Large Volume Injection and Hyphenated Techniques for Gas Chromatographic Determination of Pbdes and Carbazoles in Air

Large Volume Injection and Hyphenated Techniques for Gas Chromatographic Determination of PBDEs and Carbazoles in Air

Petter Tollbäck

Doctoral Thesis Department of Analytical Chemistry Stockholm University 2005

1 Doctoral Thesis, 2005

Petter Tollbäck [email protected] Department of Analytical Chemistry Stockholm University S-106 91 Stockholm

Cover by Holger and Vera Tollbäck Akademitryck AB, Edsbruk, Sweden 2005

2 Till Holger och Vera

3 4 List of contents Abstract ...... 7 Sammanfattning ...... 9 List of papers ...... 11 Abbreviations ...... 13 Aims ...... 15 PBDEs and carbazoles ...... 17 Polybrominated diphenyl ethers (PBDEs) ...... 17 PBDEs – environmental pollutants ...... 18 Biological effects of PBDEs ...... 19 PBDEs – an analytical challenge ...... 21 Carbazoles ...... 22 Health effects of carbazoles ...... 23 Gas chromatography ...... 25 Chromatographic resolution in GC ...... 25 Film thickness of the stationary phase ...... 26 Column length ...... 27 Inner diameter ...... 28 Stationary phases ...... 31 Injection techniques for GC ...... 33 Vaporizing and non-vaporizing injections ...... 33 Analyte peak focusing ...... 34 Cold trapping ...... 34 Retention gap focusing ...... 34 Solvent effects ...... 34 Conventional injectors ...... 36 The on-column injector ...... 36 The split/splitless injector ...... 37 The programmed temperature vaporizer (PTV) ...... 38 At-column injection ...... 39 The septum equipped temperature programmable injector . . . 39 Direct injection ...... 40 Large volume injection (LVI) ...... 41 Why large volume injection? ...... 41 Common misconceptions about large volume injections ...... 42 On-column large volume injection (OC-LVI) ...... 42 Loop-type injector/interface ...... 43

5 Concurrent solvent evaporation ...... 45 Peak deformation ...... 46 Why loop-type? ...... 48 Large volume splitless injection ...... 49 Large volume PTV injection ...... 50 Sample introduction and solvent evaporation ...... 50 Analyte trapping ...... 54 Analyte transfer ...... 54 The author’s note on the loop-type and the PTV injectors ...... 55 GC detectors ...... 57 The flame ionization detector (FID) ...... 57 The nitrogen phosphorus detector (NPD) ...... 57 The electron capture detector (ECD) ...... 58 Mass spectrometry (MS) ...... 58 Electron ionization (EI) ...... 60 Chemical ionization (CI) ...... 60 Electron capture negative ionization (ECNI) ...... 60 Sample extraction ...... 63 Static and dynamic extractions ...... 64 Soxhlet extraction ...... 64 Ultrasonication-assisted solvent extraction ...... 65 Dynamic sonication-assisted solvent extraction ...... 65 On-line coupling to GC ...... 67 Benefits of hyphenation ...... 67 Concerns about hyphenated systems ...... 68 LC-GC ...... 68 NPLC and RPLC ...... 68 Heart-cut or back-flush? ...... 69 On-line extraction-GC ...... 73 Dynamic microwave-assisted extraction (DMAE)-GC ...... 73 Pressurized hot water extraction (PHWE)-GC ...... 73 Supercritical fluid extraction (SFE)-GC ...... 74 Dynamic sonication-assisted extraction (DSAE)-GC ...... 74 Air sampling ...... 75 The sampling set-up ...... 75 Concluding remarks ...... 77 Acknowledgements ...... 79 References ...... 81

6 Abstract This thesis is based on studies in which the suitability of various gas chromatography (GC) injection techniques was examined for the determination of polybrominated diphenyl ethers (PBDEs) and carbazoles, two groups of compounds that are thermally labile and/or have high boiling-points. For such substances, it is essential to introduce the samples into the GC system in an appropriate way to avoid degradation and other potential problems. In addition, different types of gas chromatographic column system and mass spectrometric detectors were evaluated for the determination of PBDEs. Conventional injectors, such as splitless, on-column and programmed temperature vaporizing (PTV) injectors were evaluated and optimized for determination of PBDEs. The results show on-column injection to be the best option, providing low discrimination and high precision. The splitless injector is commonly used for “dirty” samples. However, it is not suitable for determination of the high molecular weight congeners, since it tends to discriminate against them and promote their degradation, leading to poor precision and accuracy. The PTV injector appears to be a more suitable alternative. The use of liners reduces problems associated with potential interferents such as polar compounds and lipids and compared to the hot splitless injector, it provides gentler solvent evaporation, due to its temperature- programming feature, leading to low discrimination and variance. Increasing the injection volume from the conventional 1-3 µL to >50 µL offers two main benefits. Firstly, the overall detection and quantification limits are decreased, since the entire sample extract can be injected into the GC system. Secondly, large volume injections enable hyphenation of preceding techniques such as liquid chromatography (LC), solid phase extraction and other kinds of extraction. Large-volume injections were utilized and optimized in the studies included in this thesis. With a loop-type injector/interface large sample volumes can be injected on-column providing low risk of discrimination against compounds with low volatility. This injector was used for the determination of PBDEs in air and as an interface for the determination of carbazoles by LC-GC. Peak distortion is a frequently encountered problem associated with this type of injector that was addressed and solved during the work underlying this thesis.

7 The PTV can be used as a large volume injector, in so-called solvent vent mode. This technique was evaluated for the determination of PBDEs and as an interface for coupling dynamic sonication-assisted solvent extraction on- line to GC. The results show that careful optimization of the injection parameters is required, but also that the PTV is robust and yields reproducible results. PBDEs are commonly detected using mass spectrometry in electron capture negative ionization (ECNI) mode, monitoring bromine ions (m/z 79 and 81). The mass spectrometric properties of the fully brominated diphenyl ether, BDE-209, have been investigated. A high molecular weight fragment at m/z 486/488 enables the use of 13C-labeled BDE-209 as an internal surrogate standard.

8 Sammanfattning Den här avhandlingen fokuserar på injektionstekniker för gaskromatografi (GC), med avseende på två grupper av ämnen, som är termiskt labila och/ eller har höga kokpunkter: polybromerade difenyletrar (PBDE) och carbazoler. I många fall är provintroduktionen den mest kritiska delen av en GC-analys. Vidare har GC-kolonnsystemet och den masspektrometriska detektionen av PBDE undersökts. Konventionella tekniker så som splitless-, PTV- (programmable temperature vaporizing) och on-columninjektion har utvärderats och optimerats med avseende på haltbestämning av PBDE. Mest tillförlitlig visade sig on- columninjektionen vara, med låg diskriminering och hög precision. För komplicerade matriser används vanligen splitlessinjektion. Denna teknik är dock inte lämplig för PBDE-kongener med hög bromeringsgrad. Resultaten presenterade i den här avhandlingen visar att precisionen är låg, till följd av diskriminering eller nedbrytning. Istället förslås PTV- injektorn som ett mer lämpligt alternativ. Denna förångningsinjektor är robust mot matrisrester, till exempel lipider och polära ämnen. Temperaturprogrammeringen möjliggör en mer kontrollerad lösningsmedelsförångning jämfört med splitlessinjektorn, vilket resulterar i lägre diskriminering och högre precision. Att öka injektionsvolymen från de konventionella 1-3 µL till över 50 µL ger två vinster. Detektions- och kvantifieringsgränserna sänks, eftersom hela eller en större del av provextraktet kan injiceras på GC-systemet. Storvolymsinjektioner möjliggör också direktkoppling av upparbetningstekniker, till exempel vätskekromatografi (LC) och olika extraktionstekniker. Storvolymsinjektioner har optimerats och utnyttjats i stor del av arbetet. Med en loopinjektor kan stora volymer injiceras on-column, vilket ger liten risk för diskriminering. Denna injektor har använts för bestämning av PBDE i luft samt som interface för bestämning av carbazoler på LC-GC. Deformation av kromatografiska toppar är ett vanligt fenomen då denna typ av injektor används. Detta problem har undersökts och avhjälpts i det arbete som denna avhandling baseras på. PTV-injektorn kan också användas för injektion av stora volymer. Tekniken har utvärderats för bestämning av PBDE samt som interface för

9 koppling av dynamisk ultraljudsextraktion till GC. Resultaten visar att en noggrann optimering är nödvändig, men också att PTV-injektorn är robust och ger reproducerbara resultat. PBDE detekteras vanligen med masspektrometri i sk “electron capture negative ionization (ECNI) mode”, där bromjonerna (m/z 79 och 81) registreras. Det masspektrometriska mönstret för den fullt bromerade difenyletern BDE-209 har undersökts. Upptäckten av ett fragment vid m/z 486/488 gör att kol-13-märkt BDE-209 kan användas som intern standard, vilket ökar noggrannheten vid bestämning av BDE-209.

10 List of papers I Influence of the injection technique and column system on gas chromatographic determination of polybrominated diphenyl ethers (PBDE) Jonas Björklund, Petter Tollbäck, Christian Hiärne, Eva Dyremark and Conny Östman Journal of Chromatography A 1041, 201-210, 2004 - The author is responsible for a major part of the experimental work and for writing a substantial part of this paper.

II Large volume injection GC-MS in electron capture negative ion mode utilizing isotopic dilution for the determination of polybrominated diphenyl ethers in air J. Björklund, P. Tollbäck and C. Östman Journal of Separation Science 26, 1104-1110, 2003 - The author is responsible for setting up the injection system, investigating the peak distortion phenomena and for writing a substantial part of this paper.

III Large-volume programmed-temperature vaporizer injection for fast gas chromatography with electron capture and mass spectrometric detection of polybrominated diphenyl ethers P. Tollbäck, J. Björklund and C. Östman Journal of Chromatography A 991, 241-253, 2003 - The author is responsible for a major part of the experimental work and for writing a substantial part of this paper.

IV Coupled LC-GC-NPD for determination of carbazole-type PANH and its application to personal exposure measurement P. Tollbäck, H. Carlsson and C. Östman Journal of High Resolution Chromatography 23 (2), 131-137, 2000 - The author is responsible for all the experimental work and partly for writing this paper.

V Dynamic sonication assisted solvent extraction coupled on-line to GC-MS for the determination of PBDEs in air P. Tollbäck and C. Östman In manuscript - The author is responsible for all the experimental work and for writing this paper.

11 VI Mass spectrometric characteristics of decabromo-diphenyl ether and the application of isotopic dilution in the electron capture negative ionization mode for the analysis of polybrominated diphenyl ethers J. Björklund, P. Tollbäck and C. Östman Journal of Mass Spectrometry 38 (4), 394-400, 2003 - The author is responsible for experimental work and for writing a substantial part of this paper.

Papers not included in this thesis

Enhanced detection of nitroaromatic explosive vapors combining SPE-air sampling, SFE and Large Volume Injection-GC R. Batlle, H. Carlsson, P. Tollbäck, A. Colmsjö and C. Crescenzi Analytical Chemistry 75 (13), 3137-3144, 2003

Automated rotary valve injection for polybrominated diphenyl ethers in gas chromatography J. Björklund, P. Tollbäck, E. Dyremark and C. Östman Journal of Separation Science 26, 594-600, 2003

Evaluation of gas chromatographic injection techniques for PBDE. P. Tollbäck, J. Björklund and C. Östman Organohalogen Compounds 61, 49-52, 2003

Determination of high molecular weight PBDE by isotopic dilution in ECNI-MS. J. Björklund, P. Tollbäck and C. Östman Organohalogen Compounds 61, 163-166, 2003

Evaluation of the gas chromatographic column system for the determination of polybrominated diphenyl ethers J. Björklund, P. Tollbäck and C. Östman Organohalogen Compounds 61, 239-242, 2003

12 Abbreviations BDE BromoDiphenyl Ether BDE-47 2,2’,4,4’-tetraBDE BDE-49 2,2’,3,4-tetraBDE BDE-99 2,2’,4,4’,5-pentaBDE BDE-100 2,2’,4,4’,6-pentaBDE BDE-119 2,3’,4,4’,6-pentaBDE BDE-153 2,2’,4,4’,5,5’-hexaBDE BDE-154 2,2’,4,4’,5,6’-hexaBDE BDE-183 2,2’,3,4,4’,5’,6-heptaBDE BDE-191 2,3,3’,4,4’,5’,6-heptaBDE BDE-197 2,2’,3,3’,4,4’,6,6’-octaBDE BDE-203 2,2’,3,4,4’,5,5’,6-octaBDE BDE-206 2,2’,3,3’,4,4’,5,5’,6-nonaBDE BDE-207 2,2’,3,3’,4,4’,5,6,6’-nonaBDE BDE-208 2,2’,3,3’,4,5,5’,6,6’-nonaBDE BDE-209 2,2’,3,3’,4,4’,5,5’,6,6’-decaBDE BTBPE Bis(2,4,6-TriBromoPhenoxy) Ethane DBC DiBenzoCarbazole (7-H-dibenzo(c,g)carbazole) DecaBDE DecaBromoDiphenyl Ether ECD Electron Capture Detector ECNI Electron Capture Negative Ionization EI Electron Ionization (previously Electron Impact) FID Flame Ionization Detector GC Gas Chromatography HeptaBDE HeptaBromoDiphenyl Ether HexaBDE HexaBromoDiphenyl Ether LC Liquid Chromatography LVI Large Volume Injection MS Mass Spectrometry NonaBDE NonaBromoDiphenyl Ether NPD Nitrogen Phosphorus Detector NPLC Normal Phase Liquid Chromatography

13 OC On-Column OctaBDE OctaBromoDiphenyl Ether PAC Polycyclic Aromatic Compound PAH Polycyclic Aromatic Hydrocarbon PANH Polycyclic Aromatic Nitrogen-containing Heterocyclic PBDE Polybrominated Diphenyl Ether PentaBDE PentaBromoDiphenyl Ether PTV Programmed Temperature Vaporizer/Vaporizing RPLC Reversed Phase Liquid Chromatography RSD Relative Standard Deviation SIM Selected Ion Monitoring SPE Solid Phase Extraction TetraBDE TetraBromoDiphenyl Ether Th Thompson, unit for m/z TriBDE TriBromoDiphenyl Ether

14 Aims The main objectives of the work underlying this thesis were to investigate, optimize and evaluate large volume injection for gas chromatographic analysis of thermally labile compounds and compounds with high boiling points (referred to, for convenience, as high-boiling compounds hereafter). The developed techniques were applied to the determination of polybrominated diphenyl ethers (PBDEs) and carbazole-type polycyclic aromatic nitrogen- containing heterocyclics (PANHs) in air. More traditional methods were also investigated in the course of the work. In addition, large volume injectors were utilized as interfaces for coupling clean-up techniques and gas chromatography on-line. These techniques have been developed to improve detection limits and accuracy. Automated, closed systems also reduce the risk of contamination, a common problem when analyzing PBDEs. Determination of the thermally labile decaBDE congener has proven to be troublesome. Conventional gas chromatographic set-ups generally result in severe discrimination against this compound, leading to poor precision and accuracy. The results presented in this thesis may therefore also assist the development of accurate and reproducible analytical methods for determination of PBDEs in environmental samples.

15 16 PBDEs and carbazoles In this thesis the development and use of sensitive methods for determining selected air-borne pollutants are described. The following paragraphs are intended to give a brief introduction to the investigated compounds.

Polybrominated diphenyl ethers (PBDEs) Pollution with polybrominated diphenyl ethers, Figure 1, has been referred to as the new PCB problem. Considering the similarities between PBDEs and PCBs in structure, function and environmental occurrence the comparison is obvious. However, there are also differences, in biological activity and physical properties, for instance. For more extensive discussion of PBDEs as environmental pollutants, the reader is referred to recent reviews that have been published on this topic [1-3].

%U 2 2

%U %U [ \ %U %U %U

3%'( %'(

%U %U %U 2 2 %U %U

%U %U %U %U %U %U %U %U %U %U %'( %'(

Figure 1. General structure of PBDEs and three selected BDE congeners.

PBDEs constitute a group of additive flame-retardants that are predominantly found in electronic equipment, furniture and textiles. They are blended into or adsorbed onto the materials to reduce the flammability. Thus, they are not covalently bonded to the host material. Their flame-retarding properties are based on the elimination of free radicals formed during combustion processes [4].

17 Three technical PBDE mixtures (penta-, octa- and deca-BDE) are commercially used for flame-retarding purposes. The estimated world demand for PBDEs in 1999 was 67,000 tonnes, of which the deca-BDE mixture accounted for about 80 %. The results of a characterization of these products by Sjödin et al. are summarized in Table 1 [5]. As can be seen, synthesis of the technical mixtures is not specific, yielding about twenty congeners ranging from triBDE to decaBDE. Regulations against the use of the penta-BDE and octa-BDE mixtures within the European Union have shifted production towards the deca-BDE mixture in this part of the world. However, the USA and Japan are still using the low molecular weight mixtures.

Table 1. Composition of the three technical PBDE-mixtures.

Composition (%) PBDE-mixture Tri- Tetra- Penta- Hexa- Hepta- Octa- Nona- Deca- Penta-BDE 0-1 24-38 50-62 4-8 Octa-BDE 10-12 43-44 31-35 9-11 0-1 Deca-BDE 0.3-3 97-98

PBDEs – environmental pollutants Being additive flame retardants, PBDEs could be suspected to migrate from their host polymer to the surroundings. The first evidence of PBDEs in the environment was reported in 1979 by Zweidinger [6] and in 1981 by Andersson and Blomquist [7]. The latter authors discovered a number of BDE congeners in fish from the river Viskan in Sweden. Since then PBDEs have been found in a wide range of environmental compartments, such as sediments [8-11] and sewage sludge [12-15], as well as in various biota. Numerous papers describe the occurrence of PBDEs in marine animals, for example fish [8, 16, 17], seabirds [18] and mammals [19-22]. PBDEs have also been found in human blood [23, 24], adipose tissue [25-28] and human milk [29-34]. Recently high levels of PBDEs have been found in food, particularly fatty fish, sausage and cheese [35]. Zweidinger et al. were the first to report the presence of PBDEs in air,

18 detecting BDE-209 close to a manufacturing plant for brominated flame retardants [6]. Interest in analyzing air with regard to PBDEs has increased in recent years. The publications on this subject are summarized in Table 2. In 1995 Watanabe et al. reported concentrations in the air as high as 3.1 ng/m3 in rural sites in Osaka, Japan [36]. Jaward et al. presented a large-scale investigation of the outdoor air in 22 countries, mainly in Europe [37]. The highest amounts were found in the UK, which has been both the largest producer and user of PBDEs in Europe. Levels of PBDEs in air have also been determined in Sweden [2, 38], USA [39], Canada [40-42], Chile [43], Norway and the UK [44-46] Sjödin et al. determined several BDE congeners including BDE-209 in different indoor environments, and found increased levels in blood from people exposed to high concentrations of PBDEs in their work environment (an electronics recycling plant) [51]. Thomsen et al. found 7-59 pg/m3 of BDE-49 and BDE-99 in laboratory air, which were responsible for blank problems. Harrad et al. measured the PBDE levels in indoor domestic and work environments [45]. They calculated the daily exposure via the respiratory system to be about 7 ng. This short summary of the occurrence of PBDEs in the environment illustrates the global breadth of their distribution as pollutants.

Biological effects of PBDEs Information about the biological effects of PBDEs is sparse, but suggests that their bioactivity is low. Their acute toxicity, measured as LD50, has been reported to be 0.5-5 g/kg body weight [4]. Analyses involving oral administration to rats have shown that the low molecular weight congeners are easily absorbed; after five days 86 % of the dose was still retained, and the half-lives were 20-30 days for tetraBDE and 45-119 days for hexaBDE [52, 53]. The bioavailability of decaBDE is low, due to its large size and only 1-10 % is absorbed [54, 55]. The rats’ excretions showed that the PBDEs are metabolized to a large degree. An interesting finding is that following exposure to BDE-209 the levels of hexa- to nonaBDE in rainbow trout were significantly increased [56]. The low molecular weight BDE congeners have been shown to inhibit the binding of the thyroid hormone thyroxine (T4) to transthyretin [57].

19 Table 2. Publications of determination of PBDEs in air. Ref. b 2004 [41] Year 2004 [50] 2004 [40] 2001 [47] 2001 [48] 2003 [49] 2004 [45] 2004 [42]

3 10 × ) 3

3 10 1979 [6] 1979 6 × -170 3 10 × Outdoor: n.d.-4.4 (low-high) Indoor: 76-2088 Outdoor: 39-48 Other environm. n.d. – 87 (Individual congeners) 0.8-2.5 2004 [46] 0.22-37 2004 [44] 0.22-37 2004 Recycling plant: 100-70 plant: Recycling [39] BDE-47: 12-59 BDE-99: 7-29 Air: n.d. Dust: 2-23 g/g 4-77 2004 Indoor: 60-15509 Outdoor: 10-33 4.4 Outdoor: 4.8 Indoor: 42.1 calculated. n.d. = not detected. quartz filter 88-3100 1995 [36] PUF adsorbents membrane devices devices membrane Active, glass microfiber filter and filter microfiber glass Active, two PUF adsorbents Active, glass fiber filter and two PUF adsorbents to glassware Passive, adsorption adsorbent adsorbent Passive, PUF n.d (<6) PUF adsorbents 2004 [43] Active, glass fiber filter and XAD-2 adsorbent Organic films from building surfaces collected. Passive, PUF Indoor: 2-3600 c

Active, glass fiber filter and two c c Passive, Semipermeable Semipermeable Passive, c Passive, PUF 0.06-43 2004 [37] c Concentration of BDE-209 in air not d technique Sampling (pg/m Sum. Levels a

c, d c, Year of publication. Date for previous publication in conference proceedings and sampling may be earlier. b v. BDE-congeners nonaBDE, octaBDE, 209 154, 183, 190) 190 181, 166, 154, 153, 138, 119, 100, 209 183, 154, 153, 196, 191, 184, 183, 156, 154, 153, 138, 209 206, 207, 197, Not specified if more congeners than detected in samples were investigated. Congeners in parenthesis not were detected in samples. Sampling site type Sample USA Japan In Sweden Outdoor air Outdoor air air Indoor Norway BDE-209 USA TetraBDE to hexaBDE air Indoor USA 99, 47, 85, 100, 128,153, 154, 183, Indoor air and dust Chile 99, 28, 47, 100, 153,154, 183 Outdoor air 47, 99, 100 UK and Ireland Active, Outdoor air air Outdoor Canada Active, Glass fiber filter Active,SPE 47, 99, 100,Europe 153, 154, (190), 209 17, 28, 32, 35, 37, 47, 66, 71, 75, 85, 99, outdoorair and Indoor n.d. – 25*10 UK 153, 138, 100, 99, 71, 47, 85, 66, 28, (17, 33, 17, 28,99, 47, 100, 153, 154, 183 air Outdoor Active, quartz filter and XAD-2 Canada Active, quartz filter and XAD-2 NorwayUK and air and outdoor Indoor air Outdoor 154 153, 100, 99, 47, Indoor and outdoor air 49, 28, 47,99, 75, 100, 153, 154 Canada 17, 28, 47, 66, 71, 85, 99, 100, 153, 154 Sweden 99,49, 28, 47, 100, 153, 154, 183 air and outdoor Indoor 126, 33, 85, 100, 28, 66, 47, 99, 77, 17, a and two filter fiber glass Active, air Indoor c 119, 85, 100, 47, 66, 99, 28, 49, 71, 17,

20 Considering the structural similarities of T4 and PBDEs, competition between them for the binding sites is not surprising. Meerts et al. proved 11 BDE congeners to have estrogenic effects [58]. The ubiquitous BDE-100 was among those with the highest activity. The carcinogenic effect of BDE-209 is low [59], possibly due in part to the low uptake of this compound. Unfortunately, the carcinogenicity of the smaller BDE congeners, which are absorbed to a much higher extent, has not yet been tested. All toxicological studies of PBDEs to date have been based on oral intake of the compounds. The toxic effects of exposure through inhalation have not yet been investigated.

PBDEs - an analytical challenge There are 209 possible BDE congeners. However, unlike the PCBs only about twenty are found in technical mixtures and environmental samples. Their chromatographic separation is consequently not a major problem in PBDE analysis. The high molecular weight BDE congeners put extraordinary demands on the analytical procedure. These compounds have high boiling points, readily adsorb to glass surfaces and degrade thermally. Consequently, determination of nona- and decaBDE is particularly troublesome and rarely presented in the literature. In a Round-Robin study initiated by de Boer et al. large discrepancies were found in the concentrations of BDE-209 reported by the participating laboratories [60]. Most of the methods used involved splitless injection, which is not well suited for thermally labile or high-boiling compounds. Short columns were used for determining decaBDE in most cases, but often with thick stationary phases, which could promote degradation, as discussed in Paper I. As mentioned earlier, the aims of the studies this thesis is based upon were to develop accurate and sensitive techniques for the determination of PBDEs, and to investigate and evaluate different set-ups and conditions for the GC analysis of compounds with high boiling points and thermal lability. From this perspective BDE-209 is an excellent model substance.

21 Carbazoles Carbazoles constitute a group of polycyclic aromatic nitrogen-containing heterocyclics (PANHs), Figure 2, belonging to the large group of polycyclic aromatic compounds (PACs).

N N H H Carbazole Benzo(def)carbazole

N N N H H H Benzo(a)carbazole Benzo(b)carbazole Benzo(c)carbazole

N N H H Dibenzo(c,g)carbazole Dibenzo(a,i)carbazole

Figure 2. Structure of investigated carbazoles.

The determination of carbazoles is of interest to various industrial sec- tors, including the oil industry, since they are constituents of many fossil fuels. Carbazoles have been identified, for example, in crude oils [61], shale oils [62], fuels [63] and tar [64]. Furthermore, the benzocarbazole ratio, a/(a+c), can be used as a marker for the origin of oils, and Larter et al. found a correlation between the relative and absolute concentrations of these benzocarbazoles and the distance from an oil source to its reservoir [65]. These relationships could be used to help find new petroleum accumulations.

22 Carbazoles are also formed during incomplete combustion of organic matter and have been found in tobacco smoke [66], sewage sludge [67], outdoor air [68] and indoor air (Paper IV). Given their biological activity, carbazoles are of both environmental and health concern.

Health effects of carbazoles The effects of carbazoles on a biological system are highly dependent on their structure. For example carbazole may cause skin disorders [69], but conflicting results regarding its carcinogenicity have been published [70, 71]. On the other hand, a large amount of evidence has been reported that con- firms the mutagenic and carcinogenic effects of 7H-dibenzo[c,g]carbazole (DBC) [72]. DBC has been shown to form large amounts of DNA-adducts in liver and skin [73-75], Warshawsky and Barkley found it to be as carcinogenic as benzo(a)pyrene (BaP) in mice [76, 77], and Sellakumar and Shubik reported it to be an even more potent carcinogen than BaP in the respiratory tract of hamsters [78, 79].

23 24 Gas chromatography The separation of compounds in gas chromatography is based on their partitioning between a stationary, liquid phase, e.g. modified polysiloxane, and a mobile, gaseous phase, i.e. hydrogen, nitrogen or helium. On a non- polar column, such as 100 % dimethyl polysiloxane, analytes are separated according to their vapor pressure or boiling points. Further retention mechanisms can be added and selectivity modified by introducing more polar functionalities to the stationary phase, for example phenyl-, cyano- or triflouromethyl groups.

Chromatographic resolution in GC Today, most GC analyses are carried out using open tubular capillary columns, which provide high separation efficiency. The resolution between two peaks is a function of the number of theoretical plates (N), the separation factor (α) and the retention factor (k) for the last eluting of the two peaks, according to Purnell’s equation:

α − = N ⎛ 1⎞⎛ k ⎞ Rs ⎜ ⎟⎜ ⎟ (1) 16 ⎝ α ⎠⎝ k +1⎠ This equation is based on isothermal conditions, as is all basic gas chromatographic theory. However, a temperature program is often used, which alters the partitioning coefficient (K) during the chromatographic separation. In these cases the calculated values should be considered guidelines rather than absolute values. The number of theoretical plates is a function of the column length (L) and the height equivalent to a theoretical plate (H). L N = (2) H If we assume infinitely sharp initial bands, the separation is limited by band broadening during the separation process.

25 H can be described by the van Deemter equation:

B H = A + + Cµ µ (3) where µ is the linear flow rate. For open tubular capillary columns the eddy or multiple path diffusion term, A, equals zero and the longitudinal diffusion term, B, becomes insignificant at higher flows. Hence, band broadening is a function of the resistance to mass transfer, i.e. the C term, which can be divided into two separate terms: resistance to mass transfer in the stationary phase, Cs and resistance to mass transfer in the mobile phase, Cm. Hence, for capillary GC the height equivalent to a theoretical plate (Equation 3) can be approximated as:

= µ + µ H Cs Cm (4)

Film thickness of the stationary phase For gas chromatographic separation of different compounds, they must be thoroughly dissolved in the stationary phase and sufficient binding sites must be available to retain them. A thick stationary phase film is used to trap and separate volatile analytes. Thin films may cause column overload at high sample loadings due to the low number of sites in the stationary phase. The film thickness of the stationary phase also contributes to the band broadening in the stationary phase (Cs):

2kd 2 C = f s + 2 Ds 3(1 k)

where k is the retention factor for a given analyte, df the stationary phase film thickness and Ds the diffusion coefficient for the analyte. Band broadening in the stationary phase is proportional to the square of the film thick-

26 ness. For columns with very thin stationary phases the Cs term becomes insignificant compared to the Cm term. For PBDE analysis, relatively thick films have traditionally been used to prevent interactions with residual silanol groups, since these active sites could induce degradation. However, in Paper I results are presented showing that increased film thickness actually has a negative effect on the yields of labile congeners. With thicker films the retention times increase and the analytes spend more time in the column at an elevated temperature. In the comparison of two 15 m columns, the retention time for BDE-209 increased from 23.3 to 25.6 minutes, resulting in a 60 % smaller peak area, with a 0.25 µm film than with a 0.1 µm film. From a chromatographic perspective, films that are as thin as possible should be used for the determination of PBDEs, since these analytes have low volatility and are most often found in low concentrations.

Column length According to Equation 2 the number of theoretical plates (N) increases with column length (L) and the resolution is proportional to the square root of N. The retention time (tR) on the other hand, is directly proportional to the column length, according to:

L(1+ k) t = (5) R µ

This means that doubling the column length only increases the resolution by a factor of 1.4, while the retention time increases by a factor of two. Papers I and III describe investigations on the influence of column length on the yields of the thermally labile BDE congeners. The time spent in the column at an elevated temperature increases with column length, resulting in increased degradation of the sensitive BDE congeners. Thus, longer columns give poor yields and longer analysis times, but only minor improve- ments in resolution.

27 Inner diameter Since all other terms in the van Deemter equation are virtually insignificant, H is determined mainly by the resistance to mass transfer in the mobile phase and between the mobile and stationary phases, Cm, which can be described by:

r 2 (1+ 6k +11k 2 ) C = m + 2 (6) Dm 24(1 k)

where r is the radius of the capillary column and Dm the diffusion coefficient for the analyte. The retention factor (k) can be described as

K k = β (7) where K is the equilibrium coefficient at a given temperature and β the volumetric mobile phase/stationary phase ratio. β can be approximated as:

β = r (8) 2d f

Equations 7 and 8 give:

2d f (9) k = K r

From Equation 1 the resolution can be calculated as a function of the column radius, with the help of Equations 2, 4, 6 and 9. The relative resolution for a 100 µm and a 250 µm column is plotted in Figure 3. The resolutions are calculated for equilibrium coefficients of 2000 and 10000 and are relative to that of a 30-meter column, with an inner diameter of 250 µm. For example, a 30 m (i.d. = 250 µm) column corresponds to a 4.5 m (i.d. = 100 µm) column in terms of resolution under isothermal conditions.

28 3.5 250 µm K=10000

3 100 µm K=10000 250 µm K=2000

2.5 100 µm K=2000

1.5

1 Relative resolution 0.5

0 0 5 10 15 20 25 30 35 40 45 50 &ROXPQOHQJWK P

Figure 3. Resolution vs. column length for two inner diameters, 100 µm and 250 µm at two equilibrium coefficients (K). The resolutions are relative to that of a 30 meter column (i.d. = 250 µm).

The retention time depends on the column’s length and radius, the mobile phase flow rate and the equilibrium coefficient. Equations 5 and 9 give:

2d L(1+ K f ) t = r R µ

The equilibrium coefficient (K) is temperature dependent and thus changes during the oven temperature program. For columns of different radii but equal resolution retention times are increased as the bore increases, e.g. retention times are between 6.7 and 2.7 times longer with a 30 m, 250 µm i.d. column than with a 4.5 m, 100 µm i.d. column, Figure 4. Hence, by using columns with smaller inner diameters the analysis times can be reduced while maintaining resolution. This theory have been exploited in practice by the use of narrow bore columns and fast-GC [80-82]. Paper III describes the use of narrow bore columns (i.d. = 100 µm) for the separation of PBDEs. The peak widths were approximately a third of those obtained with conventional columns

P P 5 5 W W

Figure 4. Calculated retentions time ratios between a 30 m (i.d. = 250 µm) column and a 4.5 m (i.d. = 100 µm) column for different equilibrium

coefficients (K). df= 0.1 µm.

(i.d. = 250 µm), and a retention time as short as 6.5 minutes for the last eluting component (BDE-209) was achieved. Such analyses should be classified as fast-GC according to the definitions proposed by Dagan and Amirav [82, 83]. No additional increase in resolution was observed when going from an inner diameter of 100 µm to 50 µm, possibly because extra-column band broadening (for example due to the injection) affects the separation to a greater extent than the in-column band broadening at these diameters. High inlet pressure is an encountered problem with narrow-bore columns, especially when long columns are used to improve resolution. However, in fast-GC shorter columns are used, and the pressures applied are similar to those used for corresponding conventional columns. Nevertheless, narrow bore columns may place extra demands on the other parts of the GC system. Due to low volumetric flows the splitless times should be increased, or a pressure pulse can be applied during the splitless time. Paper III describes the combination of large volume injection via a PTV injector with fast-GC. Initial attempts to combine these techniques using a loop-type injector failed due to severe peak distortion, probably as a result of the large differences in the inner diameter of the retention gap and the column. In addition, the narrow bore columns proved to be incompatible with the TSQ7000 mass

30 spectrometer for determination of the high molecular weight BDE congeners. This was most likely due to the long transfer line used in that particular instrument, causing the analytes to spend relatively long times at an elevated temperature, and thereby increasing thermal degradation.

Stationary phases A large number of stationary phases are available for gas chromatography and an appropriate selection is required to obtain the best resolution and chromatography of the investigated analytes. Paper I reports an investigation of a number of different column brands and types with respect to the separation of PBDEs. It is shown that the choice of column has a major impact on the yield of BDE-209, presumably due to differences in the number or kind of catalytic sites. The DB-1 (100 % dimethyl polysiloxane) and DB-5 (95 % dimethyl and 5 % phenyl polysiloxane) columns proved to be the best choices, whereas an HP-1 column completely degraded decaBDE. Detailed information about the differences of these stationary phases could help to explain the results, but the manufacturing processes are unfortunately confidential. The retention of the BDE congeners was similar or identical on the different investigated non-polar stationary phases. An interesting feature of the semi-polar DB-200 column, which contains 35 % trifluoropropyl, is its ability to separate bis(2,4,6-tribromophenoxy) ethane (BTBPE), a brominated flame retardant, and BDE-191. On non-polar columns, such as the DB-5, these compounds co-elute. The yields obtained using the DB-200 columns were acceptable, but not as high as those obtained with the DB-1 and -5 columns, possibly due to its thicker stationary phase film (0.25 µm).

31 32 Injection techniques for GC While columns and detectors are often investigated and evaluated, the choice of injection technique is often dictated by the injectors available, which in many cases are split/splitless injectors. However, for many types of compounds sample introduction is the most critical step of the whole GC analysis, and yields may be poor if an inappropriate method is used. Thermally labile compounds are easily degraded in hot injectors, placing additional demands on the injection technique if high sensitivity and reproducibility are required. In the studies this thesis is based upon, a large number of injection techniques were evaluated for high-boiling and thermally labile compounds. Thermal degradation was shown to increase with temperature, time and number of catalytic sites (e.g. free silanol groups). These parameters should in all cases be kept as low as possible.

Vaporizing and non-vaporizing injections GC injection techniques can be divided into two fundamentally different categories: vaporizing and non-vaporizing. In non-vaporizing injections the sample is introduced directly into the column as a liquid. Thus, the transfer of analytes to the column is efficient and discrimination low or non-existent. On the other hand, the risk of contamination and column deterioration is considerable. In vaporizing injectors the sample is introduced into a heated chamber, often a glass liner, in which the solvent and analytes are vaporized due to the elevated temperature. This injection process involves three consecutive steps: – Sample introduction: Normally 1-3 µL of sample is introduced into the injector liner utilizing a syringe. Parameters such as injection speed have been shown to be critical [84]. – Solvent vaporization: The solvent is vaporized in the liner and allowed to enter the column. – Analyte transfer: As long as the solvent remains liquid the solutes cannot be heated above the solvent’s boiling point and are therefore transferred to the GC column after full vaporization of the solvent. As the process depends on vaporization, high-boiling compounds may be discriminated against. Matrix residues, which are potentially harmful to the column, are to a large extent deposited on the liner walls.

33 Analyte peak focusing The injected sample solutes are diluted in a large volume of liquid or vapor. In a vaporizing injector the transfer of analytes to the column is often slow. Consequently, the initial bands are broadened in space and time. However, due to focusing of the start bands in the column, gas chromatography generally generates very sharp, narrow peaks, with widths of 1-10 seconds. It is important to consider and understand these refocusing processes. In practice, the combined effects of several of these processes, rather than just one, are usually observed.

Cold trapping In vaporizing injectors the initial bands are partly focused by cold trapping. The difference in temperature between the injector and the GC oven forces the analytes to condense in the first part of the column or the retention gap (if used). This requires the difference between the GC oven temperature and the elution temperature of the analyte to be sufficiently large, 60-90°C [85].

Retention gap focusing Uncoated pre-columns or retention gaps are widely used in on-column and large volume injections to refocus the initial bands, which are broadened in space during sample introduction. The subject has been thoroughly investigated by Grob and co-workers. The analytes move much faster in the retention gap compared to the coated column, due to the large difference in their retention power. Solutes elute at very low temperatures in a retention-gap, but are almost completely immobilized when they reach the stationary phase. Elution from the coated column requires much higher temperatures. Consequently, the analytes are strongly retarded when they reach the coated column and the bands are narrowed. This effect is also known as phase ratio focusing [86-88].

Solvent effects In both vaporizing and non-vaporizing injections the solvent plays an important role in the focusing process. There are two types of solvent effects: solvent trapping and phase soaking.

34 Solvent trapping is best explained using an uncoated column as an example. The solvent/analyte mixture is either injected directly or vaporized and recondensed in the column, Figure 5. The solvent forms a film on the capillary walls, the flooded zone and is subsequently vaporized from the rear end of the film (A). High-boiling analytes are deposited in the column along the flooded zone, whereas volatile compounds are vaporized and re-trapped in the solvent (B and C) until all solvent is vaporized, leaving a narrow initial band (D).

Figure 5. The solvent effect. Volatile compounds (dots) are focused during solvent evaporation, $ 6DWXUDWHGFDUULHUJDV whereas high-boiling compounds (solid gray) are distributed along the flooded zone.

A. The solvent film is formed containing both % volatile and high-boiling compounds. The solvent evaporates from the rear end. The carrier gas is rapidly saturated with solvent vapors. B. The high-boiling analytes are retained on the & capillary walls. Volatile analytes are going into vapor phase and subsequently trapped in the remaining solvent. ' C. The volatile compounds are concentrated in

the last remaining solvent. 9RODWLOHDQDO\WHV D. High-boiling compounds are spread along the +LJKERLOQJDQDO\WHV entire flooded zone. Volatile compounds are 6ROYHQW deposited at the point were the last remaining solvent evaporated.

This is obviously a simplified description, for several reasons. Firstly, the dividing line between volatile and high-boiling solutes is diffuse. Secondly, some compounds are weakly retained by the solvent and will be only partially subject to solvent trapping. Hence, the process is more complex than this theory suggests, and has been subject to extensive investigation, by Grob [88], for example. Phase soaking occurs in the coated column ahead of the flooded zone, Figure 6. The solvent evaporates from the solvent plug at the beginning of the column and is retained in the stationary phase. The stationary phase swells together with the solvent, yielding a soaked zone, in which the retention

35 power is strongly increased. Consequently the solutes are slowed down or trapped as they reach this zone.

Figure 6. Phase soaking. The solvent Carrier Gas soaks the stationary phase and volatile analytes are trapped (A). The solvent is A also retarded in the soaked zone and moves therefore slower at the rear end B than in the front end, where the retardation is lower. The analytes move

together with the solvent (B) until the C velocity of the rear end of the solvent (soaked zone) becomes greater than the Volatile analytes velocity of the analytes (C). Solvent Stationary Phase

The soaking solvent heavily overloads the column, forming a triangular profile, Figure 6A. The front end of the solvent zone moves faster than the rear end, since solvent evaporating from the rear end is slowed down by the soaked stationary phase. Hence, the solvent is trapped by itself. As the solvent spreads in the column, the rear end accelerates until it finally reaches the same velocity as the front end. A given trapped solute will move together with the solvent in a similar manner as in solvent trapping, until the velocity of the rear end of the solvent exceeds the migration speed of the solute. The solute is then left behind, focused in space (C) [88].

Conventional injectors

The on-column injector The non-vaporizing on-column injection is the simplest technique for introducing samples in GC. The sample is injected directly into the column or a retention gap, in which the analytes are focused due to the solvent and retention gap effects. Virtually complete analyte transfer is achieved and no discrimination occurs. Since the injector is not heated, the risk of thermal degradation is minimal. Hence, on-column injection is theoretically the most reliable technique.

36 However, on-column injection is traditionally considered to be difficult to handle in practice, even when using auto-samplers. Needle breakages, damage to columns’ stationary phases and peak broadening have all been major concerns, limiting the practical application of the technique. Even though modern set-ups are much less prone to these problems, on-column injection is often not considered robust. In addition, because of the lack of liners the columns may be damaged by sample matrix residues, such as polar compounds, lipids etc. A retention gap, acting as a guard-column, can be used to increase the life span of the analytical columns, but it needs to be replaced regularly. For determination of PBDEs the on-column injector proved to be superior in terms of discrimination, Paper I.

The split/splitless injector The most common injection technique for GC is to use a split/splitless injector, introduced in the late 1960s by Kurt Grob [89]. This vaporizing injector has gained popularity due to its robustness and capacity for handling “dirty” samples and has become the most widely available and commonly used type 6HSWXP of GC injector. &DUULHUJDV 6HSWXP For trace analysis this injector is LQOHW SXUJHOLQH operated in splitless mode. The sample is )HUUXOHVHDO introduced into a glass liner, normally /LQHU heated to 250-350°C, Figure 7. The 6SOLWOLQH analytes and solvent are vaporized and subsequently transferred to the column as vapors. The split valve is opened, 6SOLWSRLQW typically after 1-3 minutes, and remaining solvent vapors (and analytes) are discarded through the split exit. *&FDSLOODU\ The transfer of analytes to the column is usually high (>80 %), but discrimination against high-boiling Figure 7. Schematic diagram of compounds is a recognized problem. the splitless injector. Potential solutions to this problem include

37 increasing the injector temperature, prolonging the splitless time and applying a pressure pulse during analyte transfer. The use of a pressure pulse compresses the vapor cloud increases the flow into the column, which has comparable effects to prolonging the splitless time. However, even when applying these techniques the split/splitless injector has several disadvantages. Firstly, in spite of the high temperature, the vaporization of the solvent is many cases slower than could be expected. Injected as droplets the sample actually “bounces around” in the liner and proportions of the analytes may travel beyond the split point, or even beyond the liner [90]. Such injector flooding leads to poor transfer of analytes, especially those with high boiling points, to the column. Due to the uncontrolled vaporization large differences in absolute and relative peak areas are often observed, leading to irreproducible results, even when internal standards are used. Liners with obstacles, such as an “inverted cup” [91] or glass wool [92] have been proposed as solutions to the problem. The concept is based on retaining and transferring heat to the solvent. However, the efficiency of this approach has been questioned [93]. Ironically, the obstacles themselves may also cause discrimination, due to irreversible adsorption to active sites. Grob and Biedermann suggest that injection through a hot needle provides the best evaporation conditions, causing the sample to be nebulized in the liner as a thermospray [93]. Secondly, the split/splitless liner represents an “ideal” environment for thermal degradation, providing both heat and catalytic sites on a relatively large glass surface. Since splitless liners are relatively wide the transfer is slow, often taking 1-2 minutes, providing the last of the three requisites for thermal degradation: time. Hence, splitless injection is not suitable for thermally labile compounds such as BDE-209, Paper I.

The programmed temperature vaporizer (PTV) Although the programmed temperature vaporizer has been in use for more than thirty years, it has only recently gained favor among manufacturers and users [94]. A PTV was used in the studies reported in Papers I, III and V. The temperature-programming feature of the PTV allows more controlled injection compared to the traditional splitless injector. When introducing the sample at a temperature around the boiling point of the

38 solvent (±20°C) gentle solvent evaporation is achieved, and the analytes are trapped on the liner walls or on an adsorbent above the split point. By rapidly increasing the temperature the analytes are transferred to the column. Compounds with low volatility are deposited in the liner, so there is less column contamination and deterioration than when on-column injectors are used. The PTV gives significantly higher yields of high-boiling and thermally labile compounds [95]. Analyses of such compounds also benefit from the use of increased pressure during transfer. Operated in temperature programmed pulsed splitless mode (TPPSL) the results are comparable with those obtained with the on-column injector for most of the BDE congeners, Paper I.

At-column injection In this thesis the term at-column injection is used for techniques such as direct injection and use of septum-equipped temperature programmable injectors (SPIs). Their common denominator is the use of tapered liners in which the column can be tightly fitted in a similar way as a press-fit connector. The transfer of analytes to the column is increased compared to a splitless injection, and the use of liners decreases the risk of contamination and column degradation compared to on-column injection. Since the analytes may enter the column both as vapors and dissolved in a liquid solvent, the at-column injection is a combination of vaporizing and non-vaporizing processes.

The septum equipped temperature programmable injector (SPI). An SPI was used in Paper I, for PBDE analysis. With this system, the sample is injected into the cold liner (typically at 50-80°C) close to the column entrance and partly transferred to the column as a liquid. By rapidly increasing the temperature, residues of analytes deposited on the liner walls are transferred to the column. The SPI exhibited high reproducibility and low discrimination. However, since this injector was not available in combination with MS, it was not evaluated for determination of PBDEs in real samples. The SPI has been discontinued, but SPI-like injections can be performed using tapered liners for programmed temperature vaporizers (PTVs).

39 Direct injection At-column injection can also be applied using a common splitless injector, i.e. by direct injection, in which the sample is introduced into a hot, tapered liner. As in normal splitless injection, the solvent vaporization is a slower process than may be expected, despite the high temperature. Consequently, the sample is only partly vaporized and liquids may reach the lower part of the liner. However, analytes both in gaseous phase and dissolved in the solvent will be pushed into the column by the carrier gas, since there is no split point. The solutes are focused on the column or the retention gap by cold trapping and solvent effects. Remaining solvents can be vented out through a drilled hole in the liner [96-98]. The behavior of PBDEs in direct injection systems was studied in the investigation of injection techniques described in Paper I. The results were promising, but the technique was not investigated and optimized thoroughly, and hence it was not included in the paper. Figure 8 shows the discrimination profile for direct injection and splitless injection relative to on-column injection. The yield of the high-boiling and thermally labile BDE-209 was high, but so too was its standard deviation. The precision could possibly be improved by further optimization.

140

120 Direct Injection 100 On-column

40 Splitless Relative Response (%)

0 BDE-7 BDE-49 BDE-99 BDE-153 BDE-203 BDE-209

Figure 8. The relative response for six BDE congeners when using direct injection and splitless injection, normalized to on-column injection.

40 Large volume injection (LVI) Several techniques for increasing the injection volume tolerance of the GC system have been described in the literature. Commercially available instruments for some of these techniques have been developed, as interest in them is increasing among analytical chemists, but only devices built in- house are available for some of the others. Like their smaller volume counterparts, large volume injections can be divided into two categories: non- vaporizing and vaporizing. Non-vaporizing large volume injectors introduce the sample directly into the GC column system, and they can be categorized according to either the instrumental set-up or the solvent evaporation procedure involved. In this thesis the former distinction is used, giving two main types: the on- column-LVI and the loop-type injector/interface. These techniques have been developed and studied in particular by Grob and co-workers [99, 100]. The programmed temperature vaporizer can also be used for large volume injections in solvent vent mode. This technique has been investigated by Mol et al. [101] and Engewald et al. [97], for example. In the studies underlying this thesis loop-type interfaces and large volume PTV injections were used.

Why large volume injection? A feature common to all of the traditional GC injection techniques is that limited sample volumes can be introduced into the system: normally 1-3 µL. This is a major drawback, which restricts the detection and quantification limits of the analytical methods. The sample volume after extraction and clean-up can rarely be decreased to less than about 50-100 µL, with a linear volume-concentration relationship, since the analytes may be deposited on the walls of the vessel. More importantly, the sample composition may be altered if the solvent volumes are too low, since the analytes may have differing affinities for the glass or plastic surfaces of the sample container. Consequently, only 1-6 % of the sample is usually introduced into the GC system with a traditional injection technique. Using large volume injections, larger fractions of the sample can be analyzed and there is no need for analyte enrichment by sample volume reduction.

41 In addition, use of injection volumes as large as 500-1000 µL enables hyphenation with preceding techniques, such as LC, SPE or other types of extraction.

Common misconceptions about large volume injections Various large volume injection techniques have been introduced and evaluated since the late 1970’s, but they are still often met with suspicion. Commonly raised concerns are that the procedure will result in increased contamination of liners, retention-gaps and columns, and the fact that the signal-to-noise ratio is not necessarily increased with concentration. However, this applies not only to large volume injections but also to off-line sample volume reductions. Provided that the same percentage of the sample is injected, the sample matrix affects all injection techniques in the same manner independently of injection volume. Thus, eliminating these problems requires attention to the clean-up methods and the detection, to avoid contamination and interfering peaks as well as to improve the signal to noise ratio, rather than adjustment of the injected volume. For large volume injections careful optimization and evaluation are required. On the other hand this also often applies to traditional injectors. While the former techniques are often well investigated, split/splitless and on-column injectors are usually operated with the same settings, regardless of the analytes, solvents, matrix etc. In many cases much could be gained by optimizing the standard injection techniques.

On-column large volume injection (OC-LVI) Large volume on-column injection systems are based on traditional on-column injectors. The sample is introduced via an auto-sampler or an LC- pump into a retention gap where the solvent is vaporized [102]. Normally the OC-LVI is associated with partially concurrent solvent evaporation, in which some of the solvent is evaporated continuously during the sample introduction [103]. Volatile analytes are retained in the residual solvent and subsequently refocused by the solvent effects. Consequently, the sample introduction rate must be slightly higher than its evaporation rate. This can be ensured by adjusting the injection speed together with the GC oven temperature and carrier gas flow.

42 Note that the solvent evaporation occurs from the rear of the flooded zone, )URP $XWRVDPSOHURU Figure 9. /&SXPS The on-column-LVI system is usually equipped with an early solvent vapor exit, which serves two purposes: to 5HWHQWLRQ JDS increase the evaporation rate and to protect the detector from large amounts &DUULHUJDVDQG of solvent vapor [104]. In this design the VROYHQWYDSRUV retention gap (an uncoated pre-column) is connected to a coated pre-column )ORRGHG ]RQH followed by a three-way connector, joining the pre-column, the vapor exit and the analytical column. During the solvent evaporation stage the solvent vapor exit is open and the solvent vapors are Figure 9. Partially concurrent solvent evaporation. Volatile analytes are eliminated. With this set up the vapor flow trapped in the liquid film and rate can be increased 20-50 fold [100]. refocused by the solvent effect. Volatile analytes are trapped in the remaining solvent or the soaked stationary phase of the pre-column.

Loop-type injector/interface The loop-type interface consists of a 6- or 10-port valve equipped with a loop, in which the sample is loaded, or a fraction from a preceding step (e.g. LC, SPE or other extraction) is trapped, Figure 10. By switching the valve the sample is pushed by the carrier gas into the retention gap, where the solvent is evaporated. While the solvent evaporates an increase in pressure can be observed, and a subsequent pressure drop indicates that the solvent has evaporated. If a vapor exit is used, it is closed some 10-30 seconds later to ensure that all solvent vapors have left the column system. A restrictor is mounted on the vapor exit valve to purge the solvent vapor exit and thus prevent remaining solvents interfering with the subsequent analysis by back- flow. Once the injection is finished and the solvent has been evaporated a normal GC separation is performed [105].

43 B

I CD A H L

E F GK

Figure 10. The loop-type injector/interface. A: Gas flow regulator, B: Injection valve equipped with an injection loop and an injection port or a connection to preceding technique (e.g. LC), C: Carrier gas line, D: Transfer line, E: Retention gap, F: Pre-column, G: Analytical column, H: Vapor exit, I: Vapor exit valve with restrictor, J: Detector, K: GC oven, L: Long carrier gas line placed inside the GC oven.

The carrier gas can never completely empty the part of the retention gap outside the thermostatically controlled oven, instead it leaves solvent residues on the capillary walls. The remaining solvent leaks into the column system during the analysis, resulting in a broad, tailing solvent peak that interferes with the analyte peaks if a general detection system, such as FID or fullscan EI-MS is applied. To avoid this, separate carrier gas and transfer lines are used, Figure 10C and D. The carrier gas is diverted to the transfer line during injection and to the carrier gas line during analysis. The injection valve is equipped with a restrictor to enable purging of the transfer line. With a more selective detector a common line from the injection valve into the GC oven and the column system can be used, Paper II. Once appropriate conditions have been established, large volumes, e.g. from 100 µL to several mL, can be injected without modifying the parameter settings.

44 Concurrent solvent evaporation The large amounts of solvents injected are managed through a process called concurrent solvent evaporation [106], Figure 11. When the solvent reaches the thermostatically controlled GC oven, it starts to vaporize. The solvent is then pushed back by its own vapor pressure, preventing it from reaching the coated GC column, provided that the injection temperature, i.e. the GC oven temperature, is kept at the pressure-corrected boiling point of the solvent, or higher. At these settings the evaporation site in the retention gap is focused in space within about 0.5 m. Therefore a short retention gap can be used, normally 2-3 meters, which gives sufficient buffer space to avoid column flooding.

&DUULHUJDV

Figure 11. Fully concurrent solvent evaporation. The solvent evaporation occurs at the front end of the 5HWHQWLRQ solvent plug. Solvent trapping does not occur to any JDS large extent, so volatile compounds are either trapped in the pre-column or lost through the vapor exit. )ORRGLQJ VROYHQW

6ROYHQW YDSRUV

Since the evaporation occurs from the front end of the solvent plug, no solvent trapping is achieved, which affects volatile analytes, see deformation of early eluting peaks, below. Similarly to OC-LVI, a solvent vapor exit is often used together with a loop-type injector, to increase the evaporation rate. The retention gap (Figure 10E) is connected to a pre-column (F), which in turn is connected to the solvent vapor exit (H) and the analytical column (G) with a Y-connector. Analytes with high boiling points will be deposited in the retention gap, whereas volatile compounds are trapped in the pre-column. The trapping capacity of the pre-column is however limited since no significant phase soaking occurs, and volatile analytes may be lost unless a co-solvent is used.

45 Peak deformation One of the drawbacks of the loop-type is the risk of peak deformation, three types of which may occur, as investigated in Paper II and discussed below.

Peak splitting As described above, due to inappropriate temperature settings the sample may fully or partly flood the coated (pre-) column. A too low temperature generates an insufficient pressure in front of the solvent plug, which consequently moves forward and finally floods the column. Different portions of the sample will then have different starting points in the retention gap and the column, resulting in each peak being split into two or more peaks. In some cases a too high injection temperature may also produce this effect, due to uncontrolled evaporation shooting the solvent into the coated GC column. The remedy is to adjust the injection temperature so that it is at the pressure-corrected boiling point of the solvent. An extreme case of the peak splitting phenomenon is illustrated in Figure 12.

53oC

55oC

54oC

Figure 12. Peak splitting. The selected part of three PAH chromatograms show peak splitting for flourantene, pyrene and benzo(a)fluorine. Gaussian peaks are obtained at 54°C, whereas the peaks are distorted at erroneous settings, i.e. 53 and 55°C.

46 Broadening of early eluting peaks In concurrent solvent evaporation the solvent evaporates from the front end of the solvent plug. At this point the temperature in the oven is at least as high as the boiling point of the solvent. As the vapor moves into the coated columns its boiling point decreases due to the pressure drop along the column system. Hence, little re-condensation of the solvent occurs and thus the soaking of the stationary phase in the coated pre-column is poor, limiting the retention capacity. Consequently, volatile compounds start eluting during the injection and solvent evaporation, resulting in band broadening. Volatile compounds can also be lost through the vapor exit together with the solvent vapors, especially if high injection temperatures are used. To prevent the described effects the use of co-solvents has been proposed. Grob and Müller used n-heptane in n-pentane to trap and focus early eluting compounds [107]. The higher boiling solvent (n-heptane) forms a thin film on the capillary walls in front of the evaporation site, trapping volatile analytes. For the same reasons, Noij and Kooi injected 100 µL of n-decane before the injection of the sample, which was dissolved in a mixture of MtBE and ethyl acetate [108]. In the study presented in Paper II n-dodecane was added to the primary solvent (n-hexane) to sharpen the peaks of the early eluting analytes. A concentration of 0.1 % was enough to trap the lowest boiling analytes, i.e. monoBDE and phenanthrene. Due to the large difference in boiling points between the solvents (∆t = 147°C) n-dodecane in this case acts as a keeper rather than a co-solvent. Dodecane did not evaporate to any significant extent during the solvent evaporation, but was eluted after closure of the vapor exit, during the GC oven temperature program, giving a late eluting solvent peak. However, the amount reaching the detector was low; e.g. only 1 µL in a total volume of 1 mL. A keeper does not require the injection to be re-optimized, nor does it affect the elution strength of the solvents in a preceding LC separation. In addition, the risk of the small remaining volume of dodecane flooding the coated column is small. This keeper technique cannot be applied when analyzing volatile compounds. Corresponding peaks (up to C14 in an alkane series) will simply be lost in the dodecane solvent peak. This is an obvious drawback, but for semi-volatile compounds such as those investigated in the work underlying

47 this thesis (the low molecular weight PANHs, PAHs and PBDEs), adding a keeper is a simple and efficient way to obtain sharp, early eluting peaks. If higher concentrations of dodecane are used, the pre-column may no longer be necessary since the retaining effect of the keeper may be enough to trap the low-boiling analytes [100]. Excluding the pre-column increases the solvent evaporation rate and thus reduces the injection time. It also simplifies the column system.

Trailing peaks In these studies, the most common type of peak deformation was a trailing peak following the main peak of all eluted compounds. The source of this peak deformation was identified by separately analyzing the contents of the carrier gas line, the transfer line, the retention gap, the pre-column, the vapor exit and the analytical column after an injection. The trailing peaks proved to originate from the carrier gas line. When a loop-type interface with separate transfer and carrier gas lines is used, some of the solvent and the solutes contained therein will inevitably shoot up through the carrier gas line during injection. The analytes are then deposited in the capillary outside the GC oven and subsequently eluted, probably due to indirect heating of the carrier gas line. To solve this problem the first Y-press-fit connector was moved inside the oven and a 5 m carrier gas line, Figure 10L, was used. Even though some of the sample may enter the carrier gas line it is prevented from going beyond the thermostatically controlled GC oven with this set-up, and will accordingly be focused on the pre-column. If a selective detector is used, the transfer and carrier gas line can be combined into a common line. Trailing peaks are also avoided with this design. However, even though not detected, tailing solvent peaks may affect the detection and lead to increased contamination of the detector, which can reduce both the lifespan of a mass spectrometer’s ion source filament and the sensitivity the detector.

Why loop-type? The loop-type interface is well suited for large volume injections when determining PBDEs and carbazoles. Since neither the most low-boiling

48 PANHs nor PBDEs should be considered volatile, the risk of losses through the vapor exit is low. The set-up is simple; no pump or auto-sampler is required for offline methods and the pump flow in hyphenated techniques is variable. In addition, PBDEs have proven to be thermally degraded and may be adsorbed by active sites in retention gaps, which should therefore be kept as short as possible, Paper I. On-column-LVI is beneficial for volatile compounds and requires a stable, non-pulsed and predefined sample introduction flow. In addition the retention gap is usually several meters long.

Large volume splitless injection A hot splitless injector can be used for injecting large volumes of samples. The liquids are kept in place by a packing material, preventing them from reaching below the split point. The cooling effect of the evaporating solvent retains the volatile analytes at the site of evaporation, which requires a packing material of low thermal mass, e.g. glass wool. After full solvent vaporization the packing material is heated to the injector temperature and the analytes are transferred to the GC column. The solvent vapors can be discarded by the overflow technique, through the septum purge line. The septum purge is closed prior to full vaporization of the solvent [109]. The process resembles that of a large volume PTV injection. The solvent vapors may also be discarded by concurrent solvent recondensation, through the column. For this, the GC oven is kept at a temperature below the boiling point of the solvent, which is recondensed in the GC capillary. With this set-up the vapors enter the column at the same rate as they are formed in the injector [110]. Both these techniques utilize packing materials, primarily glass wool, making large volume splitless injection unsuitable for determination of PBDEs, as described in Paper III. Different cup-liner designs are promising, but are not yet established alternatives for preventing the liquid from flooding the inlet [111].

49 Large volume PTV injection The programmed temperature vaporizer, operated in a so-called solvent vent mode, can be used for large volume injections. The sample is introduced into a liner at a low temperature (e.g. 50-80°C) with the split valve open, and the solvent is evaporated and discarded, but the analytes are trapped on the liner walls or on a packing material. The analytes are then transferred to the column by closing the solvent split and rapidly increasing the injector temperature, typically to 275-350°C. The robustness of this type of injector and its ability to handle dirty samples [112, 113] prompted further investigation. Large volume PTV injections were successfully used in the studies presented in Paper III and V for the determination of PBDEs. Although evaluation and careful optimization was required, it proved to be a suitable technique even for BDE-209, in contrast to earlier assertions [114].

Sample introduction and solvent evaporation Sample introduction may be accomplished with either multiple injections or continuous injection [97]. In multiple injections mode, several small portions are injected at fixed intervals. This method is simple and fully compatible with common auto-samplers. After the injection of a small volume (normally 5 µL) time is allowed for full evaporation of the solvent to prevent flooding of the injector. Multiple injections do not allow hyphenation. Furthermore, a complete sample introduction cycle must be performed for each portion. Irreproducibility in the injection may be cumulative, resulting in poor precision, and rates of wear of septa and injection ports are drastically increased. If the sample is injected continuously the solvent is evaporated concurrently. A continuous injection may be performed using either an auto- sampler or a pump, and it allows hyphenation. As with all modes of vaporizing injection, the solvent evaporation must occur above the split point in the liner. If sample components are deposited below this point transferring analytes is difficult, as they are not covered by the main stream of the carrier gas. Adsorption and thermal degradation may also be induced by the active sites outside the liner. In extreme cases material is lost through the split line together with solvent still in liquid state. The solvent evaporation is affected by four parameters: the split flow,

50 the injector temperature, the sample introduction flow rate and the evaporation surface area [115-117]. To promote solvent evaporation the first two of these parameters should be kept high. Solvent evaporation obviously increases with temperature, while an increased solvent vent flow more rapidly discards the solvent vapors, Papers III and V. For both multiple and continuous injections the ratio of the sample introduction to the evaporation rates is important. The yields of the congeners were strongly affected by the sample introduction rate. At both too low and too high settings the response decreased. To investigate this further, the fate of the solvent was visually observed by continuously introducing a solution of perylene into a glass liner illuminated with UV-light. The injection followed three main scenarios depending on the sample introduction, solvent evaporation and flooding characteristics, Figure 13.

Figure 13. Solvent evaporation in a multibaffled PTV liner. Injections of solvent containing perylene were performed at 80°C and monitored under UV-light. E A: Schematic diagram of the liner, with G needle (E) and GC column and splitpoint (F).

B: Sample introduction flow rate: 100 µL/min. The solvent leaves the needle as droplets (G) and evaporates at the first baffle before the next droplet has left the needle (H). H I C: Sample introduction flow rate: 300 µL/min. The solvent leaves the needle as a non-visible spray and evaporates continuously at the first baffles. A liquid film is formed, which traps volatile analytes (I).

D: Sample introduction flow rate: 500 µL/min. The sample introduction rate exceeds the F solvent evaporation rate, resulting in J flooding (J). ABCD

51 A high sample introduction flow rate led to insufficient solvent evaporation and, hence, to injector flooding, as illustrated in Figure 13J, resulting in losses of material and discrimination, as described above in the discussion about the splitless injector. Too low injection flow rates also led to losses of solutes. Staniewski and Rijks suggested this was due to the absence of a liquid solvent film in the liner, causing the solutes to be weakly retained and consequently lost [118]. This theory was supported by the visual observations. At 100 µL/min the solvent leaves the needle as droplets, each of which are evaporated in the liner before the next leaves the needle, Figure 13G and H. This rationale satisfactorily explains the behavior of BDE-47, but not that of compounds with high boiling points such as BDE-209. The effect of low introduction flow rates could not be fully explained by the results of the studies this thesis is based upon. Optimal injection parameter settings depend on the solvent boiling point. As illustrated in Figure 14, n-pentane and n-hexane have different optimal injection flow rates, since they have different boiling points: 36 and 69°C, respectively. When using n-hexane, the highest yields are obtained at an injection flow rate of 300 µL/min, at which the evaporation is optimal, forming a steady film in the liner baffles, Figure 13I. The injection flow rate is not only important for the set-up used in Papers III and V. Figure 15 shows the relative responses of six BDE congeners for fast multiple

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Figure 14. Detector response for BDE-209 as a function of sample introduction flow rate. The optimal settings differ for n-pentane and n-hexane due to the different boiling points.

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Figure 15. Relative response for six selected BDE congeners injected with a conventional syringe. Grey bars: fast multiple injections (10x10 µL). White bars: slow continuous injection (100 µL/min). injections and a continuous injection at 100 µL/min, made using a syringe. With the latter technique the yield and precision were clearly lower for BDE-209. With auto-samplers small injection volumes are easier to inject at high speeds, so multiple injections in this case are preferable for the determination of PBDEs. Obstacles in the flow path, e.g. baffles and cups, prevent the solvent from being flushed through the liner. Glass wool, optionally combined with packing materials such as Tenax, is commonly used in PTV liners for large volume injections. Apart from trapping volatile compounds, the packing material hinders the solvent and provides an increased surface area from which the evaporation and heat exchange can take place. On the other hand, compounds with high boiling points may be difficult to desorb, resulting in poor yields in the subsequent transfer. Thermal degradation may also be catalyzed at active sites in the materials [98]. Mol et al. investigated a number of different liners and packing materials with respect to degradation and

53 discrimination [119]. Tenax and glass wool in some cases caused complete loss of the analytes. For high-boiling and thermally labile compounds the cup-liner gave the highest recoveries. In study III different liners were investigated for large volume PTV injections of PBDEs. Cup-liners were unfortunately not available for the PTV used (a Gerstel CIS-4). Instead, an empty multi-baffled liner proved to be the best choice, providing low discrimination and high precision.

Analyte trapping During solvent evaporation volatile analytes can be lost through the split exit together with the solvent. There are several ways to handle this problem. By means of partial concurrent solvent evaporation during speed-controlled injection a small portion of the solvent can be kept in a liquid state, in which volatile analytes are trapped. If multiple injections are to be used the technique is more complicated, as a small amount of solvent has to remain liquid after each sample introduction. As discussed in the previous paragraph, packing materials, such as Tenax and glass wool, are more commonly used to trap the volatile analytes [120-122], but they are accompanied with clear risks of discrimination and thermal degradation [119]. These problems are addressed in Paper III. The yield of BDE-209 was significantly reduced when utilizing glass wool or sintered glass liners as trapping agents compared to an empty liner. Instead, n-dodecane was used as a keeper to retain the low boiling point monoBDE and triBDE. As for the loop-type injector, an addition of 0.1 % n-dodecane in the primary solvent (n-hexane) was enough to trap these compounds. Co-solvents have also been utilized by Termonia et al., who showed that 15 % n-octane in n-hexane can be used to trap low-boiling compounds, such as biphenyl [123].

Analyte transfer Once the solvent has been eliminated the analytes are transferred to the column, by closing the split valve, increasing the temperature and (optionally) applying a pressure pulse. Ideally the transfer temperature should be as high as possible to desorb and transfer even analytes with high boiling points. On the other hand, the risk of degradation of thermally labile compounds

54 increases with temperature. In Paper III a temperature of 325°C was shown to be optimal for transferring the high molecular weight BDE-209. As in splitless injection, the transfer rate can be increased if a pressure pulse is applied during the splitless time. This was shown to increase the yield of BDE-209 up to four fold. The transfer proved to be fast, even for the high-boiling BDE-209. After approximately 35 seconds the maximum yield was obtained. The short transfer time is not surprising considering the small inner diameter of the liner and the applied pressure pulse.

The author´s note on of the loop-type and the PTV injectors The advantages and disadvantages of the loop-type and the PTV injectors are summarized in Table 3. The loop-type injector exhibited low discrimination against high molecular weight congeners and high precision with regard to absolute and relative responses of the PBDEs. However, the retention gaps of this set-up had a limited life span even when analyzing relatively clean air samples. The junctions of the capillary columns system require special attention and operator experience to avoid leaks. The discrimination against high molecular weight congeners, using PTV injector, was low but significant. The transfer efficiency of BDE-209 was in the range of 76-80 %. On the other hand the PTV is rugged and robust towards sample matrix residues. Approximately 100 large volume injections of air sample extracts were carried out, without need for replacing the liner. Hence, the choice of injection technique stands between low detection limits and robustness.

Table 3. Advantages and disadvantages of loop-type and PTV injection.

Loop-type injector PTV Discrimination against high-boiling compounds None Low Thermal degradation None Low Robust towards matrix residues Low High Ruggedness Intermediate High Instrumental complexity High Intermediate Commercially available No Yes

55 56 GC detectors Numerous types of gas chromatography detectors have been developed to meet the wide range of GC demands and applications. The requirements for sensitivity are met by virtually all GC detectors, with detection limits in the fg-ng range. Selectivity towards compounds containing specific elements, for example Br and N, enables detection and quantification in the presence of chromatographically interfering species. In addition, since the chemical background is vastly reduced, detection limits are in most cases improved. High-selectivity systems improve the determination of certain compound classes, but similarly reduce the amount of information that can be obtained about the sample. In general, GC detectors do not provide structural data, with the exception of mass spectrometers. In this section the detectors utilized in the work underlying this thesis are discussed.

The flame ionization detector (FID) The flame ionization detector is the most generally applicable and one of the most widely used types of detector for gas chromatography. The critical parts of the FID are the hydrogen/air flame and the collector. Most carbon- containing compounds eluting from the GC column are ionized in the flame and a signal is generated as the ions are picked up by the collector [124]. The FID is fairly sensitive and has a wide linear range. In addition it is easy to use and requires minimal maintenance. The major disadvantage of the FID is its poor selectivity, which places high demands on the sample clean-up. In the studies this thesis is based on the flame ionization detector was used only as a reference detector.

The nitrogen phosphorus detector (NPD) The nitrogen phosphorus detector (or thermionic detector, TID) is selective towards nitrogen- and phosphorus-containing compounds. As with the FID, the NPD is supplied with hydrogen, air and nitrogen, but due to the low flows no flame is ignited. Instead, the formation of ions takes place in a

57 boundary layer, formed around a heated alkali salt bead. The mechanism of the NPD is not fully understood, but it is believed to involve decomposition of the eluting compounds to CN, PO and PO2 [125, 126]. These radicals form ions by extracting electrons from the alkali salt present in the bead. A detector signal is generated as the ions are picked up by the collector. The gas flows are critical for the detector’s performance. Fine-tuning, especially of the hydrogen gas flow, gives selectivity towards phosphorus- or nitrogen-containing compounds. In Paper IV these settings were optimized with respect to sensitivity and selectivity for detecting carbazole-type PANH.

The electron capture detector (ECD) The electron capture detector is widely used in environmental analysis, due to its selectivity towards halogenated compounds, such as PCBs and PBDEs. A nickel-63 foil emits electrons (β-radiation), which interact with the make- up gas (normally nitrogen) to produce thermal electrons, i.e. electrons with lower energy.

β → β + - + N2 *+ N2 + e where the kinetic energy of β* is lower than that of β. The thermal electrons produce a constant current between two electrodes in the detector. Effluents with electronegative functional groups, e.g. halogens and nitro groups, capture thermal electrons causing a drop in the current, which is registered as the detector signal. Even though the ECD is based on indirect detection, it is very sensitive. The limits of detection for PBDEs are in most cases in the sub-picogram region. ECDs were used for evaluation of different injectors in studies I, III and V.

Mass spectrometry (MS) The mass spectrometer provides more information, selectivity and in many cases more sensitivity than any other type of GC detector. Although more expensive and complex than the alternatives, MS is rapidly gaining ground and may even be the most widely used detection technique for GC today.

58 In gas chromatography the analytes are eluted in a gas flow, which makes the mobile phase easy to discard and simplifies hyphenation to MS. The mass spectrometer consists of three fundamental parts. The ion source is a more or less closed vessel where the effluents are ionized. A filament placed just outside the ion source emits electrons, which are essential for the ionization. Generally, different ion sources are used for electron ionization and chemical ionization. The analyzer may be any one of various types, but all have the same purpose: to separate the ions according to their mass to charge ratio (m/z). High-resolution instruments (e.g. time of flight, double focusing sector and fourier transform ion cyclotron resonance analyzers) provide a resolution higher than 5000 (m/∆m) and the option of accurate mass measurements. However, these systems are expensive and in many cases more difficult to operate than simpler alternatives. In the studies this thesis is based upon, a quadrupole analyzer was used, which together with the associated ion-trap constitutes low-resolution MS. In quadrupole systems, ions with different m/z values are discriminated by applying both a direct potential (U) and an altering potential (V) between four rods. The altering field gives the ions entering the quadrupole chamber a trajectory, which is either stable or unstable, depending on the settings. Only ions with stable trajectories can pass through the chamber and be detected. The resolution is determined by the U/V ratio, whereas the discrimination between ions with different m/z ratios is determined by the absolute settings of U and V. The quadrupole is relatively inexpensive and robust, but has low mass resolution (∆(m/z) is generally 1 Th) and is comparably slow. The latter disadvantage can be alleviated if the analytes are eluted in extremely narrow peaks, as they are with narrow bore columns (Paper III) and flash GC. In these cases fullscan spectra may not be representative, so selected ion monitoring (SIM) has to be applied. The detector is usually an electron or photo multiplier. When an ion collides with such a detector, a number of electrons (or photons) are released. These in turn collide with a dynode, releasing more electrons that collide with the next dynode, releasing more electrons, and so on, finally generating a measurable current: the detector signal.

59 Electron ionization (EI) In electron ionization the effluent from the GC column is bombarded with highly energetic electrons, normally 70 eV, emitted from the ion source filament. The process results in radical formation followed by fragmentation of the analytes due to the excess energy:

M + e- → M•+ + 2e- M•+ → A+ + B• and A• + B+ M•+ → A•+ + B and A + B•+

EI generally gives a high level of structural data and unique fragmentation patterns for each compound or group of compounds. The process is reproducible and to a large extent independent of the instrument, so generic databases can be utilized for spectra interpretation. Electron ionization mass spectrometry was used in study IV to confirm the identity of the carbazoles determined in real air samples by LC-GC-NPD.

Chemical ionization (CI) Chemical ionization is used as an alternative or supplement to EI. It is a softer technique yielding less fragmentation, which may give additional structural information as well as being beneficial for sensitivity and selectivity. The CI process involves the ionization of a reagent gas, e.g. methane, isobutane or ammonia. Compared to EI, the CI ion source is more closed to provide a higher ion source pressure. The electrons emitted from the filament collide with the reagent gas and the products formed react with the effluents from the GC column. Ionization and fragmentation depend on the structure of the analyte.

Electron capture negative ionization (ECNI) In ECNI-MS the reagent gas serves as a buffer, slowing down the electrons emitted from the filament (1). These thermal electrons are captured by the analytes in either an associative (2) or a dissociative manner (3).

60 - → - e + CH4 eth + CH4* (1) - → - AB + eth AB (2) - → - AB + eth A + B (3) where the kinetic energy of CH4* is higher than that of CH4. Brominated compounds such as the PBDEs investigated in studies I-III, V and VI mainly undergo dissociative electron capture, forming free bromine ions. Hence, PBDEs have traditionally been determined by ECNI, monitoring m/z 79 and 81. The technique has excellent selectivity towards brominated compounds and low detection limits. However, the structural information obtained is minimal and since only bromine ions are detected isotopic dilution cannot be applied. The mass spectrometric properties of PBDEs are presented in Paper VI. The investigation revealed that BDE-209 shows a different fragmentation pattern from those of BDEs with lower molecular weight. By uptake of one electron and cleavage of the ether bridge a large fragment at m/z 486/488 is - formed, corresponding to C6Br5O , Figure 16. Monitoring this fragment allows the use of isotopic dilution for quantification. 13C-labeled BDE-209 shows the same fragmentation pattern, with ions at m/z 492/494. To avoid interferences in the mass spectra, m/z 486 and 494 should be recorded for the native BDE-209 and the 13C-labeled BDE-209, respectively. If assuming that discrimination and degradation, for instance, affect the native BDE-209 and the 13C-labeled BDE-209 in a similar manner, the latter compound is more reliable as internal surrogate standard than for example the commonly used BDE-119 for the problematic determination of BDE-209, as described in Paper II and VI. In addition, by monitoring higher m/z fragments the signal to noise ratio is increased, since interfering ions are less likely to be present in the higher mass region than in the m/z 79 - 81 region. The detection limit for BDE-209 was consequently decreased. - Nona- and octaBDE showed similar behavior, yielding C6Br5O or - C6Br4O fragments. The abundance of these ions in relation to the bromine ions was lower than for BDE-209. Nevertheless, increased or similar signal

61 to noise ratios were observed when analyzing real samples as well as a technical PBDE mixture for congeners 197, 206, 207, 208 and an unidentified octaBDE. As for BDE-209, the presence of these fragments makes isotopic dilution applicable to these compounds too [127].

100 488.2 486.2 Br Br 90 78.5 471 O 80.5 Br Br 486 80 488

Br Br Br Br 70 Br 407 Br 60

50 490.2 484.2

Relative Abundance 40

407.2 20

147.4 10 160.4 326.9 471.3 174.4 391.2 563.6 641.2 719.2 797.5 0 100 200 300 400 500 600 700 800 900 m/z

Figure 16. Mass spectrum of BDE-209. Two large fragments are formed; at m/z 79 and 81 (Br-) and around m/z 486/488. Monitoring the latter fragment enables isotopic dilution.

62 Sample extraction Although some samples can be analyzed without clean-up, most analytical methods involve extraction of target analytes in some way from the sample matrix. A large variety of techniques is available for different applications. One of the most important distinctions between them concerns the state (gaseous, liquid or solid) of the samples they are designed to handle. This section focuses on techniques used for extracting target analytes from the particles collected on glass fiber filters and analyzed in the studies associated with this thesis. The aim of any extraction is to transfer target compounds from a matrix (which may have virtually any degree of complexity) to an extraction medium, e.g. an organic solvent. In some cases selective extractions of one or a group of compounds are possible, but normally the process is more general and subsequent chromatographic steps and/or selective detection are required. All forms of extraction depend on the partitioning between the sample matrix and the extraction media. Like any chemical reaction the extraction processes may be under either thermodynamic or kinetic control. Since an extraction includes several steps, e.g. transfer of target compounds to matrix surfaces, desorption and diffusion in the extraction media, the overall process may involve a combination of thermodynamically and kinetically controlled steps. Briefly, a thermodynamically controlled extraction depends on the coefficient of the matrix-solvent-equilibrium for a given compound, i.e. the partitioning coefficient, while a kinetically controlled extraction is limited by the desorption rate from the matrix to the solvent [128]. Heating the extraction media is a common way to increase recoveries. In general, higher temperatures favor partitioning of the analytes to the solvent both thermodynamically and kinetically, because the solvent’s capacity for dissolving target compounds usually increases with temperature and the viscosity of the solvent is reduced, increasing the mobility of dissolved compounds [129]. Consequently, the resistance to mass transfer is decreased. Heated solvents also have enhanced ability to swell and permeate the matrix [130].

63 Static and dynamic extractions Static extractions are performed in a closed vessel with a predefined volume of solvent. The recovery of a given analyte depends on the partitioning coefficient between the matrix and the extraction medium as well as the extraction rate and time. The extraction is often time consuming and the target compounds must be partitioned into the solvent to a high extent. Consecu- tive extractions are normally performed to increase recoveries. In dynamic extractions the extraction media are flushed through a sample-containing vessel. Since fresh solvent is continuously supplied, the equilibrium will be driven towards the solvent and the partitioning coefficient becomes less important. If thermodynamically controlled, a dynamic extraction will consume less solvent and time. However, if kinetically controlled and an extended time is required, dynamic extraction may actually consume more solvent than the corresponding static method, unless the dynamic extraction is combined with static steps. Dynamic extractions normally require rather sophisticated instruments. The equipment is not usually disposable, which may lead to memory effects. On the other hand, dynamic extractions generally enable extraction profiles to be monitored and on-line transfer to chromatographic analysis systems.

Soxhlet extraction Even though introduced as early as 1879, Soxhlet extraction is still one of the most commonly used extraction techniques. The set-up is simple, consisting of a round flask, a cooler and the extractor, which allows many extractions to be performed in parallel without significantly increasing costs. The sample is placed in the extractor and the flask is filled with solvent, which is heated to its boiling point. The sample is continuously flushed with fresh solvent that has condensed in the cooler, and extracted compounds are enriched in the flask. Soxhlet extraction is thus dynamic, but can at the same time be run for many hours, without increasing solvent consumption. However, the technique has several drawbacks. Generally, elevated temperature yields higher recoveries from complex matrices, but in Soxhlet extraction the solvent is cold.

64 Consequently, the extraction is slow, commonly with extraction times of 8-48 h. There is also a risk of adsorption to active sites on the large glass surfaces and degradation of labile compounds as the extract in the flask is heated for several hours. For these reasons Soxhlet extraction was not selected as a reference method in Paper V, although this is a common approach. Soxhlet extraction was used in the analysis of carbazoles in air samples, Paper IV. During evaluation of the method using spiked filters, benzo(b)carbazole proved to be degraded if the temperature of the heater was too high, illustrating one of the mentioned drawbacks of the technique.

Ultrasonication-assisted solvent extraction Ultrasonication-assisted extraction is today one of the most popular extraction methods, due to its simplicity and the possibility it offers to run many samples in parallel. The sample is placed in a vessel, e.g. a test tube, and a solvent with suitable properties is added. The extraction is performed under ultrasonication to enhance the mass transfer of target analytes to the solvent and to disrupt the matrix physically. In most cases two or more consecutive extractions are carried out to increase the recoveries. Ultrasonication-assisted solvent extraction was used by Sjödin et al., for example, for the determination of flame retardants, including PBDEs, in indoor air [47]. Ultrasonication-assisted solvent extraction was used in studies I-III, V and VI for the extraction of PBDEs from glass fiber filters, and in study V to evaluate dynamic sonication-assisted extraction.

Dynamic sonication-assisted solvent extraction Paper V presents a dynamic sonication-assisted extraction (DSAE) method for determination of PBDEs in air. The technique was developed by Sanchez and co-workers for the extraction of organophosphate esters from glass fiber filters [131]. The set-up is simple, robust and can be assembled from unmodified equipment that is available in most laboratories. The sample is loaded into an extraction cell, lowered into a thermostatically controlled ul- trasonic bath and the pressurized extraction medium is flushed through the cell using a HPLC pump.

65 In this study indoor air particles collected on glass fiber filters were used. The results show that an elevated temperature significantly increased the recoveries for BDE congeners of low and intermediate molecular weight, i.e. 47, 154 and 183. Slightly lower recoveries were obtained for BTBPE with ultrasonication and for BDE-154 and BDE-209 with ultrasonication in combination with an elevated temperature. This study was performed on filters cut into small pieces. When extracting large, intact parts of a filter both the recoveries and the precision were improved by ultrasonication. A conclusion drawn from these results was that ultrasonication is important for mixing the sample matrix and solvent. Channeling in the solid sample may lead to poor solvent penetration of parts of the matrix decreasing the extraction efficiency and reproducibility. The use of ultrasonication solved these problems. The average recovery from real samples using the DSAE set-up was found to be 95 %. This was determined by first performing a dynamic extraction followed by exhaustive static extraction of the same sample. The results were reproducible with an average RSD of 2.8 %. The recoveries could possibly be increased by adding a static, stopped-flow step to provide more time for the extraction.

66 On-line coupling to GC Throughout much of the history of analytical chemistry, automation and hyphenation have been intensively researched. For instance, chromatographic and mass spectrometric systems have been successfully coupled, and widely applied. Methods for multidimensional chromatography and on-line clean- up are also attracting interest and in some cases commercially available systems have been introduced. The high separation efficiency and wide variety of sensitive and selective detectors available make gas chromatography the first choice analytical method for many applications. However, the analysis is often preceded by sample preparation steps, of varying complexity, for example extraction, group isolation and removal of polar material and lipids. Besides decreasing the detection limits, one of the most attractive features of large volume injections in GC is the possibility it offers for hyphenation with clean-up techniques. Liquid chromatography [132], solid phase extraction [133] and an array of other extraction procedures [134] have been successfully coupled to gas chromatography.

Benefits of hyphenation Whether the technique preceding the gas chromatographic analysis is LC, SPE or another form of extraction, a larger portion of the sample will be introduced into the GC column than if an off-line method and conventional GC are used. This is a result of the large volumetric loading capacity of the clean-up method and the large-volume GC interface. Moreover, apart from carry-over the risk of contamination in a closed system is minimal and limited to contaminants in the solvents used, which can easily be tested for impurities. On-line techniques also minimize losses, which can be severe with off-line methods as a result of accidental spillages, incomplete sample transfers from one container to another and co-evaporation with the solvent during concentration steps. The overall detection and quantification limits are consequently decreased, making hyphenated techniques well suited for trace analysis. A fully automated instrument also offers better control over the complete analytical method. Parameters such as solvent and gas flow rates, temperatures, event times and fraction collection can be precisely regulated and may be

67 recorded. The sample preparation is simplified and time consuming manual clean-up steps can be reduced to loading an auto-sampler. This makes on- line methods more reliable, reproducible and time efficient than their off- line counterparts.

Concerns about hyphenated systems The risk of memory effects is significantly higher in a system consisting of tubing and valves rather than disposable glassware and pipettes. Careful washing procedures must be employed and system blanks regularly checked. If extractions are coupled directly to GC the entire sample will be injected and analyzed. This leaves no option for repeated runs and further investigations unless the sample is divided into portions, which may be possible with samples of matrices such as blood or sediment, but more difficult with air samples, as described in Paper V.

LC-GC One of the most interesting applications of large volume injections for GC is to couple LC on-line to GC. The combination of the two chromatographic methods offers a technique with truly orthogonal separation systems. The first on-line coupled LC-GC instruments were described in the mid-1980s [102, 135, 136]. Since then the technique has been developed, in particular by Grob, to include a large variety of interfaces and target compounds as well as LC parameter settings, such as mobile phases and flows [100]. A detailed description of LC-GC is beyond the scope of this text, but has been thoroughly covered elsewhere [99, 100, 132]. A common concern for all attempts to hyphenate these techniques is the interfacing problem. Even capillary LC with flows at about 10 µL/min yields fraction volumes that are too large for conventional GC injections. Hence, large volume GC injections, described above, are crucial elements of LC-GC.

NPLC and RPLC The non-polar, volatile solvents used as mobile phases in normal phase LC (NPLC) make hyphenation to GC relatively straightforward. Its compatibility with GC has made NPLC (or normal phase SPE) the most widely used clean-

68 up method for GC analysis, so many existing methods can be easily transferred to on-line techniques. A variety of solvents with low boiling points, such as n-pentane, n-hexane, dichloromethane and methyl-tert-butyl ether, can be chosen as mobile phases without interfering with even the most volatile analytes [100, 132]. Coupling reversed phase LC (RPLC) to GC places higher demands on the interface [137]. Water is not a suitable solvent for GC analysis, due to the hydrolysis of siloxane bonds it induces, causing rapid deterioration of the analytical GC column. For non-vaporizing interfaces, retention gaps with special qualities and/or conditions are required, especially for the on-column/ retention gap technique. Due to the low vapor pressure of water, analysis of volatile compounds is virtually impossible. Co-solvents have been suggested to improve the capacity of trapping low-boiling analytes [138]. Vaporizing injectors, such as the PTV, is less sensitive towards water-containing samples. However, the trapping of volatile analytes and injector flooding are problems that need to be addressed [139].

Heart-cut or back-flush? Transferring heart-cut fractions eluting from an LC column minimizes the requirements for the LC system both chemically and technically. The LC effluent is introduced into the GC system as long as the target analytes are eluting. The heart-cut technique has several inherent drawbacks if large groups of compounds are to be transferred. The fraction volume may be rather large and cannot be decreased without losses of LC resolution and/or sample capacity. Since the analytes are separated in space before introduction into the GC-column, they are subject to changing conditions during the large volume injection, i.e. changes in sample introduction time, solvent effects and temperature, due to the cooling effect of the evaporating solvent. In addition, it is essential to transfer the entire fraction. Even so, both early and late eluting compounds may be discriminated against, as an effect of retention time drifts and tailing, for the late eluents. Some of these problems can be overcome by the back-flush technique, i.e. using an additional valve to flush the eluents back through the column. Assuming equal conditions in both directions of the LC column all

69 components that are still in the column when the flow is reversed will elute as a single peak. The volume of the back-flush fraction is generally smaller than that of the corresponding heart-cut fraction. Since the solutes are evenly distributed within the back-flush peak only a portion of it can be transferred, if desired. This also reduces the risk of discrimination against late eluting, potentially tailing analytes. The elution strength of the mobile phase must remain constant to ensure equal retention times in both directions. Therefore, the back flush approach is not compatible with gradient elution. Paper IV describes an NPLC-GC-NPD system for the determination of carbazole-type PANHs. The isolation of this group of compounds is based on hydrogen bonding between the carbazoles and the nitrogen of the dimethylamino propyl groups of the stationary phase [140, 141]. A secondary retention mechanism is electron donor/acceptor complex formation, the strength of which depends on the respective analytes’ numbers of fused aromatic rings. The column was end-capped in-situ with hexamethyldisilazane, to avoid hydrogen bonding, between acridines and free silanol groups, Figure 17.

&DUED]ROH $FULGLQH

1 1 + + 1 2 6L

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Figure 17. Retention mechanism for carbazoles and acridines in the dimethylamino propyl column. The retention mechanism of carbazoles is based on the hydrogen bonding to the stationary phase. Unless the column is end-capped acridines interact with residual silanol groups.

The method described in Paper IV includes a pre-separation of PAHs and aliphatic compounds from the PANHs. In later investigations the crude extract was introduced into the LC-GC-NPD system, while maintaining selectivity and gas chromatographic resolution.

70 The method was further developed to include the acridine-type PANHs (unpublished results). A schematic diagram of this “double back-flush” system is shown in Figure 18. The first eluting analytes, in this case the acridines, were trapped in the loop by switching the trapping valve (B). The loop has to be as large as, or larger than, the analyte band. At the same time the back- flush valve (A) was switched to facilitate a back-flush of the last eluting

+ & ( 13'

$ +3/& $XWR 89 3XPS VDPSOHU GHWHFWRU

Figure 18. Schematic diagram of the double back-flush LC-GC-NPD. A: Back-flush valve, B: Trapping valve equipped with a loop in which the first eluting analytes are trapped, C: Injection valve, D: Injection loop-valve, equipped with two loops for the two fractions, E: Vapor exit valve. compounds, i.e. the carbazoles. After a 2-minute delay the trapping valve (B) was switched again and the acridines were back-flushed through the column. The applied double back-flush technique resulted in two Gaussian shaped peaks, with evenly distributed analytes, Figure 19. The fractions were trapped in two loops mounted on a multi-position valve (Figure 18D) and subsequently injected and analyzed separately on the GC-NPD system, Figure 20. For the carbazoles the elution volume was significantly decreased and a more robust injection procedure was achieved for the acridine fraction, compared to when applying the heart-cut technique. The average RSD for the relative response was 2.9 % and below 5 % for all analytes except dibenz(a,i)acridine (6.2 %). No cross contamination between the two groups was detected.

71 $FULGLQHV &DUED]ROHV $ [ 56 ABSORBANCE

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Figure 19. LC-UV chromatograms of carbazoles and acridines. A: The LC separation without back-flush, B: LC separation with double back-flush. The acridines and carbazoles are eluted as two uniform fractions.

1 +

1 1 + + 1 + 1 +

1 1 1 + + + .0$SIGNAL

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Figure 20. GC-NPD chromatograms of carbazoles and acridines. The two groups are injected and separated one by one.

72 On-line extraction-GC Coupling extraction to analytical separation and detection, optionally via clean- up steps, enables automation of a major part of the analytical chain [134]. Ideally such a system reduces the manual work to sampling and data interpretation. On-line coupling to GC requires the extraction media to be flushed out from the extraction vessel and into the interface. Consequently, hyphenated extraction-GC systems are based on dynamic extraction or a combination of dynamic and static extraction steps. For solid samples, dynamic microwave- assisted extraction (DMAE), pressurized hot water extraction (PHWE), supercritical fluid extraction (SFE) and dynamic sonication-assisted extraction (DSAE) have all been successfully coupled to gas chromatography.

Dynamic microwave-assisted extraction (DMAE)-GC Ericsson and Colmsjö presented a dynamic microwave-assisted extraction procedure coupled to SPE for determining PAH in sediment [142, 143]. The extraction efficiency was enhanced by the increased temperature. The transformation of the energy (from radiation to heat), requires solvents and/ or sample matrix to have dipole-moments, which are affected by the microwaves. A major benefit of using microwaves is that the sample matrix itself can be heated and not dependent on indirect heating from hot solvents. The technique was later coupled on-line to GC for determination of organophosphate esters in air using a PTV-injector as the interface [144].

Pressurized hot water extraction (PHWE)-GC Pressurized hot water extraction coupled on-line to GC has been developed mainly by Reikkola and co-workers [145-147]. The temperature is increased up to 325°C and elevated pressure keeps the water in its liquid state. This decreases the dielectric constant and increases the solubility of non-polar compounds in the water. Since water is not suitable for direct transfer to GC the solvent needs to be exchanged. As the water is cooled down its polarity increases and lipophilic compounds can easily be trapped on an SPE column and eluted with organic solvents [145] or extracted by membrane extraction into an organic acceptor

73 phase [146-148]. Kuosmanen et al. determined brominated flame retardants, including PBDEs up to hepta-brominated congeners, in sediment by PHWE- SPE-GC using an on-column interface [149].

Supercritical fluid extraction (SFE)-GC In supercritical fluid extraction, the extraction medium is easily eliminated since the most commonly used fluid, CO2, is vaporized when leaving the pressurized system. The restrictor is connected to the GC injector, which is cooled by the expanding gas, thereby increasing the analyte trapping efficiency. The extraction medium is eliminated either through a split exit or through the column. SFE has been hyphenated to GC by split/splitless [150, 151], on-column [152] and PTV [153] interfaces. Modifiers such as methanol and dichloromethane are commonly used to increase recoveries, but may cause problems due to flooding effects if conditions are not optimized.

Dynamic sonication-assisted extraction (DSAE)-GC DSAE coupled on-line to GC was first described by Sanchez et al. for the determination of organophosphate esters in air [154]. So far only PTVs have been used as interfaces, but other large volume injection systems could be applied. Paper V presents a DSAE-GC-MS method for the determination of PBDE in air, as described earlier. Due to the selectivity of the ECNI/MS detection, the extract could be transferred to the GC without any pre-separation steps other than removing particles using a frit at the exit of the extraction cell. Only one interfering matrix peak was observed. The previously proposed solid-phase extraction step used in the off-line method described in Papers I-III, V and VI, was unable to remove this unidentified compound. The reproducibility for spiked filters was high, with an average RSD of 4.8 %. For real samples the average RSD was higher (16 %), possibly due to irreproducible sample homogenization. The on-line DSAE-GC-MS method benefits from the low risk of contamination, unlike the off-line method, which showed high and irreproducible background levels, in particular for BDE-49 BDE-99, BDE-183 and BDE-209, making the results uncertain.

74 Air sampling The methods described in this thesis are primarily designed for the determination of environmental pollutants in air. From a clean-up perspective, air can be considered a relatively simple matrix. The polar and lipid contents are commonly low and samples can therefore be analyzed without extensive sample pre-treatment. In the studies presented in Paper V the crude extracts were transferred to the GC inlet, without affecting the peak shape or the lifespan of either the inlet liners or columns. On the other hand, analytical methods for air samples are compara- tively difficult to evaluate representatively. Samples of matrices such as blood, soil and sediment can be spiked with target analytes, and absolute recoveries from extraction and purification steps can easily be calculated. Generating an atmosphere containing a known amount of gaseous and particulate phase analytes is far more difficult, though successful attempts have been reported [155, 156]. Furthermore, the problematic issue of the gaseous and particulate phase distribution of semi-volatile compounds has to be addressed. Both PBDEs and carbazoles are almost entirely found in the particulate phase of the air. This means the compounds are more or less adsorbed to air-borne dust and particles, which constitutes the actual matrix. Hence, spiking filters used for collecting the particulate matter does not representatively mimic real samples. In such investigations very high recoveries are normally obtained. When extracting spiked glass fiber filters with the dynamic sonication- assisted solvent extraction technique presented in Paper V, the average recoveries were 98 %. For this reason the extraction was mainly evaluated using real samples and relative recoveries since the actual amounts of PBDEs were not known.

The sampling set-up The analyzed air samples were collected using the set-up shown in Figure 21. This device was developed by Östman et al. for the determination of PAHs in air and consisted of an anodized aluminum sampler head containing a glass fiber filter and two polyurethane foam plugs (PUFs) in series [157]. The sampler was connected to a pump, normally adjusted to pump 3 L/min.

75 Particles were collected on the filter and semi-volatile compounds were adsorbed on the first PUF. The second PUF was a back-up adsorbent for breakthrough determinations. The phase distribution determined using this set-up should be treated with caution. During sampling, compounds initially adsorbed to particles could be desorbed and again adsorbed on the PUFs, thus giving erroneous indications about the native distribution. All compounds discussed in this thesis were found to be in the particulate phase, with the exception of carbazole, 12 % of which was found in the gaseous phase.

Figure 21. The adsorbent holder for air sampling. F: Glass fiber filter, S: Sampling PUF, B: Back-up PUF.

76 Concluding remarks This thesis deals with gas chromatographic injection techniques for high- boiling and thermally labile compounds, with particular focus on large-volume injections. A number of methods have been investigated for the determination of polybrominated diphenyl ethers. Hopefully, the reader will have gained knowledge about the principles and applicability of both conventional and “new” techniques, especially for PBDE analysis, but also for compounds with similar physical properties and chromatographic behavior. Sensitive and robust methods need to be developed for determination of flame-retardants, especially decaBDE, due to concerns about their possible health and environmental effects. One aim of the work presented here was to assist the development of such techniques. Splitless injectors are commonly employed for the analysis of complex matrices such as biological samples. The results in Paper I prove this technique to be irreproducible and to discriminate against the high molecular weight BDE congeners. PTVs are proposed as alternatives, when vaporizing systems are required. In studies I, III and V this injection technique was successfully used for introducing PBDEs to the GC-column, for both conventional and large injection volumes. Dynamic extraction coupled on-line to GC-MS is a convenient approach for the determination of polybrominated diphenyl ethers in air, as shown in Paper V. The closed system minimizes losses and the risk of contamination, which are common problems during PBDE analysis. The technique would be of particular use for screening PBDEs in different environments. The LC-GC-NPD technique presented in Paper IV could be a power- ful tool both for environmental purposes, e.g. for determining the carcinogenic dibenzo(c,g)carbazole and for the oil industry to establish benzocarbazole ratios. The mass spectral characteristics of BDE-209 presented in Paper VI enable the use of isotopic dilution for quantification. With this technique environmental chemists may improve the accuracy of determinations of this compound. In future research the developed arsenal of methods will be applied to the determination of PBDEs in both indoor and outdoor air.

77 The increasing number of large volume injection techniques and their applications confirm the need to overcome the limitations of conventional methods. However, a common problem for all reported large volume techniques is balancing the sample introduction and solvent evaporation rates. In the future, the scope for developing an injector that does not require these parameters to be fine-tuned will be explored.

78 Acknowledgements Jag vill först och främst tacka min handledare Conny Östman för att han alltid stöttat mig och mina idéer. Lycka till med allt. Ett stort tack till min vapendragare Jonas Björklund, för vetenskapliga projekt, moraliskt stöd och omständliga historier. Utan dig hade den här avhandlingen inte blivit skriven. Och vad hade du tänkt att jag ska göra nu när du sticker? Tack till Jonas Rutberg för oändligt tålamod med alla ’data’-frågor, layout av den här boken och discoveryreferat. Jonas och Jonas ska också tackas som bra kompisar och musiker. Suck on my lung ‘cause I’m losin’ my snail to a bucket full of leafs. Jag vill även tacka mina rumskamrater (som alla hann före mig): Magnus E, för midsomrar, grisfötter och gemensamma projekt som aldrig blev av; Ove, för brandfacklor, dammsugare och utflykter samt Sindra, för KÖL- promenader och för att hon alltid lyssnar (till skillnad från undertecknad). Tack till Håkan, som var min handledare för länge, länge sedan. Du, vi borde köra igång nåt alifatprojekt. En hälsning till det gamla gardet: Gunnar, Bettan, Ludde (denne gigant), Åsa (tack för middagar m.m.), Joanna, Kakan, Petr och Homer S... Magnus A – det trodde ni aldrig. Speciellt tack till Gunnar och Bettan som gjorde C-kursen så trevlig att jag stannade. Min generation doktorander; ni har gjort det roligt att gå till jobbet. Tack: Stina M, för alla bisarra skämt, man aldrig riktigt vänjer sig vid. Jenny, för kemometrihjälp, javakurs och paniska barnhistorier. Stina C, för midsomrar och den delade avhandlingsvåndan. Magnus Å, för hjälp med kemometrin. Typiskt att projektet inte blev av. Malin, för CE- och kemometrisnack och dom där oväntade syrligheterna. Helena I, för det ständigt goda humöret och engagemanget i Q-ToF:en. Leila, för smittande skratt och granatäpplen. Helena H, för moderlig uppmuntran när allt känts hopplöst. Anders Ch, för PTV-diskussioner och trummande fingrar i korridoren. Thorvald, lycka till med gåsen. Yvonne, för den coolaste disputationen någonsin. Nana, for help with the Q-ToF and for beautiful English.

79 Samt Lina, Christoffer, Ragnar, Bodil och Kent, som jag aldrig riktigt hann lära känna, men önskar lycka till med avhandlingar och jobb. Tack till Yassar för ett väl genomfört exjobb. Och för godis. Carlo, thanks for interesting scientific discussions, but of course most of all for Italian cakes and stories. It’s absolutely crazy! Ramon and Cristina, I miss you around here. Hope to see you soon. Ralf ska ha en eloge för hjälp med matten. Du är ju en snäll kille under den hårda ytan. Lena, Anita och AnneMarie; det är ni som gör att den här institutionen funkar. Rune tackas för hjälp med injektorprototyper. Nu vet jag hur vi ska göra. Jag vill också tacka nuvarande och gammal personal: Anders, Bosse, Sven, Ulrika, Björn, Ulla, Eva, Roger, Rolf och Bengt-Ove. Tack till dem som tog sig tid att läsa igenom avhandlingen (innan den trycktes): Conny, Jonas (båda), Bosse, Ralf, Ulrika, Björn, Håkan och Anders. Pol – I hope we can turn SUPF into something really interesting Günther Weissmann, lycka till med SUPF! I also want to acknowledge John Blackwell for proof reading this thesis. Kompisar utanför institutionen: Ludde, Tjalle, Andreas, Henrik, Stefan och Ebba, tack för vidgade vyer. John, Ian, Mani and Reni, thanks for the soundtrack. Jag vill också tacka mina systrar Malin och Karin med familjer. Hade jag fått lite av er begåvning hade det här gått dubbelt så snabbt. Mina föräldrar kan jag knappast tacka för naturvetenskapen, men för allt annat; uppmuntran, Sonderedssomrar, som fantastiska farföräldrar, ekonomisk hjälp, etc. Sist och mest vill jag tacka min älskade familj, min fru Johanna och mina barn Holger och Vera. Ni har avdramatiserat det här avhandlandet. Johanna, när det är din tur ska jag försöka vara lika förstående som du varit.

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