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Large Volume Injection and Hyphenated Techniquesfor Gas Chromatographic Determination of
PBDEs and Carbazoles in Air
Petter Tollbäck
Doctoral ThesisDepartment of Analytical Chemistry
Stockholm University2005
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Doctoral Thesis, 2005
Petter Tollbä[email protected] of Analytical ChemistryStockholm UniversityS-106 91 Stockholm
© 2005 Petter TollbäckISBN 91-7155-014-3 pp 1-92, Paper V
Cover by Holger and Vera TollbäckAkademitryck AB, Edsbruk, Sweden 2005
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Till Holger och Vera
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List of contentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Sammanfattning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9List of papers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Aims . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15PBDEs and carbazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Polybrominated diphenyl ethers (PBDEs) . . . . . . . . . . . . . . . . . . . . . . 17PBDEs – environmental pollutants . . . . . . . . . . . . . . . . . . . . . . . . 18Biological effects of PBDEs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19PBDEs – an analytical challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Carbazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Health effects of carbazoles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Chromatographic resolution in GC . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Film thickness of the stationary phase . . . . . . . . . . . . . . . . . . . . . . . . 26Column length . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Inner diameter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Injection techniques for GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Vaporizing and non-vaporizing injections . . . . . . . . . . . . . . . . . . . . . . 33Analyte peak focusing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Cold trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Retention gap focusing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Solvent effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Conventional injectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36The on-column injector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36The split/splitless injector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37The programmed temperature vaporizer (PTV) . . . . . . . . . . . . . . 38At-column injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
The septum equipped temperature programmable injector . . . 39Direct injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Large volume injection (LVI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Why large volume injection? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Common misconceptions about large volume injections . . . . . . . 42On-column large volume injection (OC-LVI) . . . . . . . . . . . . . . . . 42Loop-type injector/interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
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Concurrent solvent evaporation . . . . . . . . . . . . . . . . . . . . . . . . . 45Peak deformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46Why loop-type? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Large volume splitless injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49Large volume PTV injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Sample introduction and solvent evaporation . . . . . . . . . . . . . . 50Analyte trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54Analyte transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
The author’s note on the loop-type and the PTV injectors . . . . . . 55GC detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
The flame ionization detector (FID) . . . . . . . . . . . . . . . . . . . . . . . . . . 57The nitrogen phosphorus detector (NPD) . . . . . . . . . . . . . . . . . . . . . 57The electron capture detector (ECD) . . . . . . . . . . . . . . . . . . . . . . . . . 58Mass spectrometry (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Electron ionization (EI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Chemical ionization (CI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Electron capture negative ionization (ECNI) . . . . . . . . . . . . . . 60Sample extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Static and dynamic extractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Soxhlet extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Ultrasonication-assisted solvent extraction . . . . . . . . . . . . . . . . . . . . . 65Dynamic sonication-assisted solvent extraction . . . . . . . . . . . . . . . . . 65
On-line coupling to GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Benefits of hyphenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Concerns about hyphenated systems . . . . . . . . . . . . . . . . . . . . . . . . . . 68LC-GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
NPLC and RPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Heart-cut or back-flush? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
On-line extraction-GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Dynamic microwave-assisted extraction (DMAE)-GC . . . . . . . . . 73Pressurized hot water extraction (PHWE)-GC . . . . . . . . . . . . . . . 73Supercritical fluid extraction (SFE)-GC . . . . . . . . . . . . . . . . . . . . . 74Dynamic sonication-assisted extraction (DSAE)-GC . . . . . . . . . . 74
Air sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75The sampling set-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
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AbstractThis thesis is based on studies in which the suitability of various gas chroma-tography (GC) injection techniques was examined for the determination ofpolybrominated diphenyl ethers (PBDEs) and carbazoles, two groups ofcompounds that are thermally labile and/or have high boiling-points. Forsuch substances, it is essential to introduce the samples into the GC systemin an appropriate way to avoid degradation and other potential problems. Inaddition, different types of gas chromatographic column system and massspectrometric detectors were evaluated for the determination of PBDEs.
Conventional injectors, such as splitless, on-column and programmedtemperature vaporizing (PTV) injectors were evaluated and optimized fordetermination of PBDEs. The results show on-column injection to be thebest option, providing low discrimination and high precision. The splitlessinjector is commonly used for “dirty” samples. However, it is not suitable fordetermination of the high molecular weight congeners, since it tends todiscriminate against them and promote their degradation, leading to poorprecision and accuracy. The PTV injector appears to be a more suitablealternative. The use of liners reduces problems associated with potentialinterferents such as polar compounds and lipids and compared to the hotsplitless 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 µLoffers two main benefits. Firstly, the overall detection and quantification limitsare decreased, since the entire sample extract can be injected into the GCsystem. Secondly, large volume injections enable hyphenation of precedingtechniques such as liquid chromatography (LC), solid phase extraction andother kinds of extraction. Large-volume injections were utilized and optimizedin the studies included in this thesis.
With a loop-type injector/interface large sample volumes can be in-jected on-column providing low risk of discrimination against compoundswith low volatility. This injector was used for the determination of PBDEsin air and as an interface for the determination of carbazoles by LC-GC.Peak distortion is a frequently encountered problem associated with thistype of injector that was addressed and solved during the work underly-ing this thesis.
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The PTV can be used as a large volume injector, in so-called solvent ventmode. This technique was evaluated for the determination of PBDEs and asan interface for coupling dynamic sonication-assisted solvent extraction on-line to GC. The results show that careful optimization of the injection pa-rameters is required, but also that the PTV is robust and yields reproducibleresults.
PBDEs are commonly detected using mass spectrometry in electroncapture negative ionization (ECNI) mode, monitoring bromine ions (m/z 79and 81). The mass spectrometric properties of the fully brominated diphe-nyl ether, BDE-209, have been investigated. A high molecular weight frag-ment at m/z 486/488 enables the use of 13C-labeled BDE-209 as an internalsurrogate standard.
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SammanfattningDen 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 avPBDE undersökts.
Konventionella tekniker så som splitless-, PTV- (programmable tem-perature vaporizing) och on-columninjektion har utvärderats och optimeratsmed avseende på haltbestämning av PBDE. Mest tillförlitlig visade sig on-columninjektionen vara, med låg diskriminering och hög precision. Förkomplicerade matriser används vanligen splitlessinjektion. Denna teknikär dock inte lämplig för PBDE-kongener med hög bromeringsgrad.Resultaten presenterade i den här avhandlingen visar att precisionen ärlåg, till följd av diskriminering eller nedbrytning. Istället förslås PTV-injektorn som ett mer lämpligt alternativ. Denna förångningsinjektor är ro-bust mot matrisrester, till exempel lipider och polära ämnen.Temperaturprogrammeringen möjliggör en mer kontrolleradlösningsmedelsförångning jämfört med splitlessinjektorn, vilket resulterar ilägre diskriminering och högre precision.
Att öka injektionsvolymen från de konventionella 1-3 µL till över 50 µLger två vinster. Detektions- och kvantifieringsgränserna sänks, eftersom helaeller en större del av provextraktet kan injiceras på GC-systemet.Storvolymsinjektioner möjliggör också direktkoppling avupparbetningstekniker, till exempel vätskekromatografi (LC) och olikaextraktionstekniker. Storvolymsinjektioner har optimerats och utnyttjats i stordel av arbetet.
Med en loopinjektor kan stora volymer injiceras on-column, vilket gerliten risk för diskriminering. Denna injektor har använts för bestämning avPBDE i luft samt som interface för bestämning av carbazoler på LC-GC.Deformation av kromatografiska toppar är ett vanligt fenomen då denna typav injektor används. Detta problem har undersökts och avhjälpts i det arbetesom 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
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koppling av dynamisk ultraljudsextraktion till GC. Resultaten visar att ennoggrann optimering är nödvändig, men också att PTV-injektorn är robustoch ger reproducerbara resultat.
PBDE detekteras vanligen med masspektrometri i sk “electron capturenegative ionization (ECNI) mode”, där bromjonerna (m/z 79 och 81)registreras. Det masspektrometriska mönstret för den fullt bromeradedifenyletern BDE-209 har undersökts. Upptäckten av ett fragment vid m/z486/488 gör att kol-13-märkt BDE-209 kan användas som intern standard,vilket ökar noggrannheten vid bestämning av BDE-209.
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List of papersI Influence of the injection technique and column system on gas
chromatographic determination of polybrominated diphenylethers (PBDE)Jonas Björklund, Petter Tollbäck, Christian Hiärne, Eva Dyremark andConny ÖstmanJournal of Chromatography A 1041, 201-210, 2004- The author is responsible for a major part of the experimental work and for writinga substantial part of this paper.
II Large volume injection GC-MS in electron capture negative ionmode utilizing isotopic dilution for the determination ofpolybrominated diphenyl ethers in airJ. Björklund, P. Tollbäck and C. ÖstmanJournal of Separation Science 26, 1104-1110, 2003- The author is responsible for setting up the injection system, investigating the peakdistortion phenomena and for writing a substantial part of this paper.
III Large-volume programmed-temperature vaporizer injection forfast gas chromatography with electron capture and massspectrometric detection of polybrominated diphenyl ethersP. Tollbäck, J. Björklund and C. ÖstmanJournal of Chromatography A 991, 241-253, 2003- The author is responsible for a major part of the experimental work and for writinga substantial part of this paper.
IV Coupled LC-GC-NPD for determination of carbazole-type PANHand its application to personal exposure measurementP. Tollbäck, H. Carlsson and C. ÖstmanJournal 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-lineto GC-MS for the determination of PBDEs in airP. Tollbäck and C. ÖstmanIn manuscript- The author is responsible for all the experimental work and for writing this paper.
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VI Mass spectrometric characteristics of decabromo-diphenyl etherand the application of isotopic dilution in the electron capturenegative ionization mode for the analysis of polybrominateddiphenyl ethersJ. Björklund, P. Tollbäck and C. ÖstmanJournal of Mass Spectrometry 38 (4), 394-400, 2003- The author is responsible for experimental work and for writing a substantial partof this paper.
Papers not included in this thesis
Enhanced detection of nitroaromatic explosive vapors combiningSPE-air sampling, SFE and Large Volume Injection-GCR. Batlle, H. Carlsson, P. Tollbäck, A. Colmsjö and C. CrescenziAnalytical Chemistry 75 (13), 3137-3144, 2003
Automated rotary valve injection for polybrominated diphenylethers in gas chromatographyJ. Björklund, P. Tollbäck, E. Dyremark and C. ÖstmanJournal of Separation Science 26, 594-600, 2003
Evaluation of gas chromatographic injection techniques forPBDE.P. Tollbäck, J. Björklund and C. ÖstmanOrganohalogen Compounds 61, 49-52, 2003
Determination of high molecular weight PBDE by isotopicdilution in ECNI-MS.J. Björklund, P. Tollbäck and C. ÖstmanOrganohalogen Compounds 61, 163-166, 2003
Evaluation of the gas chromatographic column system for thedetermination of polybrominated diphenyl ethersJ. Björklund, P. Tollbäck and C. ÖstmanOrganohalogen Compounds 61, 239-242, 2003
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AbbreviationsBDE BromoDiphenyl EtherBDE-47 2,2’,4,4’-tetraBDEBDE-49 2,2’,3,4-tetraBDEBDE-99 2,2’,4,4’,5-pentaBDEBDE-100 2,2’,4,4’,6-pentaBDEBDE-119 2,3’,4,4’,6-pentaBDEBDE-153 2,2’,4,4’,5,5’-hexaBDEBDE-154 2,2’,4,4’,5,6’-hexaBDEBDE-183 2,2’,3,4,4’,5’,6-heptaBDEBDE-191 2,3,3’,4,4’,5’,6-heptaBDEBDE-197 2,2’,3,3’,4,4’,6,6’-octaBDEBDE-203 2,2’,3,4,4’,5,5’,6-octaBDEBDE-206 2,2’,3,3’,4,4’,5,5’,6-nonaBDEBDE-207 2,2’,3,3’,4,4’,5,6,6’-nonaBDEBDE-208 2,2’,3,3’,4,5,5’,6,6’-nonaBDEBDE-209 2,2’,3,3’,4,4’,5,5’,6,6’-decaBDEBTBPE Bis(2,4,6-TriBromoPhenoxy) Ethane DBC DiBenzoCarbazole (7-H-dibenzo(c,g)carbazole)DecaBDE DecaBromoDiphenyl EtherECD Electron Capture DetectorECNI Electron Capture Negative IonizationEI Electron Ionization (previously Electron Impact)FID Flame Ionization DetectorGC Gas ChromatographyHeptaBDE HeptaBromoDiphenyl EtherHexaBDE HexaBromoDiphenyl EtherLC Liquid ChromatographyLVI Large Volume InjectionMS Mass SpectrometryNonaBDE NonaBromoDiphenyl EtherNPD Nitrogen Phosphorus DetectorNPLC Normal Phase Liquid Chromatography
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OC On-ColumnOctaBDE OctaBromoDiphenyl EtherPAC Polycyclic Aromatic CompoundPAH Polycyclic Aromatic HydrocarbonPANH Polycyclic Aromatic Nitrogen-containing Heterocyclic PBDE Polybrominated Diphenyl Ether PentaBDE PentaBromoDiphenyl EtherPTV Programmed Temperature Vaporizer/VaporizingRPLC Reversed Phase Liquid ChromatographyRSD Relative Standard DeviationSIM Selected Ion MonitoringSPE Solid Phase ExtractionTetraBDE TetraBromoDiphenyl EtherTh Thompson, unit for m/zTriBDE TriBromoDiphenyl Ether
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AimsThe main objectives of the work underlying this thesis were to investigate,optimize and evaluate large volume injection for gas chromatographic analysisof thermally labile compounds and compounds with high boiling points(referred to, for convenience, as high-boiling compounds hereafter). Thedeveloped techniques were applied to the determination of polybrominateddiphenyl ethers (PBDEs) and carbazole-type polycyclic aromatic nitrogen-containing heterocyclics (PANHs) in air. More traditional methods were alsoinvestigated in the course of the work. In addition, large volume injectorswere utilized as interfaces for coupling clean-up techniques and gaschromatography on-line. These techniques have been developed to improvedetection limits and accuracy. Automated, closed systems also reduce therisk of contamination, a common problem when analyzing PBDEs.Determination of the thermally labile decaBDE congener has proven to betroublesome. Conventional gas chromatographic set-ups generally result insevere discrimination against this compound, leading to poor precision andaccuracy. The results presented in this thesis may therefore also assist thedevelopment of accurate and reproducible analytical methods fordetermination of PBDEs in environmental samples.
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PBDEs and carbazolesIn this thesis the development and use of sensitive methods for determiningselected air-borne pollutants are described. The following paragraphs areintended to give a brief introduction to the investigated compounds.
Polybrominated diphenyl ethers (PBDEs)Pollution with polybrominated diphenyl ethers, Figure 1, has been referredto as the new PCB problem. Considering the similarities between PBDEsand PCBs in structure, function and environmental occurrence thecomparison is obvious. However, there are also differences, in biologicalactivity and physical properties, for instance. For more extensive discussionof PBDEs as environmental pollutants, the reader is referred to recent reviewsthat have been published on this topic [1-3].
PBDEs constitute a group of additive flame-retardants that are predominantlyfound in electronic equipment, furniture and textiles. They are blended intoor adsorbed onto the materials to reduce the flammability. Thus, they are notcovalently bonded to the host material. Their flame-retarding properties are basedon the elimination of free radicals formed during combustion processes [4].
Figure 1. General structure of PBDEs and three selected BDE congeners.
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Three technical PBDE mixtures (penta-, octa- and deca-BDE) arecommercially used for flame-retarding purposes. The estimated world demandfor PBDEs in 1999 was 67,000 tonnes, of which the deca-BDE mixtureaccounted for about 80 %. The results of a characterization of these productsby Sjödin et al. are summarized in Table 1 [5]. As can be seen, synthesis ofthe technical mixtures is not specific, yielding about twenty congeners rangingfrom triBDE to decaBDE. Regulations against the use of the penta-BDEand octa-BDE mixtures within the European Union have shifted productiontowards the deca-BDE mixture in this part of the world. However, the USAand Japan are still using the low molecular weight mixtures.
PBDEs – environmental pollutantsBeing additive flame retardants, PBDEs could be suspected to migrate fromtheir host polymer to the surroundings. The first evidence of PBDEs in theenvironment was reported in 1979 by Zweidinger [6] and in 1981 byAndersson and Blomquist [7]. The latter authors discovered a number ofBDE congeners in fish from the river Viskan in Sweden. Since then PBDEshave been found in a wide range of environmental compartments, such assediments [8-11] and sewage sludge [12-15], as well as in various biota.Numerous papers describe the occurrence of PBDEs in marine animals, forexample fish [8, 16, 17], seabirds [18] and mammals [19-22]. PBDEs havealso been found in human blood [23, 24], adipose tissue [25-28] and humanmilk [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,
Composition (%)PBDE-mixture Tri- Tetra- Penta- Hexa- Hepta- Octa- Nona- Deca-Penta-BDE 0-1 24-38 50-62 4-8Octa-BDE 10-12 43-44 31-35 9-11 0-1Deca-BDE 0.3-3 97-98
Table 1. Composition of the three technical PBDE-mixtures.
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detecting BDE-209 close to a manufacturing plant for brominated flameretardants [6]. Interest in analyzing air with regard to PBDEs has increasedin 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-scaleinvestigation of the outdoor air in 22 countries, mainly in Europe [37]. Thehighest amounts were found in the UK, which has been both the largestproducer and user of PBDEs in Europe. Levels of PBDEs in air have alsobeen 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 indifferent indoor environments, and found increased levels in blood frompeople exposed to high concentrations of PBDEs in their work environment(an electronics recycling plant) [51]. Thomsen et al. found 7-59 pg/m3 ofBDE-49 and BDE-99 in laboratory air, which were responsible for blankproblems. Harrad et al. measured the PBDE levels in indoor domestic andwork environments [45]. They calculated the daily exposure via the respiratorysystem to be about 7 ng.
This short summary of the occurrence of PBDEs in the environmentillustrates the global breadth of their distribution as pollutants.
Biological effects of PBDEsInformation about the biological effects of PBDEs is sparse, but suggeststhat their bioactivity is low. Their acute toxicity, measured as LD50, has beenreported to be 0.5-5 g/kg body weight [4].
Analyses involving oral administration to rats have shown that the lowmolecular weight congeners are easily absorbed; after five days 86 % of thedose was still retained, and the half-lives were 20-30 days for tetraBDE and45-119 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’ excretionsshowed that the PBDEs are metabolized to a large degree. An interestingfinding is that following exposure to BDE-209 the levels of hexa- tononaBDE in rainbow trout were significantly increased [56].
The low molecular weight BDE congeners have been shown to inhibitthe binding of the thyroid hormone thyroxine (T4) to transthyretin [57].
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Rec
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28, 4
7, 9
9, 1
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53, 1
54, 1
83
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BD
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BD
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-29
2001
[4
8]
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In
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and d
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47
, 99,
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A
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2003
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09
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7, 2
8, 4
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)
Pas
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2004
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c
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-37
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49,
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[37]
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, 28,
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[4
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[46]
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r ai
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77,
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99,
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, 126
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, 209
c,
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Org
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s fr
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1
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[4
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eden
In
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17, 2
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1, 8
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, 154
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, 183
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, 191
, 196
,
197,
207
, 206
, 209
Act
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filt
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XA
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sorb
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4.4×
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170×
103
2004
[5
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a C
on
gen
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aren
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is w
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t d
etec
ted
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ple
s. b Y
ear
of
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licat
ion
. Dat
e fo
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revi
ous
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licat
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pro
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ings
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c
No
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s w
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ated
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on
cen
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E-2
09 in
air
no
t ca
lcula
ted. n
.d. =
no
t det
ecte
d.
Table 2. Publications of determination of PBDEs in air.
21
Considering the structural similarities of T4 and PBDEs, competition be-tween them for the binding sites is not surprising. Meerts et al. proved 11BDE congeners to have estrogenic effects [58]. The ubiquitous BDE-100was among those with the highest activity.
The carcinogenic effect of BDE-209 is low [59], possibly due in part tothe low uptake of this compound. Unfortunately, the carcinogenicity of thesmaller BDE congeners, which are absorbed to a much higher extent, hasnot yet been tested.
All toxicological studies of PBDEs to date have been based on oralintake of the compounds. The toxic effects of exposure through inhalationhave not yet been investigated.
PBDEs - an analytical challengeThere are 209 possible BDE congeners. However, unlike the PCBs only abouttwenty are found in technical mixtures and environmental samples. Theirchromatographic separation is consequently not a major problem in PBDEanalysis.
The high molecular weight BDE congeners put extraordinary demandson the analytical procedure. These compounds have high boiling points, readilyadsorb to glass surfaces and degrade thermally. Consequently, determinationof nona- and decaBDE is particularly troublesome and rarely presented inthe literature. In a Round-Robin study initiated by de Boer et al. largediscrepancies were found in the concentrations of BDE-209 reported by theparticipating laboratories [60]. Most of the methods used involved splitlessinjection, which is not well suited for thermally labile or high-boilingcompounds. Short columns were used for determining decaBDE in mostcases, but often with thick stationary phases, which could promotedegradation, as discussed in Paper I.
As mentioned earlier, the aims of the studies this thesis is based uponwere to develop accurate and sensitive techniques for the determination ofPBDEs, and to investigate and evaluate different set-ups and conditions forthe GC analysis of compounds with high boiling points and thermal lability.From this perspective BDE-209 is an excellent model substance.
22
CarbazolesCarbazoles constitute a group of polycyclic aromatic nitrogen-containingheterocyclics (PANHs), Figure 2, belonging to the large group of polycyclicaromatic compounds (PACs).
The determination of carbazoles is of interest to various industrial sec-tors, including the oil industry, since they are constituents of many fossilfuels. Carbazoles have been identified, for example, in crude oils [61],shale oils [62], fuels [63] and tar [64]. Furthermore, the benzocarbazoleratio, a/(a+c), can be used as a marker for the origin of oils, and Larteret al. found a correlation between the relative and absolute concentra-tions of these benzocarbazoles and the distance from an oil source to itsreservoir [65]. These relationships could be used to help find new petro-leum accumulations.
N
H
N
H
N
H
N
H
Carbazole Benzo(def)carbazole
N
H
Benzo(c)carbazole
N
H
Benzo(b)carbazole
N
H
Benzo(a)carbazole
Dibenzo(c,g)carbazole Dibenzo(a,i)carbazole
Figure 2. Structure of investigated carbazoles.
23
Carbazoles are also formed during incomplete combustion of organic mat-ter and have been found in tobacco smoke [66], sewage sludge [67], outdoorair [68] and indoor air (Paper IV). Given their biological activity, carbazolesare of both environmental and health concern.
Health effects of carbazolesThe effects of carbazoles on a biological system are highly dependent ontheir structure. For example carbazole may cause skin disorders [69], butconflicting 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-adductsin liver and skin [73-75], Warshawsky and Barkley found it to be as carcino-genic as benzo(a)pyrene (BaP) in mice [76, 77], and Sellakumar and Shubikreported it to be an even more potent carcinogen than BaP in the respiratorytract of hamsters [78, 79].
24
25
Gas chromatographyThe separation of compounds in gas chromatography is based on theirpartitioning 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 separatedaccording to their vapor pressure or boiling points. Further retentionmechanisms can be added and selectivity modified by introducing more polarfunctionalities to the stationary phase, for example phenyl-, cyano- ortriflouromethyl groups.
Chromatographic resolution in GCToday, most GC analyses are carried out using open tubular capillary col-umns, which provide high separation efficiency. The resolution between twopeaks is a function of the number of theoretical plates (N), the separationfactor (α) and the retention factor (k) for the last eluting of the two peaks,according to Purnell’s equation:
⎟⎠⎞
⎜⎝⎛
+⎟⎠⎞
⎜⎝⎛ −=
11
16 kkNRs α
α(1)
This equation is based on isothermal conditions, as is all basic gas chromato-graphic theory. However, a temperature program is often used, which altersthe partitioning coefficient (K) during the chromatographic separation. Inthese cases the calculated values should be considered guidelines rather thanabsolute values.
The number of theoretical plates is a function of the column length (L)and the height equivalent to a theoretical plate (H).
HLN = (2)
If we assume infinitely sharp initial bands, the separation is limited by bandbroadening during the separation process.
26
H can be described by the van Deemter equation:
µµ
CBAH ++= (3)
where µ is the linear flow rate. For open tubular capillary columns the eddyor multiple path diffusion term, A, equals zero and the longitudinal diffusionterm, B, becomes insignificant at higher flows. Hence, band broadening is afunction of the resistance to mass transfer, i.e. the C term, which can bedivided into two separate terms: resistance to mass transfer in the stationaryphase, Cs and resistance to mass transfer in the mobile phase, Cm. Hence, forcapillary GC the height equivalent to a theoretical plate (Equation 3) can beapproximated as:
µµ ms CCH += (4)
Film thickness of the stationary phaseFor gas chromatographic separation of different compounds, they must bethoroughly dissolved in the stationary phase and sufficient binding sites mustbe available to retain them. A thick stationary phase film is used to trap andseparate volatile analytes. Thin films may cause column overload at high sampleloadings due to the low number of sites in the stationary phase.
The film thickness of the stationary phase also contributes to the bandbroadening in the stationary phase (Cs):
2
2
)1(32
kDkd
Cs
fs +
=
where k is the retention factor for a given analyte, df the stationary phasefilm thickness and Ds the diffusion coefficient for the analyte. Band broad-ening in the stationary phase is proportional to the square of the film thick-
27
ness. For columns with very thin stationary phases the Cs term becomesinsignificant compared to the Cm term.
For PBDE analysis, relatively thick films have traditionally been used toprevent interactions with residual silanol groups, since these active sites couldinduce degradation. However, in Paper I results are presented showing thatincreased film thickness actually has a negative effect on the yields of labilecongeners. With thicker films the retention times increase and the analytesspend more time in the column at an elevated temperature. In the comparisonof two 15 m columns, the retention time for BDE-209 increased from 23.3to 25.6 minutes, resulting in a 60 % smaller peak area, with a 0.25 µm filmthan with a 0.1 µm film.
From a chromatographic perspective, films that are as thin as possibleshould be used for the determination of PBDEs, since these analytes havelow volatility and are most often found in low concentrations.
Column lengthAccording to Equation 2 the number of theoretical plates (N) increaseswith column length (L) and the resolution is proportional to the square rootof N. The retention time (tR) on the other hand, is directly proportional tothe column length, according to:
µ)1( kLtR
+= (5)
This means that doubling the column length only increases the resolution bya factor of 1.4, while the retention time increases by a factor of two.
Papers I and III describe investigations on the influence of columnlength on the yields of the thermally labile BDE congeners. The time spentin the column at an elevated temperature increases with column length, re-sulting in increased degradation of the sensitive BDE congeners. Thus, longercolumns give poor yields and longer analysis times, but only minor improve-ments in resolution.
28
Inner diameterSince all other terms in the van Deemter equation are virtually insignifi-cant, H is determined mainly by the resistance to mass transfer in themobile phase and between the mobile and stationary phases, Cm, whichcan be described by:
2
22
)1(24)1161(
kDkkrC
mm +
++= (6)
where r is the radius of the capillary column and Dm the diffusion coefficientfor the analyte. The retention factor (k) can be described as
βKk = (7)
where K is the equilibrium coefficient at a given temperature and β thevolumetric mobile phase/stationary phase ratio. β can be approximated as:
fdr
2=β (8)
Equations 7 and 8 give:
rd
Kk f2= (9)
From Equation 1 the resolution can be calculated as a function of the columnradius, with the help of Equations 2, 4, 6 and 9. The relative resolution fora 100 µm and a 250 µm column is plotted in Figure 3. The resolutions arecalculated for equilibrium coefficients of 2000 and 10000 and are relative tothat 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) columnin terms of resolution under isothermal conditions.
29
The retention time depends on the column’s length and radius, the mobilephase flow rate and the equilibrium coefficient. Equations 5 and 9 give:
µ
)2
1(rd
KLt
f
R
+=
The equilibrium coefficient (K) is temperature dependent and thus changesduring the oven temperature program. For columns of different radii butequal 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 usingcolumns with smaller inner diameters the analysis times can be reduced whilemaintaining resolution.
This theory have been exploited in practice by the use of narrow borecolumns and fast-GC [80-82]. Paper III describes the use of narrow borecolumns (i.d. = 100 µm) for the separation of PBDEs. The peak widths wereapproximately a third of those obtained with conventional columns
0
0.5
1
1.5
2
2.5
3
3.5
0 5 10 15 20 25 30 35 40 45 50
Rel
ativ
ere
solu
tio
n
250 µm K=10000
100 µm K=10000
250 µm K=2000
100 µm K=2000
Figure 3. Resolution vs. column length for two inner diameters, 100 µm and250 µm at two equilibrium coefficients (K). The resolutions are relative tothat of a 30 meter column (i.d. = 250 µm).
30
(i.d. = 250 µm), and a retention time as short as 6.5 minutes for the last elut-ing component (BDE-209) was achieved. Such analyses should be classifiedas fast-GC according to the definitions proposed by Dagan and Amirav[82, 83]. No additional increase in resolution was observed when going froman inner diameter of 100 µm to 50 µm, possibly because extra-column bandbroadening (for example due to the injection) affects the separation to agreater extent than the in-column band broadening at these diameters.
High inlet pressure is an encountered problem with narrow-borecolumns, especially when long columns are used to improve resolution.However, in fast-GC shorter columns are used, and the pressures applied aresimilar to those used for corresponding conventional columns. Nevertheless,narrow bore columns may place extra demands on the other parts of the GCsystem. Due to low volumetric flows the splitless times should be increased,or a pressure pulse can be applied during the splitless time. Paper III describesthe combination of large volume injection via a PTV injector with fast-GC.Initial attempts to combine these techniques using a loop-type injector faileddue to severe peak distortion, probably as a result of the large differences inthe inner diameter of the retention gap and the column. In addition, thenarrow bore columns proved to be incompatible with the TSQ7000 mass
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 equilibriumcoefficients (K). df= 0.1 µm.
31
spectrometer for determination of the high molecular weight BDE congeners.This was most likely due to the long transfer line used in that particularinstrument, causing the analytes to spend relatively long times at an elevatedtemperature, and thereby increasing thermal degradation.
Stationary phasesA large number of stationary phases are available for gas chromatographyand an appropriate selection is required to obtain the best resolution andchromatography of the investigated analytes. Paper I reports an investigationof a number of different column brands and types with respect to theseparation of PBDEs. It is shown that the choice of column has a majorimpact on the yield of BDE-209, presumably due to differences in the numberor kind of catalytic sites. The DB-1 (100 % dimethyl polysiloxane) and DB-5(95 % dimethyl and 5 % phenyl polysiloxane) columns proved to be the bestchoices, whereas an HP-1 column completely degraded decaBDE. Detailedinformation about the differences of these stationary phases could help toexplain the results, but the manufacturing processes are unfortunatelyconfidential.
The retention of the BDE congeners was similar or identical on thedifferent investigated non-polar stationary phases. An interesting feature ofthe semi-polar DB-200 column, which contains 35 % trifluoropropyl, is itsability to separate bis(2,4,6-tribromophenoxy) ethane (BTBPE), a brominatedflame retardant, and BDE-191. On non-polar columns, such as the DB-5,these compounds co-elute. The yields obtained using the DB-200 columnswere acceptable, but not as high as those obtained with the DB-1 and -5columns, possibly due to its thicker stationary phase film (0.25 µm).
32
33
Injection techniques for GCWhile columns and detectors are often investigated and evaluated, the choice ofinjection technique is often dictated by the injectors available, which in manycases are split/splitless injectors. However, for many types of compounds sampleintroduction is the most critical step of the whole GC analysis, and yields may bepoor if an inappropriate method is used. Thermally labile compounds are easilydegraded in hot injectors, placing additional demands on the injection techniqueif high sensitivity and reproducibility are required.
In the studies this thesis is based upon, a large number of injectiontechniques were evaluated for high-boiling and thermally labile compounds.Thermal degradation was shown to increase with temperature, time andnumber of catalytic sites (e.g. free silanol groups). These parameters shouldin all cases be kept as low as possible.
Vaporizing and non-vaporizing injectionsGC injection techniques can be divided into two fundamentally different categories:vaporizing and non-vaporizing. In non-vaporizing injections the sample isintroduced directly into the column as a liquid. Thus, the transfer of analytes tothe column is efficient and discrimination low or non-existent. On the otherhand, 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 theelevated 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 havebeen shown to be critical [84].
– Solvent vaporization: The solvent is vaporized in the liner and allowedto enter the column.
– Analyte transfer: As long as the solvent remains liquid the solutes cannotbe heated above the solvent’s boiling point and are therefore transferredto the GC column after full vaporization of the solvent. As the processdepends on vaporization, high-boiling compounds may be discriminatedagainst. Matrix residues, which are potentially harmful to the column,are to a large extent deposited on the liner walls.
34
Analyte peak focusingThe 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 chromatographygenerally generates very sharp, narrow peaks, with widths of 1-10 seconds. Itis important to consider and understand these refocusing processes. Inpractice, the combined effects of several of these processes, rather than justone, are usually observed.
Cold trappingIn vaporizing injectors the initial bands are partly focused by cold trapping.The difference in temperature between the injector and the GC oven forcesthe 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 andthe elution temperature of the analyte to be sufficiently large, 60-90°C [85].
Retention gap focusingUncoated pre-columns or retention gaps are widely used in on-column andlarge volume injections to refocus the initial bands, which are broadened inspace during sample introduction. The subject has been thoroughlyinvestigated by Grob and co-workers.
The analytes move much faster in the retention gap compared to thecoated column, due to the large difference in their retention power. Soluteselute at very low temperatures in a retention-gap, but are almost completelyimmobilized when they reach the stationary phase. Elution from the coatedcolumn requires much higher temperatures. Consequently, the analytes arestrongly retarded when they reach the coated column and the bands arenarrowed. This effect is also known as phase ratio focusing [86-88].
Solvent effectsIn both vaporizing and non-vaporizing injections the solvent plays animportant role in the focusing process. There are two types of solvent effects:solvent trapping and phase soaking.
35
Solvent trapping is best explained using an uncoated column as an example.The solvent/analyte mixture is either injected directly or vaporized andrecondensed in the column, Figure 5. The solvent forms a film on the capillarywalls, the flooded zone and is subsequently vaporized from the rear end of thefilm (A). High-boiling analytes are deposited in the column along the floodedzone, 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).
This is obviously a simplified description, for several reasons. Firstly, thedividing line between volatile and high-boiling solutes is diffuse. Secondly,some compounds are weakly retained by the solvent and will be only partiallysubject to solvent trapping. Hence, the process is more complex than thistheory 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 ofthe column and is retained in the stationary phase. The stationary phaseswells together with the solvent, yielding a soaked zone, in which the retention
Figure 5. The solvent effect. Volatile compounds(dots) are focused during solvent evaporation,whereas high-boiling compounds (solid gray) aredistributed along the flooded zone.A. The solvent film is formed containing bothvolatile and high-boiling compounds. The solventevaporates from the rear end. The carrier gas israpidly saturated with solvent vapors.B. The high-boiling analytes are retained on thecapillary walls. Volatile analytes are going intovapor phase and subsequently trapped in theremaining solvent.C. The volatile compounds are concentrated inthe last remaining solvent.D. High-boiling compounds are spread along theentire flooded zone. Volatile compounds aredeposited at the point were the last remainingsolvent evaporated.
36
power is strongly increased. Consequently the solutes are slowed down ortrapped as they reach this zone.
The soaking solvent heavily overloads the column, forming a triangular profile,Figure 6A. The front end of the solvent zone moves faster than the rearend, since solvent evaporating from the rear end is slowed down by the soakedstationary phase. Hence, the solvent is trapped by itself. As the solvent spreadsin the column, the rear end accelerates until it finally reaches the same velocityas the front end. A given trapped solute will move together with the solventin a similar manner as in solvent trapping, until the velocity of the rear endof the solvent exceeds the migration speed of the solute. The solute is thenleft behind, focused in space (C) [88].
Conventional injectors
The on-column injectorThe non-vaporizing on-column injection is the simplest technique forintroducing samples in GC. The sample is injected directly into the columnor a retention gap, in which the analytes are focused due to the solvent andretention gap effects. Virtually complete analyte transfer is achieved and nodiscrimination occurs. Since the injector is not heated, the risk of thermaldegradation is minimal. Hence, on-column injection is theoretically the mostreliable technique.
Stationary Phase
Solvent
Volatile analytes
Carrier Gas
A
B
C
Figure 6. Phase soaking. The solventsoaks the stationary phase and volatileanalytes are trapped (A). The solvent isalso retarded in the soaked zone andmoves therefore slower at the rear endthan in the front end, where theretardation is lower. The analytes movetogether with the solvent (B) until thevelocity of the rear end of the solvent(soaked zone) becomes greater than thevelocity of the analytes (C).
37
However, on-column injection is traditionally considered to be difficult tohandle in practice, even when using auto-samplers. Needle breakages, damageto columns’ stationary phases and peak broadening have all been majorconcerns, limiting the practical application of the technique. Even thoughmodern set-ups are much less prone to these problems, on-column injectionis often not considered robust. In addition, because of the lack of liners thecolumns may be damaged by sample matrix residues, such as polarcompounds, lipids etc. A retention gap, acting as a guard-column, can beused to increase the life span of the analytical columns, but it needs to bereplaced regularly.
For determination of PBDEs the on-column injector proved to besuperior in terms of discrimination, Paper I.
The split/splitless injectorThe most common injection technique for GC is to use a split/splitlessinjector, introduced in the late 1960s by Kurt Grob [89]. This vaporizinginjector has gained popularity due to its robustness and capacity for handling“dirty” samples and has become the mostwidely available and commonly used typeof GC injector.
For trace analysis this injector isoperated in splitless mode. The sample isintroduced into a glass liner, normallyheated to 250-350°C, Figure 7. Theanalytes and solvent are vaporized andsubsequently transferred to the columnas vapors. The split valve is opened,typically after 1-3 minutes, and remainingsolvent vapors (and analytes) arediscarded through the split exit.
The transfer of analytes to thecolumn is usually high (>80 %), butdiscrimination against high-boilingcompounds is a recognized problem.Potential solutions to this problem include
Figure 7. Schematic diagram ofthe splitless injector.
38
increasing the injector temperature, prolonging the splitless time and applyinga pressure pulse during analyte transfer. The use of a pressure pulsecompresses the vapor cloud increases the flow into the column, which hascomparable effects to prolonging the splitless time. However, even whenapplying these techniques the split/splitless injector has several disadvantages.Firstly, in spite of the high temperature, the vaporization of the solvent ismany cases slower than could be expected. Injected as droplets the sampleactually “bounces around” in the liner and proportions of the analytes maytravel beyond the split point, or even beyond the liner [90]. Such injectorflooding leads to poor transfer of analytes, especially those with high boilingpoints, to the column. Due to the uncontrolled vaporization large differencesin absolute and relative peak areas are often observed, leading to irreproducibleresults, even when internal standards are used. Liners with obstacles, such asan “inverted cup” [91] or glass wool [92] have been proposed as solutions tothe problem. The concept is based on retaining and transferring heat to thesolvent. However, the efficiency of this approach has been questioned [93].Ironically, the obstacles themselves may also cause discrimination, due toirreversible adsorption to active sites. Grob and Biedermann suggest thatinjection 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 forthermal degradation, providing both heat and catalytic sites on a relatively largeglass surface. Since splitless liners are relatively wide the transfer is slow, oftentaking 1-2 minutes, providing the last of the three requisites for thermaldegradation: time. Hence, splitless injection is not suitable for thermally labilecompounds such as BDE-209, Paper I.
The programmed temperature vaporizer (PTV)Although the programmed temperature vaporizer has been in use formore than thirty years, it has only recently gained favor amongmanufacturers and users [94]. A PTV was used in the studies reported inPapers I, III and V.
The temperature-programming feature of the PTV allows morecontrolled injection compared to the traditional splitless injector. Whenintroducing the sample at a temperature around the boiling point of the
39
solvent (±20°C) gentle solvent evaporation is achieved, and the analytes aretrapped on the liner walls or on an adsorbent above the split point. By rapidlyincreasing the temperature the analytes are transferred to the column.Compounds with low volatility are deposited in the liner, so there is lesscolumn contamination and deterioration than when on-column injectors areused. The PTV gives significantly higher yields of high-boiling andthermally labile compounds [95]. Analyses of such compounds also benefitfrom the use of increased pressure during transfer. Operated intemperature programmed pulsed splitless mode (TPPSL) the results arecomparable with those obtained with the on-column injector for mostof the BDE congeners, Paper I.
At-column injectionIn this thesis the term at-column injection is used for techniques such asdirect injection and use of septum-equipped temperature programmableinjectors (SPIs). Their common denominator is the use of tapered liners inwhich 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 splitlessinjection, and the use of liners decreases the risk of contamination and columndegradation compared to on-column injection.
Since the analytes may enter the column both as vapors and dissolved ina liquid solvent, the at-column injection is a combination of vaporizing andnon-vaporizing processes.
The septum equipped temperature programmable injector (SPI).An SPI was used in Paper I, for PBDE analysis. With this system, the sampleis injected into the cold liner (typically at 50-80°C) close to the column entranceand partly transferred to the column as a liquid. By rapidly increasing thetemperature, residues of analytes deposited on the liner walls are transferredto the column.
The SPI exhibited high reproducibility and low discrimination. However,since this injector was not available in combination with MS, it was notevaluated for determination of PBDEs in real samples.
The SPI has been discontinued, but SPI-like injections can be performedusing tapered liners for programmed temperature vaporizers (PTVs).
40
Direct injectionAt-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, taperedliner. As in normal splitless injection, the solvent vaporization is a slowerprocess than may be expected, despite the high temperature. Consequently,the sample is only partly vaporized and liquids may reach the lower part ofthe liner. However, analytes both in gaseous phase and dissolved in the solventwill 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 trappingand solvent effects. Remaining solvents can be vented out through a drilledhole in the liner [96-98].
The behavior of PBDEs in direct injection systems was studied in theinvestigation of injection techniques described in Paper I. The results werepromising, but the technique was not investigated and optimized thoroughly,and hence it was not included in the paper. Figure 8 shows the discriminationprofile for direct injection and splitless injection relative to on-columninjection. The yield of the high-boiling and thermally labile BDE-209 washigh, but so too was its standard deviation. The precision could possibly beimproved by further optimization.
0
20
40
60
80
100
120
140
BD
E-7
BD
E-4
9
BD
E-9
9
BD
E-1
53
BD
E-2
03
BD
E-2
09
Direct Injection
On-column
Splitless
Re
lative
Re
sp
on
se
(%
)
Figure 8. The relative response for six BDE congeners when using directinjection and splitless injection, normalized to on-column injection.
41
Large volume injection (LVI)Several techniques for increasing the injection volume tolerance of the GCsystem have been described in the literature. Commercially availableinstruments for some of these techniques have been developed, as interestin them is increasing among analytical chemists, but only devices built in-house are available for some of the others. Like their smaller volumecounterparts, large volume injections can be divided into two categories: non-vaporizing and vaporizing.
Non-vaporizing large volume injectors introduce the sample directlyinto the GC column system, and they can be categorized according to eitherthe instrumental set-up or the solvent evaporation procedure involved. Inthis thesis the former distinction is used, giving two main types: the on-column-LVI and the loop-type injector/interface. These techniques have beendeveloped and studied in particular by Grob and co-workers [99, 100].
The programmed temperature vaporizer can also be used for largevolume injections in solvent vent mode. This technique has been investigatedby Mol et al. [101] and Engewald et al. [97], for example.In the studies underlying this thesis loop-type interfaces and large volumePTV injections were used.
Why large volume injection?A feature common to all of the traditional GC injection techniques is thatlimited sample volumes can be introduced into the system: normally 1-3 µL.This is a major drawback, which restricts the detection and quantificationlimits of the analytical methods. The sample volume after extraction andclean-up can rarely be decreased to less than about 50-100 µL, with a linearvolume-concentration relationship, since the analytes may be deposited onthe walls of the vessel. More importantly, the sample composition may bealtered if the solvent volumes are too low, since the analytes may have differingaffinities 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 atraditional injection technique. Using large volume injections, larger fractionsof the sample can be analyzed and there is no need for analyte enrichment bysample volume reduction.
42
In addition, use of injection volumes as large as 500-1000 µL enableshyphenation with preceding techniques, such as LC, SPE or other types ofextraction.
Common misconceptions about large volume injectionsVarious large volume injection techniques have been introduced and evaluatedsince the late 1970’s, but they are still often met with suspicion.
Commonly raised concerns are that the procedure will result in increasedcontamination of liners, retention-gaps and columns, and the fact that thesignal-to-noise ratio is not necessarily increased with concentration. However,this applies not only to large volume injections but also to off-line samplevolume reductions. Provided that the same percentage of the sample isinjected, the sample matrix affects all injection techniques in the same mannerindependently of injection volume. Thus, eliminating these problems requiresattention to the clean-up methods and the detection, to avoid contaminationand interfering peaks as well as to improve the signal to noise ratio, ratherthan adjustment of the injected volume.
For large volume injections careful optimization and evaluation arerequired. On the other hand this also often applies to traditional injectors.While the former techniques are often well investigated, split/splitless andon-column injectors are usually operated with the same settings, regardlessof the analytes, solvents, matrix etc. In many cases much could be gained byoptimizing the standard injection techniques.
On-column large volume injection (OC-LVI)Large volume on-column injection systems are based on traditional on-columninjectors. The sample is introduced via an auto-sampler or an LC- pump intoa retention gap where the solvent is vaporized [102]. Normally the OC-LVIis associated with partially concurrent solvent evaporation, in which some of thesolvent is evaporated continuously during the sample introduction [103].Volatile analytes are retained in the residual solvent and subsequently refocusedby the solvent effects. Consequently, the sample introduction rate must beslightly higher than its evaporation rate. This can be ensured by adjusting theinjection speed together with the GC oven temperature and carrier gas flow.
43
Note that the solvent evaporation occursfrom the rear of the flooded zone,Figure 9.
The on-column-LVI system isusually equipped with an early solventvapor exit, which serves two purposes: toincrease the evaporation rate and toprotect the detector from large amountsof solvent vapor [104]. In this design theretention gap (an uncoated pre-column)is connected to a coated pre-columnfollowed by a three-way connector, joiningthe pre-column, the vapor exit and theanalytical column. During the solventevaporation stage the solvent vapor exitis open and the solvent vapors areeliminated. With this set up the vapor flowrate can be increased 20-50 fold [100].Volatile analytes are trapped in the remainingsolvent or the soaked stationary phase ofthe pre-column.
Loop-type injector/interfaceThe loop-type interface consists of a 6- or 10-port valve equipped with aloop, 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 thevalve the sample is pushed by the carrier gas into the retention gap, wherethe solvent is evaporated. While the solvent evaporates an increase in pressurecan be observed, and a subsequent pressure drop indicates that the solventhas evaporated. If a vapor exit is used, it is closed some 10-30 seconds laterto ensure that all solvent vapors have left the column system. A restrictor ismounted on the vapor exit valve to purge the solvent vapor exit and thusprevent remaining solvents interfering with the subsequent analysis by back-flow. Once the injection is finished and the solvent has been evaporated anormal GC separation is performed [105].
Figure 9. Partially concurrent solventevaporation. Volatile analytes aretrapped in the liquid film andrefocused by the solvent effect.
44
The carrier gas can never completely empty the part of the retention gapoutside the thermostatically controlled oven, instead it leaves solvent residueson the capillary walls. The remaining solvent leaks into the column systemduring the analysis, resulting in a broad, tailing solvent peak that interfereswith the analyte peaks if a general detection system, such as FID or fullscanEI-MS is applied. To avoid this, separate carrier gas and transfer lines areused, Figure 10C and D. The carrier gas is diverted to the transfer line duringinjection and to the carrier gas line during analysis. The injection valve isequipped with a restrictor to enable purging of the transfer line. With a moreselective detector a common line from the injection valve into the GC ovenand 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 parametersettings.
J
E F G K
I
HA
B
C D
L
Figure 10. The loop-type injector/interface. A: Gas flow regulator, B: Injectionvalve equipped with an injection loop and an injection port or a connectionto 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 gasline placed inside the GC oven.
45
Concurrent solvent evaporationThe large amounts of solvents injected are managed through a process calledconcurrent solvent evaporation [106], Figure 11. When the solvent reachesthe thermostatically controlled GC oven, it starts to vaporize. The solvent isthen pushed back by its own vapor pressure, preventing it from reaching thecoated GC column, provided that the injection temperature, i.e. the GC oventemperature, is kept at the pressure-corrected boiling point of the solvent, orhigher. At these settings the evaporation site in the retention gap is focusedin 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 columnflooding.
Since the evaporation occurs from the front end of the solvent plug, nosolvent trapping is achieved, which affects volatile analytes, see deformation ofearly eluting peaks, below.
Similarly to OC-LVI, a solvent vapor exit is often used together with aloop-type injector, to increase the evaporation rate. The retention gap(Figure 10E) is connected to a pre-column (F), which in turn is connectedto 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 trappingcapacity of the pre-column is however limited since no significant phasesoaking occurs, and volatile analytes may be lost unless a co-solvent is used.
Figure 11. Fully concurrent solvent evaporation. Thesolvent evaporation occurs at the front end of thesolvent plug. Solvent trapping does not occur to anylarge extent, so volatile compounds are either trappedin the pre-column or lost through the vapor exit.
46
Peak deformationOne of the drawbacks of the loop-type is the risk of peak deformation, threetypes of which may occur, as investigated in Paper II and discussed below.
Peak splittingAs described above, due to inappropriate temperature settings the samplemay fully or partly flood the coated (pre-) column. A too low temperaturegenerates an insufficient pressure in front of the solvent plug, whichconsequently moves forward and finally floods the column. Different portionsof the sample will then have different starting points in the retention gap andthe column, resulting in each peak being split into two or more peaks. Insome cases a too high injection temperature may also produce this effect,due to uncontrolled evaporation shooting the solvent into the coated GCcolumn. The remedy is to adjust the injection temperature so that it is at thepressure-corrected boiling point of the solvent.
An extreme case of the peak splitting phenomenon is illustrated inFigure 12.
53oC
55oC
54oC
Figure 12. Peak splitting. The selected part of three PAH chromatogramsshow peak splitting for flourantene, pyrene and benzo(a)fluorine. Gaussianpeaks are obtained at 54°C, whereas the peaks are distorted at erroneoussettings, i.e. 53 and 55°C.
47
Broadening of early eluting peaksIn concurrent solvent evaporation the solvent evaporates from the front endof the solvent plug. At this point the temperature in the oven is at least ashigh as the boiling point of the solvent. As the vapor moves into the coatedcolumns its boiling point decreases due to the pressure drop along the columnsystem. Hence, little re-condensation of the solvent occurs and thus thesoaking of the stationary phase in the coated pre-column is poor, limitingthe retention capacity. Consequently, volatile compounds start eluting duringthe injection and solvent evaporation, resulting in band broadening. Volatilecompounds can also be lost through the vapor exit together with the solventvapors, especially if high injection temperatures are used.
To prevent the described effects the use of co-solvents has beenproposed. Grob and Müller used n-heptane in n-pentane to trap and focusearly eluting compounds [107]. The higher boiling solvent (n-heptane) formsa thin film on the capillary walls in front of the evaporation site, trappingvolatile analytes. For the same reasons, Noij and Kooi injected 100 µL ofn-decane before the injection of the sample, which was dissolved in a mixtureof MtBE and ethyl acetate [108]. In the study presented in Paper IIn-dodecane was added to the primary solvent (n-hexane) to sharpen the peaksof the early eluting analytes. A concentration of 0.1 % was enough to trapthe lowest boiling analytes, i.e. monoBDE and phenanthrene. Due to thelarge difference in boiling points between the solvents (∆t = 147°C)n-dodecane in this case acts as a keeper rather than a co-solvent. Dodecanedid not evaporate to any significant extent during the solvent evaporation,but was eluted after closure of the vapor exit, during the GC oven temperatureprogram, giving a late eluting solvent peak. However, the amount reachingthe detector was low; e.g. only 1 µL in a total volume of 1 mL. A keeper doesnot require the injection to be re-optimized, nor does it affect the elutionstrength of the solvents in a preceding LC separation. In addition, therisk of the small remaining volume of dodecane flooding the coatedcolumn is small.
This keeper technique cannot be applied when analyzing volatilecompounds. Corresponding peaks (up to C14 in an alkane series) will simplybe lost in the dodecane solvent peak. This is an obvious drawback, but forsemi-volatile compounds such as those investigated in the work underlying
48
this thesis (the low molecular weight PANHs, PAHs and PBDEs), adding akeeper is a simple and efficient way to obtain sharp, early eluting peaks.
If higher concentrations of dodecane are used, the pre-column may nolonger be necessary since the retaining effect of the keeper may be enoughto trap the low-boiling analytes [100]. Excluding the pre-column increasesthe solvent evaporation rate and thus reduces the injection time. It alsosimplifies the column system.
Trailing peaksIn these studies, the most common type of peak deformation was a trailingpeak following the main peak of all eluted compounds. The source ofthis peak deformation was identified by separately analyzing the contentsof the carrier gas line, the transfer line, the retention gap, the pre-column,the vapor exit and the analytical column after an injection. The trailingpeaks proved to originate from the carrier gas line.
When a loop-type interface with separate transfer and carrier gas lines isused, some of the solvent and the solutes contained therein will inevitablyshoot up through the carrier gas line during injection. The analytes are thendeposited in the capillary outside the GC oven and subsequently eluted,probably due to indirect heating of the carrier gas line. To solve this problemthe first Y-press-fit connector was moved inside the oven and a 5 m carriergas line, Figure 10L, was used. Even though some of the sample may enterthe carrier gas line it is prevented from going beyond the thermostaticallycontrolled GC oven with this set-up, and will accordingly be focused on thepre-column.
If a selective detector is used, the transfer and carrier gas line can becombined into a common line. Trailing peaks are also avoided with this design.However, even though not detected, tailing solvent peaks may affect thedetection and lead to increased contamination of the detector, which canreduce both the lifespan of a mass spectrometer’s ion source filament andthe sensitivity the detector.
Why loop-type?The loop-type interface is well suited for large volume injections whendetermining PBDEs and carbazoles. Since neither the most low-boiling
49
PANHs nor PBDEs should be considered volatile, the risk of losses throughthe vapor exit is low. The set-up is simple; no pump or auto-sampler is requiredfor offline methods and the pump flow in hyphenated techniques is variable.In addition, PBDEs have proven to be thermally degraded and may beadsorbed by active sites in retention gaps, which should therefore be kept asshort as possible, Paper I.
On-column-LVI is beneficial for volatile compounds and requires astable, non-pulsed and predefined sample introduction flow. In addition theretention gap is usually several meters long.
Large volume splitless injectionA hot splitless injector can be used for injecting large volumes of samples.The liquids are kept in place by a packing material, preventing them fromreaching below the split point. The cooling effect of the evaporating solventretains the volatile analytes at the site of evaporation, which requires a packingmaterial of low thermal mass, e.g. glass wool. After full solvent vaporizationthe packing material is heated to the injector temperature and the analytesare transferred to the GC column.
The solvent vapors can be discarded by the overflow technique, through theseptum purge line. The septum purge is closed prior to full vaporization ofthe solvent [109]. The process resembles that of a large volume PTV injec-tion.
The solvent vapors may also be discarded by concurrent solvent recondensation,through the column. For this, the GC oven is kept at a temperature belowthe 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 areformed in the injector [110].
Both these techniques utilize packing materials, primarily glass wool,making large volume splitless injection unsuitable for determination ofPBDEs, as described in Paper III. Different cup-liner designs are promis-ing, but are not yet established alternatives for preventing the liquid fromflooding the inlet [111].
50
Large volume PTV injectionThe programmed temperature vaporizer, operated in a so-called solvent ventmode, can be used for large volume injections. The sample is introduced intoa liner at a low temperature (e.g. 50-80°C) with the split valve open, and thesolvent is evaporated and discarded, but the analytes are trapped on the linerwalls or on a packing material. The analytes are then transferred to the columnby closing the solvent split and rapidly increasing the injector temperature,typically to 275-350°C. The robustness of this type of injector and its abilityto handle dirty samples [112, 113] prompted further investigation.
Large volume PTV injections were successfully used in the studiespresented in Paper III and V for the determination of PBDEs. Althoughevaluation and careful optimization was required, it proved to be a suitabletechnique even for BDE-209, in contrast to earlier assertions [114].
Sample introduction and solvent evaporationSample introduction may be accomplished with either multiple injections orcontinuous injection [97]. In multiple injections mode, several small portionsare injected at fixed intervals. This method is simple and fully compatiblewith common auto-samplers. After the injection of a small volume (normally5 µL) time is allowed for full evaporation of the solvent to prevent floodingof the injector.Multiple injections do not allow hyphenation. Furthermore, a complete sampleintroduction cycle must be performed for each portion. Irreproducibility inthe injection may be cumulative, resulting in poor precision, and rates ofwear of septa and injection ports are drastically increased.
If the sample is injected continuously the solvent is evaporatedconcurrently. 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 mustoccur above the split point in the liner. If sample components are depositedbelow this point transferring analytes is difficult, as they are not covered bythe main stream of the carrier gas. Adsorption and thermal degradation mayalso be induced by the active sites outside the liner. In extreme cases materialis lost through the split line together with solvent still in liquid state.
The solvent evaporation is affected by four parameters: the split flow,
51
the injector temperature, the sample introduction flow rate and the evaporationsurface area [115-117]. To promote solvent evaporation the first two of theseparameters should be kept high. Solvent evaporation obviously increases withtemperature, while an increased solvent vent flow more rapidly discards thesolvent vapors, Papers III and V.
For both multiple and continuous injections the ratio of the sampleintroduction to the evaporation rates is important. The yields of the congenerswere strongly affected by the sample introduction rate. At both too low andtoo high settings the response decreased. To investigate this further, the fateof the solvent was visually observed by continuously introducing a solutionof perylene into a glass liner illuminated with UV-light. The injection followedthree main scenarios depending on the sample introduction, solventevaporation and flooding characteristics, Figure 13.
G
E
H
F
I
J
A B C D
Figure 13. Solvent evaporation in amultibaffled PTV liner. Injections of solventcontaining perylene were performed at 80°Cand monitored under UV-light.
A: Schematic diagram of the liner, withneedle (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 thenext droplet has left the needle (H).
C: Sample introduction flow rate: 300 µL/min.The solvent leaves the needle as a non-visiblespray and evaporates continuously at the firstbaffles. A liquid film is formed, which trapsvolatile analytes (I).
D: Sample introduction flow rate: 500 µL/min.The sample introduction rate exceeds thesolvent evaporation rate, resulting inflooding (J).
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A high sample introduction flow rate led to insufficient solvent evaporationand, hence, to injector flooding, as illustrated in Figure 13J, resulting in lossesof material and discrimination, as described above in the discussion aboutthe 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 solventfilm in the liner, causing the solutes to be weakly retained and consequentlylost [118]. This theory was supported by the visual observations. At100 µL/min the solvent leaves the needle as droplets, each of which areevaporated in the liner before the next leaves the needle, Figure 13G and H.This rationale satisfactorily explains the behavior of BDE-47, but not thatof compounds with high boiling points such as BDE-209. The effect of lowintroduction flow rates could not be fully explained by the results of thestudies 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 optimalinjection flow rates, since they have different boiling points: 36 and 69°C,respectively. When using n-hexane, the highest yields are obtained at aninjection 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 rateis 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
Figure 14. Detector response for BDE-209 as a function of sampleintroduction flow rate. The optimal settings differ for n-pentane and n-hexanedue to the different boiling points.
53
injections and a continuous injection at 100 µL/min, made using a syringe.With the latter technique the yield and precision were clearly lower forBDE-209. With auto-samplers small injection volumes are easier to inject athigh speeds, so multiple injections in this case are preferable for thedetermination of PBDEs.
Obstacles in the flow path, e.g. baffles and cups, prevent the solventfrom being flushed through the liner. Glass wool, optionally combined withpacking materials such as Tenax, is commonly used in PTV liners for largevolume injections. Apart from trapping volatile compounds, the packingmaterial hinders the solvent and provides an increased surface area fromwhich the evaporation and heat exchange can take place. On the other hand,compounds with high boiling points may be difficult to desorb, resulting inpoor yields in the subsequent transfer. Thermal degradation may also becatalyzed at active sites in the materials [98]. Mol et al. investigated a numberof different liners and packing materials with respect to degradation and
Figure 15. Relative response for six selected BDE congeners injected with aconventional syringe. Grey bars: fast multiple injections (10x10 µL). Whitebars: slow continuous injection (100 µL/min).
54
discrimination [119]. Tenax and glass wool in some cases caused completeloss of the analytes. For high-boiling and thermally labile compounds thecup-liner gave the highest recoveries.
In study III different liners were investigated for large volume PTVinjections of PBDEs. Cup-liners were unfortunately not available for thePTV used (a Gerstel CIS-4). Instead, an empty multi-baffled liner proved tobe the best choice, providing low discrimination and high precision.
Analyte trappingDuring solvent evaporation volatile analytes can be lost through the split exittogether with the solvent. There are several ways to handle this problem. Bymeans of partial concurrent solvent evaporation during speed-controlledinjection a small portion of the solvent can be kept in a liquid state, in whichvolatile analytes are trapped. If multiple injections are to be used the techniqueis more complicated, as a small amount of solvent has to remain liquid aftereach sample introduction.
As discussed in the previous paragraph, packing materials, such as Tenaxand glass wool, are more commonly used to trap the volatile analytes [120-122],but they are accompanied with clear risks of discrimination and thermaldegradation [119].
These problems are addressed in Paper III. The yield of BDE-209 wassignificantly reduced when utilizing glass wool or sintered glass liners astrapping agents compared to an empty liner. Instead, n-dodecane was usedas a keeper to retain the low boiling point monoBDE and triBDE. As for theloop-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 beenutilized by Termonia et al., who showed that 15 % n-octane in n-hexane canbe used to trap low-boiling compounds, such as biphenyl [123].
Analyte transferOnce the solvent has been eliminated the analytes are transferred to thecolumn, by closing the split valve, increasing the temperature and (optionally)applying a pressure pulse. Ideally the transfer temperature should be as highas possible to desorb and transfer even analytes with high boiling points. Onthe other hand, the risk of degradation of thermally labile compounds
55
increases with temperature. In Paper III a temperature of 325°C was shownto be optimal for transferring the high molecular weight BDE-209. As insplitless injection, the transfer rate can be increased if a pressure pulse isapplied during the splitless time. This was shown to increase the yield ofBDE-209 up to four fold.
The transfer proved to be fast, even for the high-boiling BDE-209. Afterapproximately 35 seconds the maximum yield was obtained. The short transfertime is not surprising considering the small inner diameter of the liner andthe applied pressure pulse.
The author´s note on of the loop-type and the PTV injectorsThe advantages and disadvantages of the loop-type and the PTV injectorsare summarized in Table 3. The loop-type injector exhibited lowdiscrimination against high molecular weight congeners and high precisionwith regard to absolute and relative responses of the PBDEs. However, theretention gaps of this set-up had a limited life span even when analyzingrelatively clean air samples. The junctions of the capillary columns systemrequire special attention and operator experience to avoid leaks.
The discrimination against high molecular weight congeners, using PTVinjector, was low but significant. The transfer efficiency of BDE-209 was inthe range of 76-80 %. On the other hand the PTV is rugged and robusttowards sample matrix residues. Approximately 100 large volume injectionsof air sample extracts were carried out, without need for replacing the liner.
Hence, the choice of injection technique stands between low detectionlimits and robustness.
Table 3. Advantages and disadvantages of loop-type and PTV injection.
Loop-type injector PTVDiscrimination against high-boiling compounds None LowThermal degradation None LowRobust towards matrix residues Low HighRuggedness Intermediate HighInstrumental complexity High IntermediateCommercially available No Yes
56
57
GC detectorsNumerous types of gas chromatography detectors have been developed tomeet the wide range of GC demands and applications. The requirements forsensitivity are met by virtually all GC detectors, with detection limits in thefg-ng range. Selectivity towards compounds containing specific elements, forexample Br and N, enables detection and quantification in the presence ofchromatographically interfering species. In addition, since the chemicalbackground is vastly reduced, detection limits are in most cases improved.High-selectivity systems improve the determination of certain compoundclasses, but similarly reduce the amount of information that can be obtainedabout the sample. In general, GC detectors do not provide structural data,with the exception of mass spectrometers. In this section the detectors utilizedin the work underlying this thesis are discussed.
The flame ionization detector (FID)The flame ionization detector is the most generally applicable and one of themost widely used types of detector for gas chromatography. The criticalparts of the FID are the hydrogen/air flame and the collector. Most carbon-containing compounds eluting from the GC column are ionized in the flameand 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 iseasy to use and requires minimal maintenance. The major disadvantage ofthe FID is its poor selectivity, which places high demands on the sampleclean-up.
In the studies this thesis is based on the flame ionization detector wasused only as a reference detector.
The nitrogen phosphorus detector (NPD)The nitrogen phosphorus detector (or thermionic detector, TID) is selectivetowards nitrogen- and phosphorus-containing compounds. As with the FID,the NPD is supplied with hydrogen, air and nitrogen, but due to the lowflows no flame is ignited. Instead, the formation of ions takes place in a
58
boundary layer, formed around a heated alkali salt bead. The mechanism ofthe NPD is not fully understood, but it is believed to involve decompositionof the eluting compounds to CN, PO and PO2 [125, 126]. These radicalsform ions by extracting electrons from the alkali salt present in the bead. Adetector 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- ornitrogen-containing compounds. In Paper IV these settings were optimizedwith 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, dueto 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 withlower energy.
β + N2 → β*+ 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. halogensand 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 verysensitive. The limits of detection for PBDEs are in most cases in thesub-picogram region. ECDs were used for evaluation of different injectorsin studies I, III and V.
Mass spectrometry (MS)The mass spectrometer provides more information, selectivity and in manycases more sensitivity than any other type of GC detector. Although moreexpensive and complex than the alternatives, MS is rapidly gaining groundand may even be the most widely used detection technique for GC today.
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In gas chromatography the analytes are eluted in a gas flow, which makes themobile phase easy to discard and simplifies hyphenation to MS. The massspectrometer 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 areessential for the ionization. Generally, different ion sources are used forelectron ionization and chemical ionization.
The analyzer may be any one of various types, but all have the samepurpose: to separate the ions according to their mass to charge ratio (m/z).High-resolution instruments (e.g. time of flight, double focusing sector andfourier transform ion cyclotron resonance analyzers) provide a resolutionhigher than 5000 (m/∆m) and the option of accurate mass measurements.However, these systems are expensive and in many cases more difficult tooperate than simpler alternatives. In the studies this thesis is based upon, aquadrupole analyzer was used, which together with the associated ion-trapconstitutes low-resolution MS.
In quadrupole systems, ions with different m/z values are discriminatedby applying both a direct potential (U) and an altering potential (V) betweenfour rods. The altering field gives the ions entering the quadrupole chambera trajectory, which is either stable or unstable, depending on the settings.Only ions with stable trajectories can pass through the chamber and bedetected. The resolution is determined by the U/V ratio, whereas thediscrimination between ions with different m/z ratios is determined by theabsolute settings of U and V.
The quadrupole is relatively inexpensive and robust, but has low massresolution (∆(m/z) is generally 1 Th) and is comparably slow. The latterdisadvantage can be alleviated if the analytes are eluted in extremely narrowpeaks, as they are with narrow bore columns (Paper III) and flash GC. Inthese cases fullscan spectra may not be representative, so selected ionmonitoring (SIM) has to be applied.
The detector is usually an electron or photo multiplier. When an ion collideswith such a detector, a number of electrons (or photons) are released. Thesein turn collide with a dynode, releasing more electrons that collide with thenext dynode, releasing more electrons, and so on, finally generating ameasurable current: the detector signal.
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Electron ionization (EI)In electron ionization the effluent from the GC column is bombarded withhighly energetic electrons, normally 70 eV, emitted from the ion sourcefilament. The process results in radical formation followed by fragmentationof 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 fragmentationpatterns for each compound or group of compounds. The process isreproducible and to a large extent independent of the instrument, so genericdatabases can be utilized for spectra interpretation. Electron ionization massspectrometry was used in study IV to confirm the identity of the carbazolesdetermined in real air samples by LC-GC-NPD.
Chemical ionization (CI)Chemical ionization is used as an alternative or supplement to EI. It is asofter technique yielding less fragmentation, which may give additionalstructural 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 toprovide a higher ion source pressure. The electrons emitted from the filamentcollide with the reagent gas and the products formed react with the effluentsfrom the GC column. Ionization and fragmentation depend on the structureof the analyte.
Electron capture negative ionization (ECNI)In ECNI-MS the reagent gas serves as a buffer, slowing down the electronsemitted from the filament (1). These thermal electrons are captured by theanalytes in either an associative (2) or a dissociative manner (3).
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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 bromineions. Hence, PBDEs have traditionally been determined by ECNI, monitoringm/z 79 and 81. The technique has excellent selectivity towards brominatedcompounds and low detection limits. However, the structural informationobtained is minimal and since only bromine ions are detected isotopic dilutioncannot be applied.
The mass spectrometric properties of PBDEs are presented in Paper VI.The investigation revealed that BDE-209 shows a different fragmentationpattern from those of BDEs with lower molecular weight. By uptake of oneelectron and cleavage of the ether bridge a large fragment at m/z 486/488 isformed, corresponding to C6Br5O
-, Figure 16. Monitoring this fragmentallows the use of isotopic dilution for quantification. 13C-labeled BDE-209shows the same fragmentation pattern, with ions at m/z 492/494. To avoidinterferences in the mass spectra, m/z 486 and 494 should be recorded forthe native BDE-209 and the 13C-labeled BDE-209, respectively. If assumingthat discrimination and degradation, for instance, affect the native BDE-209and the 13C-labeled BDE-209 in a similar manner, the latter compound ismore reliable as internal surrogate standard than for example the commonlyused BDE-119 for the problematic determination of BDE-209, as describedin Paper II and VI.
In addition, by monitoring higher m/z fragments the signal to noiseratio is increased, since interfering ions are less likely to be present in thehigher mass region than in the m/z 79 - 81 region. The detection limit forBDE-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
62
to noise ratios were observed when analyzing real samples as well as a technicalPBDE mixture for congeners 197, 206, 207, 208 and an unidentified octaBDE.As for BDE-209, the presence of these fragments makes isotopic dilutionapplicable to these compounds too [127].
100 200 300 400 500 600 700 800 900m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
488.2486.2
78.580.5
407.2
147.4
160.4174.4 471.3326.9 391.2 563.6 641.2 719.2 797.5
484.2
490.2
O
Br
Br
Br Br
Br
Br
Br
Br
Br
Br
486
488
407
471
Figure 16. Mass spectrum of BDE-209. Two large fragments are formed; atm/z 79 and 81 (Br-) and around m/z 486/488. Monitoring the latter fragmentenables isotopic dilution.
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Sample extractionAlthough some samples can be analyzed without clean-up, most analyticalmethods involve extraction of target analytes in some way from the samplematrix. 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. Thissection focuses on techniques used for extracting target analytes from theparticles collected on glass fiber filters and analyzed in the studies associatedwith 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 groupof compounds are possible, but normally the process is more general andsubsequent chromatographic steps and/or selective detection are required.All forms of extraction depend on the partitioning between the sample matrixand the extraction media.
Like any chemical reaction the extraction processes may be under eitherthermodynamic or kinetic control. Since an extraction includes several steps,e.g. transfer of target compounds to matrix surfaces, desorption and diffusionin the extraction media, the overall process may involve a combination ofthermodynamically and kinetically controlled steps. Briefly, athermodynamically controlled extraction depends on the coefficient of thematrix-solvent-equilibrium for a given compound, i.e. the partitioningcoefficient, while a kinetically controlled extraction is limited by the desorptionrate 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 thesolvent both thermodynamically and kinetically, because the solvent’scapacity for dissolving target compounds usually increases withtemperature and the viscosity of the solvent is reduced, increasing themobility of dissolved compounds [129]. Consequently, the resistance tomass transfer is decreased. Heated solvents also have enhanced ability toswell and permeate the matrix [130].
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Static and dynamic extractionsStatic extractions are performed in a closed vessel with a predefined volumeof solvent. The recovery of a given analyte depends on the partitioning co-efficient between the matrix and the extraction medium as well as the extrac-tion rate and time. The extraction is often time consuming and the targetcompounds 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 asample-containing vessel. Since fresh solvent is continuously supplied, theequilibrium will be driven towards the solvent and the partitioning coeffi-cient becomes less important.
If thermodynamically controlled, a dynamic extraction will consumeless solvent and time. However, if kinetically controlled and an extendedtime is required, dynamic extraction may actually consume more solvent thanthe corresponding static method, unless the dynamic extraction is combinedwith 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 profilesto be monitored and on-line transfer to chromatographic analysis systems.
Soxhlet extractionEven though introduced as early as 1879, Soxhlet extraction is still one ofthe most commonly used extraction techniques. The set-up is simple,consisting of a round flask, a cooler and the extractor, which allows manyextractions 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 withfresh solvent that has condensed in the cooler, and extracted compounds areenriched in the flask.
Soxhlet extraction is thus dynamic, but can at the same time be run formany hours, without increasing solvent consumption. However, the techniquehas several drawbacks. Generally, elevated temperature yields higher recoveriesfrom complex matrices, but in Soxhlet extraction the solvent is cold.
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Consequently, the extraction is slow, commonly with extraction times of8-48 h. There is also a risk of adsorption to active sites on the large glasssurfaces and degradation of labile compounds as the extract in the flask isheated for several hours. For these reasons Soxhlet extraction was not selectedas 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 heaterwas too high, illustrating one of the mentioned drawbacks of the technique.
Ultrasonication-assisted solvent extractionUltrasonication-assisted extraction is today one of the most popular extractionmethods, due to its simplicity and the possibility it offers to run many samplesin parallel. The sample is placed in a vessel, e.g. a test tube, and a solvent withsuitable properties is added. The extraction is performed under ultrasonicationto enhance the mass transfer of target analytes to the solvent and to disruptthe matrix physically.
In most cases two or more consecutive extractions are carried out toincrease the recoveries. Ultrasonication-assisted solvent extraction was usedby 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, Vand VI for the extraction of PBDEs from glass fiber filters, and in study Vto evaluate dynamic sonication-assisted extraction.
Dynamic sonication-assisted solvent extractionPaper V presents a dynamic sonication-assisted extraction (DSAE) methodfor determination of PBDEs in air. The technique was developed by Sanchezand co-workers for the extraction of organophosphate esters from glass fi-ber filters [131]. The set-up is simple, robust and can be assembled fromunmodified equipment that is available in most laboratories. The sample isloaded into an extraction cell, lowered into a thermostatically controlled ul-trasonic bath and the pressurized extraction medium is flushed through thecell using a HPLC pump.
66
In this study indoor air particles collected on glass fiber filters were used.The results show that an elevated temperature significantly increased therecoveries for BDE congeners of low and intermediate molecular weight,i.e. 47, 154 and 183. Slightly lower recoveries were obtained for BTBPE withultrasonication and for BDE-154 and BDE-209 with ultrasonication incombination with an elevated temperature. This study was performed onfilters cut into small pieces. When extracting large, intact parts of a filterboth the recoveries and the precision were improved by ultrasonication. Aconclusion drawn from these results was that ultrasonication is importantfor mixing the sample matrix and solvent. Channeling in the solid samplemay lead to poor solvent penetration of parts of the matrix decreasing theextraction efficiency and reproducibility. The use of ultrasonication solvedthese problems.
The average recovery from real samples using the DSAE set-up wasfound to be 95 %. This was determined by first performing a dynamic ex-traction followed by exhaustive static extraction of the same sample. Theresults were reproducible with an average RSD of 2.8 %. The recoveriescould possibly be increased by adding a static, stopped-flow step to providemore time for the extraction.
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On-line coupling to GCThroughout much of the history of analytical chemistry, automation andhyphenation have been intensively researched. For instance, chromatographicand mass spectrometric systems have been successfully coupled, and widelyapplied. Methods for multidimensional chromatography and on-line clean-up are also attracting interest and in some cases commercially available systemshave been introduced.
The high separation efficiency and wide variety of sensitive and selectivedetectors available make gas chromatography the first choice analytical methodfor many applications. However, the analysis is often preceded by samplepreparation steps, of varying complexity, for example extraction, groupisolation and removal of polar material and lipids.
Besides decreasing the detection limits, one of the most attractive featuresof large volume injections in GC is the possibility it offers for hyphenationwith clean-up techniques. Liquid chromatography [132], solid phase extraction[133] and an array of other extraction procedures [134] have been successfullycoupled to gas chromatography.
Benefits of hyphenationWhether the technique preceding the gas chromatographic analysis is LC,SPE or another form of extraction, a larger portion of the sample will beintroduced into the GC column than if an off-line method and conventionalGC are used. This is a result of the large volumetric loading capacity of theclean-up method and the large-volume GC interface. Moreover, apart fromcarry-over the risk of contamination in a closed system is minimal and limitedto 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 methodsas a result of accidental spillages, incomplete sample transfers from one containerto another and co-evaporation with the solvent during concentration steps. Theoverall detection and quantification limits are consequently decreased, makinghyphenated techniques well suited for trace analysis.
A fully automated instrument also offers better control over the completeanalytical method. Parameters such as solvent and gas flow rates, temperatures,event times and fraction collection can be precisely regulated and may be
68
recorded. The sample preparation is simplified and time consuming manualclean-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 systemsThe risk of memory effects is significantly higher in a system consisting oftubing and valves rather than disposable glassware and pipettes. Carefulwashing procedures must be employed and system blanks regularly checked.
If extractions are coupled directly to GC the entire sample will be injectedand analyzed. This leaves no option for repeated runs and furtherinvestigations unless the sample is divided into portions, which may be possiblewith samples of matrices such as blood or sediment, but more difficult withair samples, as described in Paper V.
LC-GCOne of the most interesting applications of large volume injections for GCis to couple LC on-line to GC. The combination of the two chromatographicmethods offers a technique with truly orthogonal separation systems.
The first on-line coupled LC-GC instruments were described in themid-1980s [102, 135, 136]. Since then the technique has been developed, inparticular by Grob, to include a large variety of interfaces and targetcompounds as well as LC parameter settings, such as mobile phases andflows [100]. A detailed description of LC-GC is beyond the scope of thistext, but has been thoroughly covered elsewhere [99, 100, 132]. A commonconcern for all attempts to hyphenate these techniques is the interfacingproblem. Even capillary LC with flows at about 10 µL/min yields fractionvolumes that are too large for conventional GC injections. Hence, large volumeGC injections, described above, are crucial elements of LC-GC.
NPLC and RPLCThe non-polar, volatile solvents used as mobile phases in normal phase LC(NPLC) make hyphenation to GC relatively straightforward. Its compatibilitywith GC has made NPLC (or normal phase SPE) the most widely used clean-
69
up method for GC analysis, so many existing methods can be easily transferredto on-line techniques. A variety of solvents with low boiling points, such asn-pentane, n-hexane, dichloromethane and methyl-tert-butyl ether, can bechosen as mobile phases without interfering with even the most volatileanalytes [100, 132].
Coupling reversed phase LC (RPLC) to GC places higher demands onthe interface [137]. Water is not a suitable solvent for GC analysis, due to thehydrolysis of siloxane bonds it induces, causing rapid deterioration of theanalytical GC column. For non-vaporizing interfaces, retention gaps withspecial qualities and/or conditions are required, especially for the on-column/retention gap technique. Due to the low vapor pressure of water, analysis ofvolatile compounds is virtually impossible. Co-solvents have been suggestedto improve the capacity of trapping low-boiling analytes [138]. Vaporizinginjectors, such as the PTV, is less sensitive towards water-containing samples.However, the trapping of volatile analytes and injector flooding are problemsthat need to be addressed [139].
Heart-cut or back-flush?Transferring heart-cut fractions eluting from an LC column minimizes therequirements for the LC system both chemically and technically. The LCeffluent is introduced into the GC system as long as the target analytes areeluting.
The heart-cut technique has several inherent drawbacks if large groupsof compounds are to be transferred. The fraction volume may be ratherlarge and cannot be decreased without losses of LC resolution and/or samplecapacity. Since the analytes are separated in space before introduction intothe GC-column, they are subject to changing conditions during the largevolume injection, i.e. changes in sample introduction time, solvent effectsand temperature, due to the cooling effect of the evaporating solvent. Inaddition, it is essential to transfer the entire fraction. Even so, both early andlate eluting compounds may be discriminated against, as an effect of retentiontime 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
70
components that are still in the column when the flow is reversed will eluteas a single peak. The volume of the back-flush fraction is generally smallerthan that of the corresponding heart-cut fraction. Since the solutes are evenlydistributed 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 mustremain 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 determinationof carbazole-type PANHs. The isolation of this group of compounds isbased on hydrogen bonding between the carbazoles and the nitrogen of thedimethylamino propyl groups of the stationary phase [140, 141]. A secondaryretention mechanism is electron donor/acceptor complex formation, thestrength of which depends on the respective analytes’ numbers of fusedaromatic rings. The column was end-capped in-situ with hexamethyldisilazane,to avoid hydrogen bonding, between acridines and free silanol groups,Figure 17.
The method described in Paper IV includes a pre-separation of PAHs andaliphatic compounds from the PANHs. In later investigations the crude extractwas introduced into the LC-GC-NPD system, while maintaining selectivityand gas chromatographic resolution.
Figure 17. Retention mechanism for carbazoles and acridines in thedimethylamino propyl column. The retention mechanism of carbazoles isbased on the hydrogen bonding to the stationary phase. Unless the column isend-capped acridines interact with residual silanol groups.
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The method was further developed to include the acridine-type PANHs(unpublished results). A schematic diagram of this “double back-flush” systemis 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 tobe 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
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 shapedpeaks, with evenly distributed analytes, Figure 19. The fractions were trappedin two loops mounted on a multi-position valve (Figure 18D) andsubsequently injected and analyzed separately on the GC-NPD system,Figure 20. For the carbazoles the elution volume was significantly decreasedand a more robust injection procedure was achieved for the acridine fraction,compared to when applying the heart-cut technique. The average RSD forthe relative response was 2.9 % and below 5 % for all analytes exceptdibenz(a,i)acridine (6.2 %). No cross contamination between the two groupswas detected.
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 thefirst eluting analytes are trapped, C: Injection valve, D: Injection loop-valve,equipped with two loops for the two fractions, E: Vapor exit valve.
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Figure 19. LC-UV chromatograms of carbazoles and acridines. A: The LCseparation without back-flush, B: LC separation with double back-flush. Theacridines and carbazoles are eluted as two uniform fractions.
Figure 20. GC-NPD chromatograms of carbazoles and acridines. The twogroups are injected and separated one by one.
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On-line extraction-GCCoupling 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 datainterpretation.
On-line coupling to GC requires the extraction media to be flushed outfrom the extraction vessel and into the interface. Consequently, hyphenatedextraction-GC systems are based on dynamic extraction or a combination ofdynamic 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)-GCEricsson and Colmsjö presented a dynamic microwave-assisted extractionprocedure coupled to SPE for determining PAH in sediment [142, 143]. Theextraction efficiency was enhanced by the increased temperature. Thetransformation of the energy (from radiation to heat), requires solvents and/or sample matrix to have dipole-moments, which are affected by themicrowaves. A major benefit of using microwaves is that the sample matrixitself can be heated and not dependent on indirect heating from hot solvents.The technique was later coupled on-line to GC for determination oforganophosphate esters in air using a PTV-injector as the interface [144].
Pressurized hot water extraction (PHWE)-GCPressurized hot water extraction coupled on-line to GC has been developedmainly by Reikkola and co-workers [145-147]. The temperature is increasedup to 325°C and elevated pressure keeps the water in its liquid state. Thisdecreases the dielectric constant and increases the solubility of non-polarcompounds in the water.
Since water is not suitable for direct transfer to GC the solvent needs tobe exchanged. As the water is cooled down its polarity increases and lipophiliccompounds can easily be trapped on an SPE column and eluted with organicsolvents [145] or extracted by membrane extraction into an organic acceptor
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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)-GCIn supercritical fluid extraction, the extraction medium is easily eliminatedsince the most commonly used fluid, CO2, is vaporized when leaving thepressurized system. The restrictor is connected to the GC injector, which iscooled by the expanding gas, thereby increasing the analyte trapping effi-ciency. The extraction medium is eliminated either through a split exit orthrough the column.
SFE has been hyphenated to GC by split/splitless [150, 151], on-col-umn [152] and PTV [153] interfaces. Modifiers such as methanol anddichloromethane are commonly used to increase recoveries, but may causeproblems due to flooding effects if conditions are not optimized.
Dynamic sonication-assisted extraction (DSAE)-GCDSAE coupled on-line to GC was first described by Sanchez et al. for thedetermination of organophosphate esters in air [154]. So far only PTVs havebeen used as interfaces, but other large volume injection systems could beapplied.
Paper V presents a DSAE-GC-MS method for the determination ofPBDE in air, as described earlier. Due to the selectivity of the ECNI/MSdetection, the extract could be transferred to the GC without any pre-sepa-ration steps other than removing particles using a frit at the exit of the ex-traction cell. Only one interfering matrix peak was observed. The previouslyproposed solid-phase extraction step used in the off-line method describedin Papers I-III, V and VI, was unable to remove this unidentified com-pound. The reproducibility for spiked filters was high, with an average RSDof 4.8 %. For real samples the average RSD was higher (16 %), possibly dueto irreproducible sample homogenization. The on-line DSAE-GC-MSmethod benefits from the low risk of contamination, unlike the off-linemethod, which showed high and irreproducible background levels, in par-ticular for BDE-49 BDE-99, BDE-183 and BDE-209, making the re-sults uncertain.
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Air samplingThe methods described in this thesis are primarily designed for thedetermination of environmental pollutants in air. From a clean-up perspective,air can be considered a relatively simple matrix. The polar and lipid contentsare commonly low and samples can therefore be analyzed without extensivesample pre-treatment. In the studies presented in Paper V the crude extractswere transferred to the GC inlet, without affecting the peak shape or thelifespan 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 recoveriesfrom extraction and purification steps can easily be calculated. Generatingan atmosphere containing a known amount of gaseous and particulate phaseanalytes is far more difficult, though successful attempts have been reported[155, 156]. Furthermore, the problematic issue of the gaseous and particu-late phase distribution of semi-volatile compounds has to be addressed.
Both PBDEs and carbazoles are almost entirely found in the particulatephase of the air. This means the compounds are more or less adsorbed toair-borne dust and particles, which constitutes the actual matrix. Hence, spikingfilters used for collecting the particulate matter does not representativelymimic real samples. In such investigations very high recoveries are normallyobtained.
When extracting spiked glass fiber filters with the dynamic sonication-assisted solvent extraction technique presented in Paper V, the averagerecoveries were 98 %. For this reason the extraction was mainly evaluatedusing real samples and relative recoveries since the actual amounts of PBDEswere not known.
The sampling set-upThe 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 PAHsin air and consisted of an anodized aluminum sampler head containing aglass fiber filter and two polyurethane foam plugs (PUFs) in series [157]. Thesampler was connected to a pump, normally adjusted to pump 3 L/min.
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Particles were collected on the filter and semi-volatile compounds wereadsorbed on the first PUF. The second PUF was a back-up adsorbent forbreakthrough determinations.
The phase distribution determined using this set-up should be treatedwith caution. During sampling, compounds initially adsorbed to particlescould be desorbed and again adsorbed on the PUFs, thus giving erroneousindications about the native distribution. All compounds discussed in thisthesis were found to be in the particulate phase, with the exception ofcarbazole, 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.
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Concluding remarksThis thesis deals with gas chromatographic injection techniques for high-boiling and thermally labile compounds, with particular focus on large-vol-ume injections. A number of methods have been investigated for the deter-mination of polybrominated diphenyl ethers. Hopefully, the reader will havegained knowledge about the principles and applicability of both conven-tional and “new” techniques, especially for PBDE analysis, but also for com-pounds with similar physical properties and chromatographic behavior.
Sensitive and robust methods need to be developed for determinationof flame-retardants, especially decaBDE, due to concerns about their possiblehealth and environmental effects. One aim of the work presented here wasto assist the development of such techniques.
Splitless injectors are commonly employed for the analysis of complexmatrices such as biological samples. The results in Paper I prove this tech-nique to be irreproducible and to discriminate against the high molecularweight BDE congeners. PTVs are proposed as alternatives, when vaporizingsystems are required. In studies I, III and V this injection technique wassuccessfully used for introducing PBDEs to the GC-column, for both con-ventional and large injection volumes.
Dynamic extraction coupled on-line to GC-MS is a convenient approachfor the determination of polybrominated diphenyl ethers in air, as shown inPaper V. The closed system minimizes losses and the risk of contamination,which are common problems during PBDE analysis. The technique wouldbe 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 carcino-genic dibenzo(c,g)carbazole and for the oil industry to establishbenzocarbazole ratios.
The mass spectral characteristics of BDE-209 presented in Paper VIenable the use of isotopic dilution for quantification. With this techniqueenvironmental chemists may improve the accuracy of determinations of thiscompound.
In future research the developed arsenal of methods will be applied tothe determination of PBDEs in both indoor and outdoor air.
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The increasing number of large volume injection techniques and their appli-cations confirm the need to overcome the limitations of conventional meth-ods. However, a common problem for all reported large volume techniquesis balancing the sample introduction and solvent evaporation rates. In thefuture, the scope for developing an injector that does not require these pa-rameters to be fine-tuned will be explored.
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AcknowledgementsJag 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 häravhandlingen inte blivit skriven. Och vad hade du tänkt att jag ska göra nunär du sticker?
Tack till Jonas Rutberg för oändligt tålamod med alla ’data’-frågor, layoutav den här boken och discoveryreferat.
Jonas och Jonas ska också tackas som bra kompisar och musiker. Suck onmy 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, viborde 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 gjordeC-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.
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Samt Lina, Christoffer, Ragnar, Bodil och Kent, som jag aldrig riktigt hannlära 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 interestingGünther 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 minabarn 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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