCA-60 (2 ml of 30 µg/ml) in iso-octane is tested by adding 2 ml of sulphuric acid. The sulphuric acid and the iso-octane are mixed by vortex (1 min). After mixing, the solution is cooled in the refrigerator for a night. The upper layer was removed and concentrated by turbo evaporator (Zymark, Turbovap LV) and analysed by GCxGC-TOF MS analysis Acidic silica Acidic silica was prepared by adding 100 gram of silica to 78.5 gram of concentrated sulphuric acid. For the first test, the PCA elution profile was determined. A glass column filled with 30 grams of the prepared acidic silica was washed with hexane. A PCA-60 solution (0.5 ml, 280 µg/ml) was dissolved in 25 ml of hexane and brought on to the column. Elution was performed with 250 ml 20 % (v/v) dichloromethane in hexane. Six fractions were collected with the first two having a volume of 25 ml and the other fractions having a volume of 50 ml. Fractions were analysed with GC-ECNI-TOF MS Silica gel A glass column filled with 2.2 gram silica, deactivated with 1,5 % H 2 O, slurried in iso-octane was prepared. Of the technical mixture PCA-60 (30 µg/ml) 2 ml in iso-octane is added to the column. Pre-eluted is performed with 1.5 ml isooctane. Next, the column is eluted with 10 ml iso-octane, which is collected as fraction 1. Finally the column is eluted with 11 ml of 15 % diethyl ether in 37

44 isooctane, fraction 2. The fractions are concentrated in a turbo evaporator (Zymark, Turbovap LV) to 0.5 ml, and analysed by GCXGC-TOF MS. 6.3 Results & Discussion Gel Permeation Chromatography The obtained chromatograms (m/z 68-72) of fraction 4 ( min), and fraction 5 ( min) are shown in Figure 19 and in Figure 20. In both fractions PCAs were found. In fraction 4 only the short chain PCAs, with carbon plus chlorine atoms higher then 18, were found (Figure 19), but in fraction 5 all of the short chain PCAs were present (Figure 20). This experiment showed that PCAs start to elute between 14 and 16.5 minutes. When extracts of biota samples are cleaned by GPC, lipids also elute between 14.0 and 16.5 minutes, which makes this method insufficient as clean up for PCA analysis. Other fractions were not analysed because the results of fraction 4 and 5 already show that the GPC method is insufficient for PCA analysis to remove lipids. Figure 19. GPC clean up of 2 ml of PCA-60 (265 µg/ml) mixture, fraction minutes. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 38

45 Although the GPC method tested in this study was not sufficient, GPC was used by Tomy et al. (1997) to remove lipids from biota samples for PCA analysis. He reported recoveries of 54 to 88 % over the whole extraction and clean up method. The column used was a 29.5 mm i.d. x 400 mm packed with 60 gram of mesh S-XS BioBeads soaked overnight in hexane/dichloromethane (1:1 v/v). Elution was performed with 325 ml hexane/dichloromethane (1:1 v/v) with the lipids eluting in the first 150 ml. Figure 20. GPC clean up of 2 ml of PCA-60 (265 µg/ml) mixture, fraction minutes. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT Sulphuric acid Figure 21 shows the GCxGC contour plot (m/z 68-72) of this fraction. The PCA- 60 pattern can be recognized, however the intensity is very low. Some of the PCA groups are missing due to the sulphuric acid treatment or to the low intensity. First results show that sulphuric acid is not the best clean up method. 39

46 Figure 21. Sulphuric acid clean up of 2 ml of PCA-60 solution (30 µg/ml). Column combination: 30 m x 0.25 mm x 0.25 m DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT Acidic silica In the first 3 fractions PCAs were found. In fraction 4 no PCAs were present. Elution with 100 ml hexane/dcm (80:20 v/v) seems to be enough to collect all short chain PCAs. For later test 150 ml is used. A second test has been performed to determine the recovery of i) a PCA-60 solution (0.5 ml, 280 µg/ml), ii) some individual PCAs (1 ml, 10 µg/ml), and iii) PCAs spiked to 0.5 gram fish oil. The individual PCAs which are tested are, 1,1,1,3 tetrachlorotetradecane (TCTD), 1,1,1,3,9,10,hexachlorodecane (HCD) and 1,1,1,3,12,14,14,14 octachlorotetradecane (OCTD). All the solutions were weighted and after the silica clean up BDE 127 and CB 112 were added as internal injection standards. 40

47 Recoveries were calculated as follows: Based on the integrated areas of the PCAs and of the internal standards in the GCxGC chromatograms a relative response factor (RRF) is calculated as followed: RRF = (A i * C IS ) (A IS * Ci ) i = individual PCA or group of PCAs RRF i = relative response factor of PCAi A i = Peak area of PCAi C i = Concentration of PCAi in µg/ml C IS = Concentration of the internal standard in µg/ml AIS = Peak area of the internal standard The recovery can be calculated with the RRF of the PCAs with acidic silica clean up (RRF s ) compared to the RRF of the PCAs without the acidic silica clean up (RRF ref ) with the following formula: Rec = (RRF s ) * 100 % (RRF ref ) Rec = Recovery of the PCA after silica clean up (%) RRF s = Relative response factor of the PCA with acidic silica clean up RRF ref = Relative response factor of the PCA without acidic silica clean up All the areas and RRFs for the PCAs tested for acidic silica clean up are listed in appendix 1. A summary of the recoveries is given in Table 3. Table 3: Recoveries (%) calculated for acidic silica clean up of PCAs compared to BDE 127 or to CB 112. PCA m/z Int. std. BDE 127 PCA solution 41 Int. std. CB 112 Spiked fish oil Int. std. BDE 127 Int. std. CB 112 PCA * 465* 231* 760* HCD TCTD OCTD *= Not reliable No peak found Not calculated Not calculated Not calculated Not calculated

48 The recoveries found for PCA-60 are not reliable, because the internal standard CB 112, which is added, contains chlorine as well and will have a peak in the mass range The retention time of CB 112 is within the integrated time range for the PCAs. This makes that the area of the PCA-60 is summarized with the area of the internal standard CB 112 (m/z 68-72). The molecular mass of HCD is (with Cl 35 - Cl 37). At Rt 33.6 minutes a peak was found for m/z i.e. two chlorine atoms. Also a peak was found for m/z at the same retention time. This m/z is the fragment of HCD minus a chlorine atom [M-Cl] -. Recoveries calculated for both m/z are acceptable (71-92%). The molecular mass of TCTD is No peak is found in the chromatogram corresponding with this mass or with a mass of 299 [M-Cl] -. At Rt 32.8 minutes a peak was found for m/z 206 which cannot yet logically be explained by mass fragments of TCTD. Since no other compounds where injected than TCTD and the two internal standards, which have completely different retention times, the m/z 206 should be a fragment of TCTD. In the TCTD extract, which was cleaned with acidic silica, a small peak was found for m/z at the same retention time as the retention time of the m/z 206 peak, while there was no peak found for the reference TCTD extract at this retention time for m/z Calculation of the recoveries of TCTD has been performed with m/z 206. The recoveries for TCTD are lower then the recovery for HCD. To have a good overview about recovery of TCTD the experiment with acidic silica clean up should be repeated. To be sure whether the low recoveries are from analysis on the ECNI-TOF MS, the measurements could be performed on a GC-quadrupole MS with ENCI. The standard solution of OCTD (5 µg/ml) gave no peaks for m/z or for any other m/z. Due to this no conclusions can be drawn whether the acidic silica clean up is acceptable for OCTD. It is not known whether the GCxGC-TOF MS is not sensitive to detect OCTD or if the concentration of OCTD was too low to be detected. For HCD and TCTD it is possible to use acidic silica clean up, where the found recoveries are all above 50 %. To have a more exact indication of the recoveries, more recovery experiments should be performed with acidic silica clean up. The analysis could be performed with a GC- quadrupole mass spectrometer. For OCTD it is not yet known whether the clean up with acidic silica is sufficient or not. Acidic silica clean is a promising method for clean up of extracts for PCA analysis, but more PCAs, including the medium and long chain, should be validated to decide whether the acidic silica clean up is a good clean up method for the whole range of PCAs. 42

49 6.3.4 Silica gel In fraction 1 no PCAs were found. In fraction 2 the pattern of the PCA-60 solution was found (Figure 22). All the short chain PCAs seem to elute in the second fraction, and silica can be useful to separate short chain PCAs from other interfering compounds like PCBs, which all are eluting in the first fraction. Figure 22. Silica clean up of PCA-60 solution (2 ml of 30 µg/ml), fraction 2 (11 ml of 15% diethyl ether in isooctane) (m/z 68-72). Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. Testing silica clean up for medium and long chain PCAs has not yet been performed. Also quantitative testing of the silica clean up was not performed. In almost all of the experiments in this study, BDE 127 and PCB 112 were used as internal standards. With silica fractionation it is not possible to use PCB 112 as internal standard, because PCB 112 will elute in the first fraction, where all PCAs will elute in the second fraction. It is possible to use PCB 112 as an injection standard. BDE 127 will elute in the second fraction and can be used as internal standard. 43

50 In conclusion, silica fractionation seems to be sufficient for fractionation of extracts for PCA analysis, with PCAs eluting in the second fraction. It is not possible to use PCB 112 as an internal standard with silica fractionation for PCA analysis. 6.4 Conclusions The tested GPC method is insufficient for PCA analysis of biota samples and first results show that sulphuric acid is not the best clean up method for PCA analysis either. Acidic silica clean up to remove fat, in combination with a silica fractionation to separate PCAs from interfering compounds, seems to be a promising method for clean up of extracts for PCA analysis, although more tests should be performed whether this clean up is indeed sufficient for the whole range of PCAs including medium and long chain PCAs. 44

51 7. BIOMONITORING 7.1 Introduction Because of the toxicity of PCAs and the expected carcinogenity to humans, it is important to know which PCAs, short, medium or long chain, are present in the environment, in which concentrations they are present in the environment and where they can be found. To get an insight in the behaviours of PCAs it is also important to know what type of PCA patterns will appear in different type of samples and sample locations. The presence of PCAs in the environment has been reported in a number of studies (Pribylova et al., 2006, Nicholls et al., 2001, Reth et al., 2005). Although PCAs have been detected and quantified, quantification has only been performed on PCAs as a sum of PCAs and not on individual or group separated PCAs. There is no information on the quantification of PCAs with GCxGC-ECNI-TOF MS. Concentrations up to 5664 ng/g in sediments from 11 Czech rivers were found by Pribylova et al. (2006), using gas chromatography with iontrap mass spectrometric detection working in electron capture negative ionization mode (GC-ECNI-IT-MS). Quantification was performed on the sum of PCAs. In fish concentrations between mg/kg wet weight from selected industrial areas in England and Wales using GC-ECNI-IT-MS have been found (Nicholls et al., 2001). Short chain PCAs were detected in liver samples from flounder, cod and north sea dab from the Baltic sea with concentrations between 19 and 286 ng/g (wet weight) and medium chain PCAs were detected with concentrations of ng/g wet weight by Reth et al. (2005). Analyses were performed with high resolution gas chromatography coupled to low resolution mass spectrometry in the electron capture negative ionization mode. The objective of this study was to get an overview which PCAs, short, medium and long chain, are present in the environment, in which concentrations they are present in the environment, where they are found in the environment and what type of PCA pattern can be found in different types of samples and sample locations. A pilot study is performed on a number of extracts, followed by analysis of a number of biota samples. 7.2 Experimental Pilot study Four extracts of tern egg, shrimps, suspended matter and sand eel from the Western Scheldt, which had been prepared for PBDE analysis by GPC and silica, were analysed for PCAs. These samples were available from another project and could give a first impression if PCAs are present, even if these were not prepared under optimal sample treatment conditions. 45

52 Analysis are performed by comprehensive two-dimensional gas chromatography with electron capture negative ionization time-of-flight mass spectrometry as described in paragraph 3.2. The column used in the second dimension is a 3.0 m x 0.1 mm x 0.1 µm HT column. Ammonia was used as ionization gas Samples The samples listed in Table 4 have been extracted, cleaned and analysed for PCAs. Table 4: Samples extracted for PCA analysis. Lims numer Matrix Location Intake (g) 2005/0207 Eel (wild) Meuse, Eijsden /0409 Eel (wild) Ijssel, Deventer /0415 Eel (wild) Roer, Vlodrop /0435 Eel (wild) North Holland Channel /0477 Eel (wild) IJ, Amsterdam /0593 Shrimps Rijnmond /0590 Herring The Channel /0596 Herring Central North Sea /0604 Cod Central North Sea /606 Cod liver Central North Sea /0618 Mackerel Central North Sea /0623 Mussels Easter Scheldt /0629 Eel (farmed) Fish shop /0633 Eel (wild) Meuse, Keizersveer /0635 Eel (wild) Ketelmeer /0637 Eel (wild) Haringvliet-West /0639 Eel (wild) Rhine, Lobith /0641 Eel (wild) Yssel lake, Medemblik /0657 Sole Central North Sea /0661 Salmon (farmed) Norway /0663 Salmon (farmed) Scotland /1116 Eel (wild) Western Scheldt 8.53 The sample intake is listed in Table 4. Extraction was performed by Soxhlet extraction with hexane: acetone (3:1 v/v). As internal standard 1 ml of PCB

53 (1 µg/ml) was added. The extracts were cleaned with acidic silica as described in paragraph 6.2.3, and concentrated back in a turbo evaporator (Zymark, Turbovap LV) to 0.5 ml, and analysed by GCXGC-TOF MS as described in paragraph 3.2. The column used in the second dimension is a 3.0 m x 0.1 mm x 0.1 µm HT column. Ammonia was used as ionization gas. Samples 2005/0207, 2005/0409, 2005/0415, 2005/0435, 2005/0477 and 2005/0593 are analysed several times on different days on the GCxGC-ECNI- TOF MS. 7.3 Results & Discussion Pilot study In the chromatogram (m/z 68-72) of the tern egg extract a clear pattern of the short chain PCAs can be seen (Figure 23). The chromatogram (m/z 68-72) of the extract of suspended matter (Figure 24) shows the presence of PCAs. In this chromatogram the characteristic pattern of PCA s can also be seen, although the groups are less separated than in the chromatogram of the tern egg. In the shrimp extract and the extract of sand eel no PCAs are found. Figure 23. GCxGC-ECNI-TOF MS chromatograms (m/z 68-72) of a tern egg extract. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 1.0 m x 0.1 mm x 0.1 µm HT. 47

54 For these samples a clean up with GPC and silica was used. As found in paragraph 6.3.1, GPC is not a sufficient clean up because lipids are coëluting with some PCAs. Most likely the longer chain PCAs will be lost by the clean up. It is possible that PCAs were present in the shrimp and sand eel samples, but were lost by the GPC and probably more PCAs were present in the tern egg and suspended matter samples than shown. Figure 24. GCxGC-ECNI-TOF MS chromatogram (m/z 68-72) of a suspended matter extract. Column combination: 30 m x 0.25 mm x 0.25 µm DB- 1 X 1.0 m x 0.1 mm x 0.1 µm HT Samples The TOF MS seemed to be an instable instrument, that even though ammonium was used as ionization gas (see paragraph 3.2.4), the signal of the MS dropt after the first 4 samples were injected, and the ion source had to be cleaned again. With reïnjection of the samples, after cleaning the ion source, the cooling device of the modulator was not working properly as can be seen in the chromatogram (m/z ) of Figure

55 Figure 25. GCxGC-ECNI-TOF MS chromatogram (m/z ) of sample 2005/0477 measured when the cooling device of the modulator wasn t working properly. Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. When the samples were reïnjected again, the mass spectrometer was not sensitive, and the ion-source had to be cleaned again. Therefore, the other samples from Table 4 have not been analysed. Figure 26 shows the variation in area of the internal standard CB 112 (2 µg/ml) in the samples 2005/0409 and 2005/0415 on three different days of analysis. In conclusion, the GCxGC-TOF MS system in ECNI mode is not stable enough to analyse samples as a batch. 49

56 /4/ /4/ /4/ Area / /0415 Figure 26. Variation in area of the internal standard CB 112 (2 µg/ml) (m/z ) of two samples analyzed on three different days on the GCxGC- ECNI-TOF MS. 7.4 Conclusions In extracts of tern egg and suspended matter PCAs have been detected. In a shrimp extract and an extract of sand eel no PCAs were found, which can be due to insufficient sample preparation. The GCxGC-TOF MS system in ECNI mode is not stable enough to analyse samples as a batch and no results were obtained, from analysis of biota samples. 50

57 8. CONCLUSIONS AND RECOMMENDATIONS A method is developed to analyse polychlorinated alkanes in environmental and biota samples, including a Soxhlet extraction with hexane: acetone (3:1 v/v), followed by a clean- up step with acidic silica and fractionation with silica. Analysis is performed by GCxGC-ECNI-TOF MS. Using a column combination of 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT instead of a column combination of 30 m x 0.25 mm x 0.25 µm DB-1 X 1.0 m x 0.1 mm x 0.1 µm HT resulted in approved separation. When the time-of-flight mass spectrometer is used in the ECNI mode, the signal is more stable when ammonia is used instead of methane as ionization gas. In the present study 100 % of ammonia is used. It is recommended to test the use of methane with 5 or 10 % ammonia, because this mixture will be less dangerous than 100 % ammonia in case a leak will appear. Although ammonia is used as ionization gas and the signal of the time-of-flight mass spectrometer was more stable than by using methane as ionization gas, the time-of-flight mass spectrometer still was very unstable when used in the electro negative chemical ionization mode. Therefore, it is recommended to use the instrument in this mode only for qualification and not for quantification. When semi-quantitative analysis on polychlorinated alkanes is performed, it is recommended to measure first individual PCAs with varying alkane lengths and varying chlorine contents, with more than five atoms, and to find the relationship between intensity and these parameters. Next, it will be of great importants to have groups of PCAs with the same intensity separated from other groups of PCAs, so that a whole group can be quantified. Standards of individual PCAs or standards of mixtures of PCAs with known concentration of PCAs, which give the same intensities, will then be needed for quantification. Individual standards with 6 or 8 chlorine atoms in the molecule are currently available. In the present study the extraction of samples was performed by Soxhlet extraction. Accelerated solvent extraction (ASE) with hexane, as extraction solvent is an extraction method, which is also used for PCA analysis (Barber et al., 2005). It is recommended to test the possibility of using accelerated solvent extraction with acidic silica in the cell as a combined extraction and clean up as a less time consuming method. For environmental and biota samples the use of silica in combination with acidic silica clean up is a promising method. Nevertheless more PCAs, including medium and long chain PCAs, should be tested to find out whether it is a good method for analysis of the whole range of PCAs. Other clean up methods to remove lipids that have not been tested in this study, are aluminium oxide, HPLC fractionation and a multilayer silica column with 51

58 KOH or silver nitrate. Although the GCxGC-ECNI-TOF MS instrument showed to be an instable instrument, PCAs were found in an extract of tern egg and suspended matter. 52

59 ACKNOWLEDGEMENT First of all, I would like to thank the former Netherlands Institute of Fishery Research (RIVO) and the Institute of Environmental Studies (IVM) for giving the opportunity to do the master study and to do this thesis. Furthermore, I would like to thank Peter Korytar for his help with the GCxGC- TOF MS. I also would like to thank some former colleagues from RIVO for their support and help: Judith van Hesselingen en Alwin Kruijt thanks!! Christiaan Kwadijk, thank you for your support, your listening ears and for al the cops of coffee you supplied me with especially when my leg was broken. Thanks to my parents for your support and your trust in me. Mom, thank you very much for making it possible for me to write on my thesis by taking care of Niels so often! Thank you Huberthus Irth for reading the draft version of this thesis and for your comments. And finally I would like to thank Pim Leonards, for teaching me, supporting me and helping me finish this thesis. Your optimistically point of views where very supportive when things didn t work out the way I would have liked. 53

60 54

61 REFERENCES Adahchour, M.; Brandt, M.; Baier, H.U.; Vreuls, R.J.J.; Batenburg, A.M.; Brinkman, U.A.Th. Comprehensive two-dimensional gas chromatography coupled to a rapid-scanning quadrupole mass spectrometer: principles and applications. Journal of Chromatography A, 1067 (2005) Amendola, L.; Botrè, F.; Stella Carollo, A.; Longo, D.; Zoccolillo, L. Analysis of organophosphorus pesticides by gas chromatography-mass spectrometry with negative chemical ionization: a study on the ionization conditions. Analytica Chimica Acta 461, 1 (2002) Barber J.L.; Sweetman, A.J.; Thomas, G.O.; Braekevelt, E.; Stern, G.: Jones, K.C. Spatial and temporal variability in air concentration of short-chain (C10- C13) and medium-chain (C14-C17) chlorinated n-alkanes measured in the U.K. atmosphere. Environmental science & technology 39 (2005) Castells, P.; Santos, F. J.; Galceran, M. T. Solid-phase extraction versus solidphase microextraction for the determination of chlorinated paraffins in water using gas chromatography-negative chemical ionisation mass spectrometry. Journal of chromatography A 1025 (2004) CEPA, Canadian Environmental Protection Act, Priority Substances List Assessment Report, Chlorinated Paraffins, Cooley, H.M.; Fisk, A.T.; Wiens, S.C.; Tomy, G.T.; Evans, R.E.; Muir, D.C.G. Examination of the behavior and liver and thyroid histology of juvenile rainbow trout (Oncorhynchus mykiss) exposed to high dietary concentrations of C10-, C11-, C12- and C14-polychlorinated n-alkanes. Aquatic Toxicology 54 (2001) Dallüge, J.; Beens, J.; Brinkman, U. A. Th. Comprehensive two-dimensional gas chromatography: a powerful and versatile analytical tool. Journal of Chromatography A, 1000 (2003) EPA (Environmental protection Agency). Office of Toxic Substances. RM1. Washington DC. Chlorinated Paraffins. Decision Package. Environmental risk assessment (1991). Hoffmann, E. de; Stroobant.V. Mass Spectrometry Principles and Applications (2002). Iowa State university, Chemical instrumentation facility, Mass spectrometry online tutorial (2007). 55

62 Junge, M.; Huegel, H.; Marriott, P.J. Enantiomeric analysis of amino acids by using comprehensive two-dimensional gas chromatography. CHIRALITY, 19 (2007) Kimmel,J. Mass Spectrometry & Chromatography CU- Boulder CHEM 5181 Lecture 3: Ionization Techniques Part II (2006). Klimankova, E.; Holadova, K.; Hajslova, J.; Cajka, T.; Poustka, J.; Koudela, M. Aroma profiles of five basil (Ocimum basilicum L.) cultivars grown under conventional and organic conditions. Food Chemistry, 107 (2008) Korytár, P.; Leonards, P.E.G.; Boer, J. de; Brinkman, U.A.Th. Group separation of organohalogenated compounds by means of comprehensive two-dimensional gas chromatography. Journal of Chromatography A, 1086 (2005) Korytár, P.; Parera, J.; Leonards, P.E.G.; Santos, F.J.; Boer, J. de; Brinkman, U.A.Th. Characterization of polychlorinated n-alkanes using comprehensive two-dimensional gas chromatography electron-capture negative ionisation timeof-flight mass spectrometry. Journal of Chromatography A, 1086 (2005) Korytár, P. Comprehensive two-dimensional gas chromatography with selective detection for the trace analysis of organohalogenated contaminants (2006) Moore, S.; Vromet, L.; Rondeau, B. Comparison of metastable atom bombardment and electron capture negative ionization for the analysis of polychloroalkanes. Chemosphere 54 (2004) Nakagawa, S. Negative chemical ionization mass spectrometric study on the electron attachment to CF x Cl 4-x (x =1,2) and CBr y Cl 4-y (y=1,2). Chemical Physics 282 (2002) Nicholls, C.R.; Allchin, C.R.; Law, R.J. Levels of short and medium chain length polychlorinated n-alkanes in environmental samples from selected industrial areas in England ans Wales. Environmental pollution 114 (2001) Official journal of the European community L331 (2001) 1, Official journal of the European community L327 (2000) 1, OSPAR Commission. Hazardous Substances Series: Short Chain Chlorinated Paraffins. Background Document on Short Chain Chlorinated Paraffins ISBN (2001). 56

63 Parera, J.; Santos, F.J.; Galceran, M.T. Microwave-assisted extraction versus Soxhlet extraction for the analysis of short-chain chlorinated alkanes in sediments. Journal of Chromatography A 1046 (2004) Pribylova, P.; Klanova, J.; Holoubek, I. Screening of short- and medium-chain chlorinated paraffins in selected riverine sediments and sludge from the Czech Republic. Environmental Pollution 144 (2006) Reth, M.; Zencak, Z.; Oehme, M. First study of congener group patterns and concentration of short- and medium-chain chlorinated paraffins in fish from the North and Baltic Sea. Chemosphere 58 (2005) Rzepa, H.S.. Imperial College London, Chemistry, Department of Chemistry Local Teaching Pages, Handouts for Organic Chemistry Lectures ( ). Serrone, D.M.; Birtley R.D.N.; Weigand W.; Millischer R., Toxicology of Chlorinated Paraffins. Food Chem. Toxicol., 25(7) (1987) Stejnarova, P.; Coelhan, M.; Kostrhounovia, R; Parlar, H.; Holoubek, I. Analysis of short chain chlorinated paraffins in sediment samples from the Czech Republic by short-column GC/ECNI-MS. Chemosphere 58 (2005) Tomy, G. T. ; Stern, G. A.; Muir, D. C. G. ; Fisk, A. T.; Cymnalisty, C.D.; Westmore, J.B. Quantifying C 10 -C 13 polychloroalkanes in environmental samples by high-resolution gas chromatography/electron capture negative ion high-resolution mass spectrometry. Analytical Chemistry 69 (1997) Ventura, G.T; Kenig, F.; Reddy, C.M.; Frysinger, G.S.; Nelson, R.K.; Mooy, B. van; Gaines, R.B. Analysis of unresolved complex mixtures of hydrocarbons extracted from Late Archean sediments by comprehensive two-dimensional gas chromatography (GCxGC). Organic geochemistry, 39 (2008) WHO (World Health Organization) Geneva. Chlorinated paraffins. Environmental health criteria 181 (1996). 57

64 LIST OF ABBREVIATIONS 1D 2D 3D ASE BDE CB CFT CH 4 CI DCD DCDD 2 DCDD 12 DCUD DCTD DCTrD ECNI EI EPA GC GPC HCD IT MS NCI NH 3 OCTD PBDE PCA RRF Rt TCD TCDD TCN TCO TCTD TCTrD TCUD TOF WHO One-dimensional Two-dimensional Three-dimensional Accelerated solvent extraction Brominateddiphenylether Chlorobiphenyl Capillary Flow Technology Methane Chemical ionization 1,2- dichlorodecane 1,2- dichlorododecane 1,12- dichlorododecane 1,2- dichloroundecane 1,2- dichlorotetradecane 1,2- dichlorotridecane Electron capture negative ionization Electron impact ionization Environmental Protection Agency Gas chromatography/ Gas chromatograph Gel Permeation Chromatography 1,1,1,3,9,10, hexachlorodecane Ion trap Mass spectrometry Negative chemical ionization Ammonia 1,1,1,3,12,14,14,14 octachlorotetra-decane Poly brominateddiphenylether Polychloroalkane Relative response factor Retention time 1,1,1,3- tetrachlorodecane 1,1,1,3- tetrachlorododecane 1,1,1,3- tetrachlorononane 1,1,1,3- tetrachlorooctane 1,1,1,3 tetrachlorotetradecane 1,1,1,3- tetrachlorotridecane 1,1,1,3- tetrachloroundecane Time-of-flight World Health Organization 58

65 APPENDIX 1: Acidic Silica clean up test ref comp PCA-60 RRF RRF RRF RRF Recovery (%) PCA 60 ref PCA 60 sil calc. with int.std. calc. with int.std. calc. with int.std. m/z Area rt weight µg abs Area rt weight µg abs BDE 127 CB112 BDE 127 CB112 BDE 127 CB 112 BDE CB PCA ref comp HCD RRF RRF RRF RRF Recovery (%) HCD ref (M=352) HCD sil (M=352) calc. with int.std. calc. with int.std. calc. with int.std. Area rt weight µg abs Area rt weight µg abs BDE 127 CB112 BDE 127 CB112 BDE 127 CB 112 BDE CB PCA HCD ref comp TCTD RRF RRF RRF RRF Recovery (%) TCTD ref (M=338) TCTD sil (M=338) calc. with int.std. calc. with int.std. calc. with int.std. Area rt weight µg abs Area rt weight µg abs BDE 127 CB112 BDE 127 CB112 BDE 127 CB 112 BDE CB PCA TCTD?

66 ref comp OCTD RRF RRF RRF RRF Recovery (%) OCTD ref (M=478) OCTD sil (M=478) calc. with int.std. calc. with int.std. calc. with int.std. Area rt weight µg abs Area rt weight µg abs BDE 127 CB112 BDE 127 CB112 BDE 127 CB 112 BDE CB PCA x x x x x x x x x x OCTD? x x x x x x x x x x solutions spiked to fish oil comp PCA-60 RRF RRF Recovery (%) PCA 60 fat sil calc. with int.std. calc. with int.std. m/z Area rt weight µg abs BDE 127 CB112 BDE 127 CB 112 BDE CB PCA E comp HCD RRF RRF Recovery (%) HCD fat sil (M=352) calc. with int.std. calc. with int.std. Area rt weight µg abs BDE 127 CB112 BDE 127 CB 112 BDE CB PCA HCD

67 comp TCTD RRF RRF Recovery (%) TCTD fat sil (M=338) calc. with int.std. calc. with int.std. Area rt weight µg abs BDE 127 CB112 BDE 127 CB 112 BDE CB PCA E-06 1E TCTD? comp OCTD RRF RRF Recovery (%) OCTD fat sil (M=478) calc. with int.std. calc. with int.std. Area rt weight µg abs BDE 127 CB112 BDE 127 CB 112 BDE CB PCA x x x x x x OCTD? x x x x x x 61

68 APPENDIX 2 A 2D-plot of a mixture of C 10 -C 13 with 51.5 % chlorine Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 62

69 APPENDIX 2 B 2D-plot of a mixture of C 10 -C 13 with 55.5 % chlorine Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 63

70 APPENDIX 2 C 2D-plot of a mixture of C 10 -C 13 with 63 % chlorine Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 64

71 APPENDIX 2 D 2D-plot of a mixture of C 14 -C 17 with 42 % chlorine Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 65

72 APPENDIX 2 E 2D-plot of a mixture of C 14 -C 17 with 57 % chlorine Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 66

73 APPENDIX 2 F 2D-plot of a mixture of C 18 -C 20 with 30 % chlorine Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 67

74 APPENDIX 3 2D-plot of a mix of all tested mixtures of short (C 10 -C %, 55,5%, 63%), medium (C 14 -C 17 42%, 57%), and long chain (C 18 - C 20 30%) PCAs Column combination: 30 m x 0.25 mm x 0.25 µm DB-1 X 3.0 m x 0.1 mm x 0.1 µm HT. 68