Analytical pyrolysis mass spectrometry: new vistas opened by temperature-resolved in-source PYMS

International Journal o f Mass Spectrometry and Ion Processes, 118/119 (1992) 755-787 Elsevier Science Publishers B.V., Amsterdam

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Analytical pyrolysis mass spectrometry: new vistas opened by temperature-resolved in-source PYMS* J a a p J. B o o n FOM lnstttute for Atomtc and Molecular Physws, Krutslaan 407, 1098 SJ Amsterdam (Netherlands) (Received 26 August 1991)

ABSTRACT Analytical pyrolysis mass spectrometry (PYMS) is introduced and ~ts apphcatlons to the analysis of synthetic polymers, bmpolymers, bmmacromolecular systems and geomacromolecules are critically reviewed. Analytical pyrolysis reside the lomsatlon chamber of a mass spectrometer, i e. m-source PYMS, gaves a complete inventory of the pyrolysis products evolved from a sohd sample. The temperature-resolved nature of the experiment gtves a good insight into the temperature dependence of the volatthsatlon and pyrolytic dissociation processes. Chemical iomsatton techmques appear to be espectally statable for the analysis of ohgomerlc fragments released m early stages of the pyrolysis of polymer systems Large ohgomenc fragments were observed for hnear polymers such as cellulose (pentadecamer), polyhydroxyoctanoic acid (tndecamer) and polyhydroxybutync acid (henelcosamer). New in-source PYMS data are presented on artists' paints, the plant polysaccharides cellulose and xyloglucan, several microbial polyhydroxyalkanoates, wood and enzyme-&gested wood, blodegraded roots and a fossd cuticle of Mmcene age. On-hne and off-hne pyrolysis chromatography mass spectrometric approaches are also discussed. New data presented on high temperature gas chromatography-mass spectrometry of deuteno-reduced permethylated pyrolysates of cellulose lead to a better understanding of polysaccharide dissociatmn mechanisms. Pyrolysis as an on-line sample pretreatment method for orgamc macromolecules m combmatmn w~th MS techniques is a very challenging field of mass spectrometry Pyrolytic &ssocmtlon and desorptmn ~s not at all a chaotic process but proceeds according to very specific mechanisms

ANALYTICAL PYROLYSIS MASS SPECTROMETRY

Pyrolysis mass spectrometry (PYMS) is the hyphenated mass spectrometric technique in which a flash pyrolysis device is coupled directly or indirectly via a chromatographic interface to a mass spectrometer. Analytical pyrolysis is a physical analytical approach in which solid organic matter is exposed in an inert atmosphere or in vacuo to thermal energy such that structurally significant fragments are obtained. In the ideal experimental design the pyrolytic fragments of macromolecules are generated under non-isothermal conditions, escape sufficiently fast from the dissociating matrix so that overheating and further rearrangement of the pyrolysis products are prevented, and are * Paper presented at the 12th International Mass Spectrometry Conference, Amsterdam, The Netherlands, 26-30 August 1991. 0168-1176/92/$05.00

O 1992 Elsevier Science Publishers B.V. All rights reserved.

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analysed without further wall contact by soft ionisation MS techniques. Explosive thermal dissociation or flash pyrolysis of organic material dictates small sample size (micrograms or, better, nanograms), fast heating rates (non-isothermal conditions in the sample) and an open low pressure system (to achieve low partial pressures and fast escape from hot zones) in which the analytical pyrolysis takes place. All of these conditions can be met when the pyrolysis takes place inside the ionisation chamber, but in practice the analytical PYMS conditions are often quite different. Presently most PYMS instruments are not equipped for pyrolysis inside the ionisation chamber. The pyrolysis chambers and analytical systems are often interfaced in a "near-source mode" such that dropout of chemical compounds, especially polar and higher molecular weight compounds, is unavoidable. New compounds may be generated on reactive surfaces by secondary reactions in such systems. Sample sizes, which vary from 1 to 200/tg, dependent on the sensitivity of the analytical system, are often too large to prevent secondary reactions as a result of heat and mass transfer limitations. Heating rates in fact are adapted to what the MS system can handle in terms of scanning speed, resolution and/or data acquisition rate. Apart from the multiplicity of instrumental designs, there are various intrinsic factors in the pyrolysis process itself which cause deviation from ideal behaviour and make interlaboratory comparability and reproducibility difficult. The composition of pyrolysis product mixtures from macromolecular systems is changed by non-organic "additives" which influence the dissociation processes. Pyrolysis products "escape" more easily from the hot zone when only one chemical bond has to be broken compared with a breaking away from three-dimensional covalently bonded macromolecular systems or from systems with a lot of electrostatic forces involving cations or anions. In the pyrolysis of complex organic systems such as whole bacterial cells or plant cell walls molecular cage effects are to be expected in which thermally strong components of the macromolecular systems prevent the rapid escape of more labile fragments thus leading to energetic overexposure of the latter. Dissociation products from melting polymers more easily undergo condensation and rearrangement reactions leading to volatile products, which are no longer structural units of the polymer itself as has been shown by Tsuge's group [1] in some very elegant experiments with normal and perdeuterated polystyrene. Finally char formation can preferentially absorb certain compound classes and consequently can be a major obstacle in quantitative studies. The interplay of all these factors is obscure for the uninitiated and certainly limits the acceptance of analytical pyrolysis methods by the scientific community. Despite these considerations, the methodology is successfully applied in many disciplines for fingerprinting and the biennial International Conferences on Pyrolysis accurately reflect progress in the field. The proceedings of these

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conferences are published as special volumes of the Journal of Analytical and Applied Pyrolysis (1984: Vol. 8; 1986: Vol. 11; 1988, Vol. 15; 1990: Vols. 19 and 20). In this paper, I will critically review recent pyrolysis MS studies in the field of synthetic polymers, bio- and geomacromolecules, address some instrumental factors which influence the interlaboratory comparability of the data and investigate the potential of PYMS sensu lato for structural elucidation of complex organic matter. New data from our in-source PYMS work at FOM is presented. On-line P Y M S

On-line PYMS without a chromatographic interface is performed in a number of ways: i.e. in front of the ion source, near the ion source, or inside the ion source. Pyrolysis devices in front of the ion source are for example a crucible in a flame close to a molecular beam instrument [2], a resistivelyheated probe in an atmospheric pressure ion (API) source [3] or a thermobalance exiting into an API source [4] (for a review of thermal analysis MS, see ref. 5). These instruments generally use small entrance holes and skimmers for admission of the pyrolysis products. The API source has as a special feature the freedom to choose any atmosphere for pyrolysis, even reactive atmospheres for investigation of combustion conditions [3]. Pyrolysis near the ion source utilises an expansion chamber [6], an extended empty tube inlet [7], a heated glass liner [8], an all glass inlet system (AGIS) [9] or "direct" probe distillation from a glass capillary tube [10]. Compatibility of Curie point pyrolysis (5000 K s-l) with MS has been achieved by expansion chambers [6] or by reducing the temperature ramp to less than 100 K s -1 [8]. The temperature ramp of most resistive heating devices is adapted to the specifications of the MS system (scan speed and data acquisition speed) and ranges from about 2 0 K s -~ [11] to 1 K s -~ or even less [12,13]. Curie point pyrolysis systems with the higher heating rates are often interfaced to quadrupole instruments. Pyrolysis inside the ionisation chamber is employed in a modified " D C I " (direct chemical ionisation) approach in which pyrolysis takes place on a resistively-heated Pt filament [14,15]. In-source pyrolysis is a wall-free pyrolysis process which allows temperature-resolved analysis and best achieves the minimisation of the distance between area of generation of pyrolysis products and the site of their ionisation. Its reproducibility has been extensively investigated using multivariate techniques by Tas [16]. Despite small sample sizes (about l#g or less), the contamination of the ionisation chamber is a disadvantage of this approach and frequent cleaning is necessary to avoid ion optical problems. In-source PYMS gives a complete inventory of pyrolysis products and the programmed temperature nature of the experiment

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Fig. 1. In-source pyrolysis ammonia chemical ionisation mass spectrum of cellulose showing pseudomolecular ions [MNH4]+ of a series of 1,6-anhydro-oligosaccharidesranging from the monomer (m/z 180) to the dodecamer (m/z 1962). The spectrum was obtained on our JEOL DX-303 E/B sector instrumentusing a Pt/Rh filamentprobe. gives a good insight into the temperature dependence of the volatilisation and dissociation processes. Figure 1 shows a pyrolysis ammonia CIMS spectrum of cellulose, a fl(1 ~ 4) linked glucan, demonstrating that large anhydrooligosaccharides are generated under flash pyrolysis conditions [14]. These larger pyrolytic fragments are only observed under in-source PYMS conditions. Figure 2 shows an example of programmed temperature-resolved in-source pyrolysis analysis of a biodegraded periderm tissue from a side rootlet of Ericaceae (a "handpicked" plant particle of 100/~m length and 30 #m diameter) with thermal desorption of lipids at the lower temperatures (peak 1 in the total ion current (TIC) and its summary spectrum (b)) and pyrolysis of the macromolecular framework of the peatified rootlet at higher temperatures (peak 2 in the TIC and its summary spectrum (c)). The thermal desorption data on lipids from such a complex sample and from a solvent extract are very similar [17]. The mass information is indicative of aliphatic alcohols, sterols, steroid ketones and various fried-oleanane-type pentacyclic triterpenoids [18]. The summary spectrum of the peatified polymer framework shows the low voltage electron impact (EI) data for polysaccharides and biodegradatively-modified guaiacyl-syringyl lignin. Higher temperature pyrolysis in the range 800-3000°C can be obtained by resistively-heated rhenium filament in-source PYMS. Figure 3 shows the low and high temperature summation spectra of artists' "Yellow Ochre" oil paint (Talens ® van Gogh series). The low T desorption spectrum is characteristic for the polyunsaturated triglycerides (e.g. m/z 876) in the oil base with abundant m/z 262

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Fig. 3. Temperature-resolved m-source pyrolysis low voltage electron impact mass spectral data on artists' yellow oll paint showing the organic signature (A) and the inorganic signature (B). Spectrum A shows the mass spectral characteristics of the oil's triglycerides (e.g. m/z 876) with fragment ion peaks of acyl ions lndlcatwe of the fatty acids (e.g. m/z 260 and 262) and various diacylglyceryl fragment Ions in the m/z range 550-650. Spectrum B is mainly charactensed by m/z 56 from iron released from the iron hydroxide colourlng agent at high temperature (see also Fig. 4A). Data are obtained on our JEOL DX-303 E/B sector instrument using a Re filament probe with a scan cycle time of 1 s over a mass range of m/z 20-1000.

and 260 peaks from C182 and Ct83 fatty acyl moieties (see also Fig. 4A). At higher temperatures a strong m/z 56 peak is observed from iron generated from the iron hydroxide colouring agent. Figure 4 shows mass chromatograms from MS data of three yellow oil paints pigmented respectively with iron hydroxide, cadmium sulphide and a lead chromium sulphate. The iron in Fig. 4A is clearly evolved at a higher temperature than the organic matter of the paint (m/z 260 as tracer). The cadmium concentration in the "Napels Yellow" (Fig. 4B) is below threshold but zinc oxide, an opaque filler in the

J.J. Boon/Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

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paint, is seen at higher T. The temperature-resolved data on chromium lead sulphate pigmented paint (Fig. 4C) show the organic characteristics--m/z 260 for the oil and m/z 98 for dried oil paint--and pyrolysis products from the sulphate: i.e. SO at m/z 48 and SO2 at m/z 64, and lead at m/z 208 and chromium at m/z 52 that are released at higher T. The m/z 44 peak is CO2 released by decarboxylation from the oxidatively-polymerised oil components in the paint. Further investigations on the selectivity and sensitivity of metal detection in complex organic matrices following these exploratory experiments are under way. We have used this technique for characterisation of tempera and oil paint chips taken from early Renaissance paintings [19]. Other examples from our in-source PYMS work are given later on. On-line approaches are attractive for pyrolysis product profiling and temperatureresolved analysis, but the structural identification of compounds may be hampered by the complexity of the product mixture or by the incompatibility of the polarity of the compounds with direct evaporation from the probe. High resolution MS and PYMS-MS on in-source pyrolysates can partially solve the identification problem as shown by de Waart et al. [20].

Off-line P YMS Off-line microscale open system flash pyrolysis into a cold trap can be a very useful alternative especially for structural studies because the pyrolysis conditions, as well as the conditions for chemical structure analysis, can be optimised independently. After pyrolysis, the pyrolysate is derivatised, separated by chromatographic techniques and identified using MS or NMR. This very flexible approach has been utilised recently [21-23] for identification of oligomers in pyrolysates from cellulose and amylose. Microbial polyesters have been studied in detail by off-line pyrolysis fast atom bombardment mass spectrometry (FABMS) [24]. Other variations in this respect are FABMS of off-line pyrolysates of coals [25] and high resolution EIMS of off-line coal pyrolysates introduced via an AGIS [9].

Pyrolysis chromatography-mass spectrometry On-line flash pyrolysis--with Curie point or resistively-heated filament pyrolysers--coupled to gas chromatography-mass spectrometry (PYGC-MS) is very widely utilised for identification of pyrolysis products from synthetic polymers, biomacromolecules and geomacromolecules. Condensation of certain fractions of the pyrolysate, different methods of column interfacing and the chromatographic columns are important factors which reduce the interlaboratory reproducibility of PYGC-MS. A disadvantage of PYMS with a chromatographic inlet is the selectivity for apolar and medium polarity

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pyrolysis products depending on the choice of the chromatographic column. On-line derivatisation during pyrolysis [26,27] reduces this drawback to some extent. Very short column PYGC-MS with almost complete sacrifice of resolution and peak separation has been shown to pass relatively polar large molecules [28]. Possible alternatives such as pyrolysis liquid chromatography MS and pyrolysis supercritical fluid MS are still in development. Limitations on the size and polarity of the pyrolysis products are not due just to chromatographic filtering. A well-known trapping effect in Curie point pyrolysis devices is condensation of tar on the glass liners which are generally used to hold the ferromagnetic probes. This condensation can be reduced by heating of the glass liners and a very close interfacing of the GC column and the pyrolysis chamber [29] but drop-out of higher molecular weight fractions is unavoidable (although this may be a blessing in disguise because it reduces contamination of the analytical column). A similar phenomenon is known to occur in the commercially available Pyroprobe ® in which the heavier pyrolysate fractions can end up on the widely used (glass wool-plugged) quartz sample tubes inside the heating coil [30]. These fractions are subsequently repyrolysed or cracked at higher temperatures. Using 14C-labelled polymers the pyrolysate in the Pyroprobe was found to be fractionated between the quartz sampling tube, the pyrolysis probe holder, the injector system of the gas chromatograph and the capillary column of the GC mass spectrometer (68 % of a 14C-labelled polystyrene reached the chromatographic system) [31]. A modified interface for the Pyroprobe has been described which allows direct GC analysis of anhydrosugars generated by pyrolysis from polysaccharides and carbohydrate model compounds deposited on glass wool inside a quartz capillary tube [32,33]. Despite these interfacing and chromatographic filtering problems, PYGCMS is an attractive on-line method for structural characterisation of monomeric and submonomeric products of pyrolysis and for analysis of thermally extractable compounds desorbed from complex matrices or minute tissue samples. The method is unsuitable for very polar and high molecular weight pyrolysis products. PYMS STUDIES ON POLYMERS Synthetic polymers

Excellent comprehensive reviews on analytical pyrolysis of synthetic condensation polymers have been written by Montaudo and Puglisi [34,35]. Many of the proposed mechanisms of thermal dissociation have been studied by direct PYMS, in combination with linked scanning and mass-analysed ion kinetic energy spectrometry (MIKES) studies and by comparison with

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synthetic standards. Ionic processes are considered to be very important in the pyrolysis of condensation polymers, e.g. polyurethanes, polyamides, polyesters, polycarbonates, polysiloxanes, etc., involving H-transfer from NH, OH and CH groups and other exchange reactions. Free radical processes are prominent in addition polymers, e.g. polyolefins, polyvinyls and in general very apolar polymers. Free radical mechanisms are also important in condensation polymers at the high temperature end of the pyrolysis. Recent in-source PYMS work with fast scanning analysers and fast scanning data systems on synthetic polymers has shown stable high mass ions (up to 4000 Da) from perfluoroethers under negative CI conditions which could be sequenced by MS-MS (B/E geometry) [36]. The bromine chemistry in polystyrene spiked with Sb203 and fire-retarding decabromo-diphenylether was studied in the programmed temperature-resolved mode by in-source PYMS using electron attachment reactions in an argon atmosphere [37]. Because the polystyrene pyrolysis products are not ionised under these conditions, the debromination of the fire retardant and the formation of polybrominated dibenzofuran, antimonybromide and antimonyoxybromides and brominated styrene oligomers up to degree of polymerisation (DP) 15 can be very clearly observed in the negative ion mass spectra.

Biopolymers No recent comprehensive review on biopolymer pyrolysis is available [38]. Many biopolymers are condensation polymers but they have not been studied so extensively with modern pyrolysis MS techniques as have synthetic polymers. Biopolymers with hydrolysable bonds, such as glycosidic and peptide bonds, are usually analysed by other techniques. Nevertheless, their flash pyrolysis behaviour deserves attention because they are often part of more complex macromolecular systems or even whole organisms, e.g. bacteria, yeasts or plant cells, which are studied by PYMS profiling. Several protein sequence-specific pyrolysis products were identified by PYGC high resolution (HR)-MS [39] in proteins and bacteria. Other protein marker compounds released from human hair were identified using PYGCMS-MS [40]. Langhammer et al. [41] reported interesting field ionisation MS data on the thermal degradation of gelatine and silk pyrolysed in melting nylon-6,10 spiked with small amounts of Na- and K-hexanoate in off-line experiments at 330°C. The MS information points to cyclic dimers, tetramers and hexamers in the mixtures. The problem with protein pyrolysates is that many very polar compounds are formed at relatively low pyrolysis temperatures which tend to form tar fractions and do not easily reach nor pass a GC system without derivatisation. In-source PYMS, however, tends to simplify the product profile unless high resolution techniques are used to

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separate the many nominally isobaric compounds. HRMS studies on selected ions with PYDCIMS of proteins in algae have been described which lead to structure proposal by combination with MS-MS data [20]. A promising and fruitful approach in polysaccharide pyrolysis studies (see below) has been the derivatisation of the higher molecular weight polar fractions. A similar analytical approach for protein pyrolysates might give more flexibility by choosing suitable desorption/ionisation methods such as FABMS or capillary electrophoresis combined with MS to get a more complete picture of protein pyrolysates. The literature available on pyrolysis of DNA and RNA has been reviewed recently with a warning that methyl transfer reactions readily occur [42]. Early fingerprinting studies on polysaccharides by Curie point low voltage PYMS in combination with multivariate data analysis [43] and pyrolysis field ionisation mass spectrometry (PYFIMS) [44] showed sugar and linkage related differences on a fingerprint level. Recent progress in polysaccharide pyrolysis by in-source PYMS-MS, in-source PYMS and off-line pyrolysis GC-MS has been tremendous and a detailed account will be given in a separate section below. Ether-bonded systems such as lignins which result from autocatalytic free radical condensation of coniferyl and synapyl alcohols have been studied in detail with pyrolysis methods in the last five years. The pyrolysis products from lignin released from a woody matrix in cell walls do not seem to be affected by the presence of the polysaccharide matrix. The in-source PY low voltage EIMS data on sweet gum wood and its enzyme (cellulase)-digested residue in Fig. 5 demonstrated this by the similar lignin-derived ion signatures. A relatively complete picture of the identity of the monomeric pyrolysis products of lignin is available from PYGC-MS studies [29,45,46]. Dimeric and higher oligomeric units important for the linkage of the phenolic units in lignin have been observed by in-source low voltage PYMS [47,48] and by PYFIMS [49] in lignin preparations of woody tissues, biodegraded and peatifled woods, and in organic matter from soils. Their structural identification is presently the subject of study in our group [50]. Other vegetable polyphenols such as tannins have not been studied by analytical pyrolysis. Analytical pyrolysis work has been instrumental in discovering the importance of aliphatic network polymers in organisms. These polymers were first detected by regular patterns of alkanes, alkenes and alkadienes in pyrolysates of fossil remains of algae e.g. Botryococcus braunii [51], and of leaves of higher plants [52]. Metzger et al. [53] recently summarised their work on the chemical structure and biosynthesis of these macromolecular lipids in the green microalga Botryococcus braunii. Selective removal procedures have shown related hydrocarbon polymers in Agave americana cuticles [54]. Neither the precise structure of these latter polymers nor their molecular weight range is known. A possibly related group of polymers are the cutin

766

J.Y. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787 PYMS study of Cellulase d0gested xylem from Sweet Gum (Ltquidamber Styraciflua) Fresh Xylem (freezer mdled)

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767

polyesters, which consist of mid-chain and co-hydroxy C 16 and C 18 fatty acids with some degree of unsaturation. PYGC-MS data on the cutin of tomato [55] point to a thermal dissociation mechanism by fl-H transfer releasing the hydroxy fatty acids bound by two and three ester groups. Similar dissociation mechanisms occur in bacterial polyesters. Microbial polyesters

The determination of the sequence of monomeric units in a bacterial polyester, a potentially biodegradable "plastic" with different types of hydroxy fatty acids, is a difficult analytical problem. N M R can demonstrate the presence of dyad and triad sequences in polyhydroxyalkanoates (PHA) such as polyhydroxybutyric acid (PHB) and its copolymer with hydroxyvaleric acid (PHV/PHB) [56]. GC-MS studies of trimethylsilylderivatives of closed tube pyrolysis products of PHB [57] and flash pyrolysis GC-MS [58] showed monomers, dimers and trimers. Monomers from PHB and its copolymers have been seen in PYGC-MS data of bacteria [59] and in PYMS and PYGCMS of river sediments [60]. The mechanism of dissociation of PHAs is clearly by a fl-hydrogen transfer leading to molecules with a carboxyl group and an olefin end group. Direct PYMS was thought to be the technique of choice, but the mass spectrum of PHB under El and under isobutane CI conditions was surprisingly poor (highest mass observed was m[z 259 from a trimer) which was explained by Ballistreri et al. [61] to be due to decomposition processes on the probe. In contrast, series of oligomers up to 23 monomeric units could be observed in the partial methanolysate from PHB with HPLC and FABMS [61]. In search of a more rapid method of analysis, these authors prepared off-line pyrolysates, using a thermogravimetry instrument, which were investigated successfully with FABMS showing oligomers up to the decamer. The validity of this pyrolysis FABMS approach for PHB/PHV copolymers is demonstrated beautifully by comparison of off-line pyrolysate FAB/MS, methanolysis-HPLC-FABMS and N M R of the intact polymers [62]. Copolymer microstructure up to hexad level and the presence of a single copolymer or a blend ofcopolymers can be determined using these techniques together with bernouillian statistics to compare experimental and calculated data on monomer distribution [63]. Our experience with in-source PYMS of polysaccharides led us to believe that Ballistreri's [61] explanation of the decomposition of higher oligomers on the probe was unsatisfactory. If the in-source PYCIMS approach could work in a similar way on PHB and other PHA, the time consuming off-line pyrolysis FABMS method could be replaced by the faster on-line temperatureresolved analytical technique. Our in-source PYMS spectrum of PHB (Fig. 6) obtained under NH3CI conditions on our JEOL-DX-303 machine (mass

J J. Boon~Int. J Mass Spectrom. Ion Processes 118/119 (1992) 755-787

768 100

0

276

2

4!48

P e l

,

80

polyhydroxybutyric

acid

t l v e

60

534

A b u n 4{3 d a c e

620

706

20

I,I,.,LI.II.t

lit, Z00

100-

L~I , I ..........

.a 400

791 I.

600

1000

800

1050

1136 80

lZ22

60

I

I

40

20

1308 13i4

1480 1566

!~4.

.i..aJ_ |h,,.t._~ .....I.L

1910 b

.

J

~10,0

t

l

t

.I

.

L

I~OO

L

~

,

td~

I~

.

16oo

Z000 M/Z

Fig 6. In-sourcepyrolysis ammoma chemical ionlsation mass spectrum ofpolyhydroxybutyrlc aod summarised over the TIC range with the maximum intensity for ions between m/z 500 and 2000. The spectrum shows an array o f pseudomolecular ions from olefinic carboxylic acids with a mass increment of 86 mass units corresponding to butenoic acid. The m/z 1910 peak corresponds to an ohgomer with 22 monomeric umts. Data were obtained on a JEOL DX-303 E/B sector instrument using a Pt/Rh filament heated at a rate of 13.5°C s-~ with a scan cycle time of I s over a mass range of m/z 20-1000. The sample was donated by ICI-Agrochemlcal.

range observed 60-2000, cycle time 1 s) indeed demonstrates that oligomers can be observed with pseudomolecular ions up to rn/z 1910 equivalent to a homologous chain of 22 monomeric units. All observed ions are ammonia adduct ions with a nominal mass equivalent to fl-H transfer products. The temperature-resolved data on PHB (Fig. 7) show that the higher oligomers between rn/z 1000 and 2000 occur in a relatively narrow temperature range in the early stages of pyrolytic degradation. Smaller oligomers (e.g. m/z 5001000) were found to be stable over a fairly large range of higher temperatures.

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

769

' TIC

1000-2000

500-1000

100-500

'

'

'

'

i

25

'

'

'

'

i

160

'

,

,

'

i

295

'

'

'

'

I

430

'

'

'

'

i

565

,

,

,

,

i

,

,

'

~1

700 °C

Fig 7. TIC trace and mass chromatograms over the mass ranges m/z 1000-2000, 500-1000and 100-500 in temperature-resolved pyrolysis ammonia chemical ionisation mass spectrometry data of polyhydroxybutyrlc acid showing the relatwely narrow temperature (time) window, i.e. scan 34 to 38 in which higher molecular weight ohgomers are evolved from the in-source pyrolysis filament probe (see Fig 6).

It is likely that the slow heating rates in the experiments of Ballistreri et al. prevented the desorption of the larger oligomers. The more stable smaller oligomers clearly do survive the slow heating rates of the off-line pyrolysis experiments and indeed show up in the FABMS data. Our in-source PYMS study included a polyhydroxyalkanoate produced from octane by Pseudomonas oleovorans [64]. These bacteria oxidise the alkanes to the corresponding fl-hydroxy fatty acids and polymerise these to polyesters. Studies on the fatty acid compostition have shown that a shortening of the hydroxy fatty acids from C8 to C6 occurs (OH-FA: C8 = 88%, C6 = 12%). The PYMS spectrum of polyhydroxyoctanoate (Fig. 8) indeed shows that this has happened. Mass peaks indicative for the monomeric C8 and C6 olefinic fatty acids are the ammonia adduct pseudomolecular ions m/z 132 and 160. A series of peaks equivalent to pure C8 oligomers with a mass increment of 142Da is observed up to DP 13. The bacteria appear to have a preference for the C8 hydroxy fatty acid and tend to synthesise these in blocks as is evident in Fig. 8 since the homologues with only C8 units have high intensities up to DP 9 (m/z 1296). Ions from homologous oligomers with some C6 units are also prominent. These homologues have mainly one or two C6 hydroxy fatty acids mixed into the chain, e.g. m/z 870 (a hexamer of C8), 842 (a hexamer of 5 x C8 and 1 x C6), 814 (a hexamer of 4 x C8 and 2 x C6) and 786 (a hexamer of 3 x C8 and 3 x C6) occur in the ratios 10"10:7:3. At higher masses it becomes

J.J. Boon/Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

770

)2~

100

31

In s o u r c e

PY-CIMS

polyhydroxyoctanoate

80

60

444

40

~ 160

274 I

"~ 586

416

200

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,

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l i _ .

t

t

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.

•

.

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,.

•

0

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2000 M/Z

Fig. 8. In-source pyrolysis ammonia chemical ionlsation mass spectrum of polyhydroxyoctanoic acid summarised over the TIC range with maximum intensity for ions between m/z 500 and 2000. The spectrum shows an array of pseudomolecular ions from olefinic carboxylic acids (marked with an asterisk) with a mass increment of 142 mass units corresponding to octenoic acid. Since the polymer consists of two monomerlc units (fl-hydroxyhexanoic and fl-hydroxyoctanolc acid), the spectrum also shows the ions from hydroxyoctanolc ohgomers with hydroxyhexanolc acid monomers. The sample was donated by Professor B. Witholt, University of Groningen.

increasingly difficult however to determine the "purity" o f the mass peaks. P Y M S - M S is clearly necessary to give information on the purity o f these ions and to provide data on the relative position of the C6 monomers in the C8 oligomer. These new data show that the wall-free in-source approach can provide a rapid insight into the composition of these polymers. The temperatureresolved data also demonstrate that the presence of oligomers o f larger size is

J.J. Boon/Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

771

a transient phenomenon which is very difficult to observe in an off-line slow heating process such as that used by Ballistreri et al. Consequently the information in these latter experiments is limited to the smaller oligomers. Polysaccharides

Cellulose is the most studied polysaccharide analysed by pyrolysis techniques. Early studies were more concerned with aspects of flammability whereas in the last decade the conversion of cellulose-containing biomass into useful chemicals has been a major topic of research. Radlein et al. [65] give an excellent review of the current knowledge of both analytical aspects and bulk feedstock aspects of cellulose pyrolysis. Low molecular weight pyrolysis products analysed by PYGC-HRMS [66] have been explained as dissociated products from levoglucosan and cellobiosan [67]. There is also evidence that cellulose is directly fragmented into small low molecular weight products such as hydroxyacetaldehyde [68] and into higher anhydrooligosaccharides (see Fig. 1) as detected with in-source pyrolysis CIMS [69]. In-source CI spectra of cellulose are remarkably "clean" and devoid of the large amounts of submonomeric pyrolysis products seen by PYGC-MS. Much of this PYGCMS data could therefore reflect secondary dissociation reactions of the primary oligosaccharides by further thermal reactions in the pyrolysis environment, i.e. the heated walls of the on-line flash pyrolyser. The generation of oligomers only occurs at one particular moment in the temperature transient and can be observed as an "explosion" by temperatureresolved PYCIMS. After an initial appearance of smaller fragments, suddenly very large fragments (up to m/z 2000 observed in our JEOL DX-303 machine) are seen within the time frame of one or two scans, followed by release of smaller oligomeric fragments up to pentamers (see Fig. 9). Smaller fragments released from polysaccharides in close contact with the pyrolysis filament are thought to be a driving force for the "volatilisation" or propulsion of oligomers into the CI plasma [70]. The retention time differences between pyrolytic oligomers [21,71] of amylose [a(1 ~ 4)-glucan] and cellulose [fl(1 ~ 4)-glucan] analysed by HPLCMS of their benzoyl derivatives suggests preservation of the anomeric configuration of the internal glycosidic bond rather than major rearrangements or even repolymerisation [72] during thermal dissociation. High temperature GC-MS of borodeuteride-reduced permethylated off-line Curie point pyrolysates of cellulose [23] identifies the 1,6-anhydrosugar-terminated oligosaccharides up to the tetramer (Fig. 10). A second series of oligosaccharides has an acetaldehyde substituent on the reducing terminus, i.e. C4 and C3 split off from an adjacent glucose in the cellulose chain. The MS spectra of the permethylated anhydrooligosaccharides from cellulose and amylose

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

772 TIC 2610/2611 2447/2448 2286/2287 2124/2125 1962/1963 1800/1801

/

1638/1639

/

1476/1477

~

1314/1315 1152/1153

/

oo,o9, 666/667

j

~ ~ ~ f i ~ ~ ~ ~ i ~ ~ J ~ i ~ ~ ~ i i ~ ~ ~ ~ i ~ = ~ = 5 10 15 20 25 30 Scan

Fig. 9. Mass chromatograms of the pseudomolecular ions from 1,6-anhydrooligosaccharides generated by pyrolysis from cellulose The appearance of the higher ollgomers--m/z 990 is a pentamer, m/z 2610 is a pentadecamer--in a narrow temperature window (one scan) suggests an explosive dissociation process on the m-source probe. The data were obtained during tests on a JEOL SX-102 (B/E) machine with a Pt DCI probe under ammonia chemical lonisation conditions (8 kV, mass range 80-3000, cycle time 1 s) with the help of Mr. Tanaka, JEOL Tokyo.

(unpublished results) clearly prove the O-(1 ~ 4) linkage between the various sugar units. The identified products point to a transglycosidation and a retroaldolisation mechanism (Scheme 1) operating at the same time and substantially reducing the initial polymer size by in-chain cleavages. Fig. 10 (opposite) High temperature capillary gas chromatography mass spectrometry of cellulose pyrolysates obtamed under mlcroscale off-hne Curie point pyrolysis condlt]ons (510°C ferromagnetic probes). The NaBD+-reduced, permethylated pyrolysate consists of 1,6-anhydroohgosacchandes (marked by a T for transglycos]datlon) and ohgosacchandes with an acetic aldehyde on the C1 position (marked by R for retroaldolisation). The R series pyrolysis products can be traced by mass chromatography of the charactemstic ion m/z 120. All ollgomenc pyrolysis products show the m/z 305 peak Indicative of the O-(i ~ 4) linkage and m/z 187 indicative of terminal hexose units. Data were obtained on a Carlo Erba high temperature gas chromatograph under electronic flow control using an Al-clad fused silica capillary column (25 m, 0.220 mm diameter) with on-column injection and helium as carrier gas. The mass spectrometer was our JEOL DX-303 (E/B) using 70eV electron Impact conditlons and a 1 s cycle time over m/z 20-1000 These new analyses are a further extension of the earlier data published [23].

773

J.J. Boon~Int. J. Mass Spectrom. lon Processes 118/119 (1992) 755-787

t ~.,oo.~ l ?'~? O ~ H ~ ~ s 000H

F ~,,oo.. l ~.,oo.,

/. , t ~ 0 . I . a---~O O'-cxH2

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187

~

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l RI

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[ .......

Fig.10.

I I0%1

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~

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R2L

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2000

2500

3000

774

J.J Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

R e v e r s e Aldolisation Production of aldehydic ring cleavage fragments

(~H2OH ~%OR'

(~H2OH

,~---o V I

CH2OH

~ 0

6x~" ,/i ~ • "J I" " IA - - O H

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;x

o~.

1

o.

~N20H

(~H2OH

9H2OH

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CH20H

nHl(/l~O\r6~gH ..~

RO~I~" ~)hvn"

o

Mien

OH ~''

/~C~ I

~O'xj'-OR' O _ k\Y" I/I -

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;.

HI~

o.

o.

Transglycosidation Production of 1,6-anhydro oligosaccharides

CH2OH . . . . 'r ' ~ 2 ~

j jf--:-~O,, r-O"~" V~..'r'/K.bH ,~1 I

~'--"O'N~=~ O"~ ~

CH2OH

,~ - - o ,

CH2OH ~.L//]L~.=O'x~"OR'

~o~,H

1/q

6H

OYH'

.CH2OH .CH2OH .CH2~

CH2OH

J---~-~O

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O~I~

, [ /~. / l ~ O ' x J -OR'

,O H

/ O H

OH

Scheme 1. Structural information is also obtained by in-source PYCIMS-MS of specific positive and negative "parent" ions from dextran and amylose [73]. This approach is very useful for comparative studies but the MS-MS information is very difficult to translate into chemical structure information.

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

775

For example a tentatively MS-MS-identified levoglucosan coupled to an acetaldehyde unit at the 4-position [73] could not be confirmed by the high temperature (HT)GC-MS data of Lomax et al. [23] (the acetaldehyde substituent was found to be present at the C 1 position). Negative ammonia CI is as yet under-utilised but is important because it selectively ionises minor compounds sometimes significant in dissociation mechanism studies such as levoglucosenone (MW126) and 1,4-dideoxy-D-glycero-l-hexenopyranose (MW144) and their oligomers. Linkage information from in-source PYCIMS-MS has been reported for pyrolytic dimers from dextran, laminarin, amylose and agar [74]. HTGC-MS data on pyrolytic oligomers from dextran, an ~-(1 ~ 6) linked glucan, indeed corroborate the presence of O-(1 ~ 6) linked oligomers [75]. Anhydrosugars are very low in abundance in contrast to oligosaccharides with an acetaldehydic side-chain on the reducing terminal. This side-chain consists of the C6 and C5 species released by a retroaldolisation mechanism. The relatively low abundance of the anhydrosugar ions is also observed in PYCIMS data of dextran [76]. Wide mass range PYCIMS spectra [76] on all linkage types of glucans, on xyloglucan, arabinan, arabinoxylan, arabinogalactan and galactoarabinan obtained under ammonia positive ion CI and CI-addition negative ion CI give ion series pointing to similar dissociation mechanisms and in-chain cleavages as seen in cellulose and dextran. Only xylan shows extensive pyrolytic dehydration in its oligomeric ions. The pyrolysis data on xyloglucan, a glucan with O-(1 ~ 6) linked xylose and xylose-galactose side-chains, show many fragments released by a retroaldolisation mechanism presumably because the 6-0 position is substituted and cannot assist in transglycosidation reactions [77]. Figure 11 shows the structure of some of the tamarind xyloglucan pyrolysis products identified. The generation of larger oligomers from polysaccharides has been observed by Coates and Wilkins [78] in laser desorption Fourier transform mass spectrometry (FTMS) of polysaccharides from KBr discs. A strong predominance of ions was seen which we can now assign to products from retroaldolisation reactions most likely promoted by the KBr. The ions seen in these experiments are clearly generated by laser-induced pyrolysis of the polysaccharides. The strong influence of K, and of alkali metals in general, on the pyrolysis mechanism tends to reduce the formation of oligomers at higher concentrations [79]. For good analytical results on polysaccharides, a desalting procedure is of vital importance for good PYCIMS data. Crown ethers have been found to be very suitable for K ÷ removal and drastically improve the yields of oligomers [80]. A special case in this respect are pectins which only give good PYCIMS results after complete desalting using strong acids. Diads, triads and tetrads of methylated galacturonic acids were observed in the PYCIMS spectra of high degree of methylation (DM) citrus pectin [81].

776

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

Xyloglucan .,p

0

0

I

{

-~_o

Q--'~-- o T

"~k~-o

.~,

.~,, =~rX~gH

~-°T' I,.~

"~ 1

rH

.J--~-..-O o~H r,,"1

:>1- ~ 1

;~-'l ~-

(~H20H

~,,

"1" .t--"~-- 0

N~

0

or,

~---o,,ro ., OH

o.t" '

M~croscale offline flash pyrolysis Oenvatk~atJon with NaBD4 Permethylatlon High temperature GC/MS

OH

Oligomeric Xyloglucan Pyrolysis P r o d u c t s

O

H2~

/ O H

O O C3 I

~CH 2 ~)CH3 OCH3 [ , , , , ~ O x j _ O CH2CHD C H 3 ~ ~

CH3 I

(~H2OCH3 O C H 3 0 / j ~ O N j CH3

OOHa

~ I

J

~CH2 {~H3 IAL~--'-O~ r-OCH2CHD

f

G-G

G--'k L

I

f

L/1 "Xf CH30~)ICH3 H

0

OCH3

G-G--*

OCH3

G = glucose X = xylose L = galactose •k = acetic aldehyde moiety

Fig. 11. Xyloglucan and xyloglucan oligosaccharides obtained under m]croscale Curie point pyrolysis conditions (510°C ferromagnetic probes) See ref. 77.

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

777

BIOMACROMOLECULARSYSTEMS Biomacromolecular systems such as plant cells, animal cells, human cells and bacterial cells have been the subject of many PYMS studies but a thorough review is outside the scope of this paper [82]. The rapid profiling of microorganisms has been an important stimulating force for the development of the FOMautoPYMS system [83] and the later commercial instruments (VG, Extrell, Horizon Instruments, Brucker-Franszen). Tas et al. [84] have shown with PYEIMS and PYCIMS in commercial MS systems that diglyceride fragments pyrolytically released from more complex lipids in bacteria are valuable for classification of the bacterial strains despite problems with instrumental drift at higher masses. Boon et al. [85] demonstrated by Curie point PYMS and multivariate techniques that cell walls isolated from phageresistant and phage-sensitive strains of Streptococcus lactis can be discriminated. Yeasts [86], fungi [87] and algae [20] have been the subject of fingerprinting studies by in-source PYMS-(MS) combined with multivariate statistical methods. A very elegant method for rapid detection of bacterial infections in urine using Curie point PYMS has been presented by Huff et al. [88]. Analytical pyrolysis techniques have been used for microbial mat studies in field samples [89,90]. PYGC-MS profiling studies on bacteria and human cells combined with clever data analysis methods were reported by Morgan's group [91]. The usefulness of PYMS and PYGC-MS for plant cell wall characterisation in agriculture has been pointed out by Boon [92]. Tissue typing by microsampling within plants [93,94], the chemical changes and the localisation of phytoalexins in fungal-infected xylem of carnations [95], the lignification of suspension cultures [96] and enzymatic digestion of plant material [97] have also been investigated. Biodegradation, peatification and fossilisation of woody tissues of plants have been studied by Curie point PYMS, PYGC-MS and in-source PYMS [98,99,100]. Hempfling and co-workers [101,102] used PYFIMS and other techniques for characterisation of litter decomposition and soil formation in German forest soil. The influence of the environment on the health of spruce needles has been investigated by Simmleit and co-workers using PYFIHRMS [103,104]. An important role for analytical pyrolysis methods in soil science was postulated recently by Schnitzer [105]. PYMS will play an increasing role in the rapid assessment of environmental quality because it has the ability to trace organic pollutants in highly complex matrices without extensive sample pretreatment. Dissolved macromolecular environmental poisons such as chloroligninsulphonates and chlorolignins, often released into rivers and lakes by the paper and wood pulping industry, can be probed with PYGC-MS and PYMS [106,107]. The application of pyrolysis for the study of museum objects and in

778

J.J Boon/Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

archaeology has been reviewed [108]. Oudemans and Boon [109] have investigated human dietary habits in the past by analytical pyrolysis studies of chars and other organic remains in and on ancient pottery. The applications of PYMS techniques to biomacromolecular systems and their remains are manifold. Only a few examples have been mentioned here. It is essential for the correct "reading" of the fingerprinting information that the correlation between the mass spectrometric data, the chemical structure of the pyrolysis products and the chemical structure of the sample is understood. Special attention should be given to the "catalytic" effects of inorganic trace components, cations and anions, because they can drastically influence the volatilisation and chemical structure of the organic pyrolytic fragments (see for example ref. 107). GEOMACROMOLECULES

Analytical pyrolysis studies are very important in the Earth Sciences for the characterisation of kerogens in sedimentary rocks [110,111]. Kerogen is a collective term for geomacromolecules which consist of selectively preserved biopolymers [112], newly (bio)synthesised heteropolymers such as humic compounds formed during biodegradation of organic matter by microorganisms [113], and newly formed macromolecular systems during partial natural "pyrolysis" processes in the subsurface. Economically important products of these diagenetic processes are oil and coal. The thermal maturation of kerogens, a mainly hydrous closed-system pyrolysis at temperatures between 80 and 150°C over millions of years, can be simulated successfully in the laboratory at higher temperatures of 280-350°C over several days using a technique known as MSSV (microscale sealed vessel pyrolysis) developed by Horsfield et al. [114]. Under these conditions free radicals formed by cracking of the kerogen are capped by hydrogen derived from water or from the kerogen. Products are analysed by GC-MS. On-line PYGC-MS and PYMS are used for anhydrous analytical pyrolysis fingerprinting of the kerogens to determine the various moieties in geomacromolecules from aliphatic, sulphur- and nitrogen-containing pyrolysis products [115,116]. Structural relationships shown in Fig. 12 between thiophenes as flash pyrolysis products, alkylthiophene moieties in kerogens and the original carbon skeletons preserved by sulphur stabilisation of double bonds and functional groups have been proposed by Sinnighe Damste and coworkers [117,118] after an extensive world-wide survey of sulphur-containing kerogens. The origin of the aliphatic moieties is considered to be mainly from aliphatic network polymers originating from microalgae and plants [119]. These macromolecules are usually demonstrated by PYGC(-MS) because they are generally characterised by regular pattern of alkanes, alkenes and

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

779

Proposed structural relationships between geomacromolecules and their flash pyrolysis products alkylthlophene mo=etms carbon skeletons m geomacromolecules

tdentlf,ed by

PYGCMS

linear

~sopreno=d

I

~

branched

steroid

Fig. 12. Proposed structural relationships between geomacromolecules and their flash pyrolysis products. This proposal by Smnighe Damste and Eglinton is the result of an extenswe survey of sulphur-rich kerogens by PYGC-MS, of chemical studies of the speciation of sulphur compounds in kerogens and of chemical synthesis of thiophene standards (see refs 117 and 118). The figure is adapted from Eghnton et al. [118].

alkadienes. In-source Pt filament probes fail to pyrolyse these aliphatic geomacromolecules. Rhenium filament pyrolysis does the job, as is shown in Fig. 13. The data on Miocene leaf remains, rich in aliphatic network polymers, were obtained by in-source PY isobutane CIMS showing the alkanes in the pyrolysate by (M-H) + and alkenes by (MH) ÷ and (M-H) ÷ pseudomolecular ions. A regular pattern of aliphatic chains is observed up to a chain length of C35. Although this isobutane CI ionisation PYMS method has its complications, it could speed up the analysis of kerogens by a factor of 10 compared with PYGC and is easily interfaced with multivariate data analysis programs. A quantitative PYMS method for typing of kerogens was reported [120] using poly p-tert-butylstyrene as an internal standard to determine pyrolysis product yields in samples which vary greatly in their degree of thermal maturation. Earlier, Curie point PYMS was used extensively in comparative studies of coals [121,122]. Excellent PYMS work has been performed on the Argonne premium coals by Winans and co-workers using PYHRMS [9,123] and PYFABMS [25], and by Meuzelaar's group using Cu-PYMS, thermo-

780

J.J. Boon/Int J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

. t,-

"

~

en

..a

~

tr.,

0

f~

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781

gravimetry PYMS and PYFIMS for temperature-resolved studies [124]. A remarkable constancy of the coal pyrolysis product profiles is observed under conditions which minimise explosive phenomena and avoid problems with heat and mass transport (small sample size and low--100°Cmin - ~ heating rates). The question remains as to how "primary" are the pyrolysis products which are generated under these conditions. Because of the thermal maturity of coals, most of the bond cleavages have relatively high energetic barriers and there is a general lack of hydrogen in the system to stabilise free radicals. Consequently, it is very difficult to obtain a good pyrolytic conversion of high rank coals into products which are relevant for the macromolecular structure of the coals. The work of Winans demonstrates that preparative vacuum pyrolysis and in-source desorption of the pyrolysate using FAB, HRMS and MS-MS techniques is a good analytical approach for the identification of larger aromatic complexes in coal. CONCLUDING REMARKS PYMS fingerprinting studies are clearly very useful for characterisation and analysis of synthetic polymers, biopolymers and more complex bio- and geomacromolecules. Higher molecular weight oligomeric fractions generated from macromolecules are best analysed by wall-free in-source PYMS using chemical ionisation conditions in which the gas acts as the ionisation medium as well as the bath for relaxation of the analyte. Higher molecular weight fractions appear under "explosive" conditions in a narrow temperature (time) window. At the very high temperatures obtained with rhenium filaments (800-3000°C) inorganic species and metals can be analysed. Performing the pyrolysis inside the ionisation chamber presently allows temperature-resolved analysis at temperature ramps of up to 20°C s-~, but higher heating rates are desirable and possible in newer generations of mass spectrometers with faster mass analysis and data acquisition. Fingerprinting information on complex systems must be backed up by chemical structure studies of the pyrolysis products. There is an urgent need for more understanding of the mechanisms of dissociation of macromolecules. Mass spectrometry can play an important role in the preliminary identification of the structural elements obtained by pyrolysis from the macromolecular systems. There is scope for MS-MS approaches although the complexity of the ion mixture and the transient character of pyrolysis present specific difficulties and require high resolving power and very high sensitivities. Combined approaches using microscale off-line pyrolysis, derivatisation, molecular spectroscopic techniques, especially NMR, and mass spectrometry will have to provide the means for a solid structural identification of some of the pyrolytic fragments. New techniques in mass spectrometry such as

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reflection time-of-flight MS and external ion source FT-ion cyclotron resonance may cast new light on the time domain of pyrolytic reactions and explosive desorption phenomena. Laser probing experiments on the gas phase and selective ionisation of pyrolysis products using laser multiphoton ionisation MS will give us more understanding on the relative amounts of neutral and radical species. The role of mass spectrometry in characterisation of the solid phases after pyrolysis may be limited although secondary ion mass spectrometry imaging studies of the organic surface during heating could enlighten us about the mass transfer phenomena which lead to gas phase pyrolysis products. Pyrolysis as an on-line sample pretreatment method for organic macromolecules in combination with mass spectrometric techniques for analysis is a very challenging technique. New vistas have been opened by in-source PYMS techniques. The chemical structure of pyrolysis products of such labile macromolecules as polysaccharides proves that pyrolytic dissociation and desorption is not a chaotic process but proceeds according to specific mechanisms. EXPERIMENTAL

Analyses reported here have been performed on a JEOL DX-303 E/B sector instrument equipped with a post-acceleration detector (10kV). The mass range at full acceleration voltage (3kV) of this instrument is 1500Da. Experiments were performed with in-source resistively-heated filament probes using Pt/Rh or Re wire (thickness 100 #m). Heating rates for the Pt/Rh probe were 15.5°C s- 1under EI and 13.5°C s- ~under CI conditions up to 800°C. For the Re probe higher heating rates are used to an upper limit of 3200°C with a power supply built at FOM-AMOLF. Ionisation conditions were low voltage (16eV) El, isobutane or ammonia CI. Mass spectra were obtained over a mass range of 2000 Da under 1 s cycle time conditions. ACKNOWLEDGEMENTS

The author thanks the technical staff of the FOM Institute for Atomic and Molecular Physics, the group technicians G.B. Eijkel, A. Tom, B. Brandt-de Boer, J. Pureveen and J. Commandeur, and the various Ph.D. students, post-doctoral researchers and guest scientists for their contributions to the field of analytical PYMS under his supervision over the last eight years. The author's research program on the Mass Spectrometry of Macromolecular Systems is part of the research program of FOM (Foundation for Fundamental Research on Matter), a subsidiary of the Dutch Organisation for Scientific Research (NWO). Donna Mehos and Frans Saris are gratefully acknowledged for critical reading of the manuscript.

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REFERENCES 1 H. Ohtanl, T. Yuyama, S. Tsuge, B. Plage and H.R. Schulten, Eur. Polym. J., 26 (1990) 893. 2 T.A. Mllne and M.N. Soltys, J. Anal Appl. Pyrol., 5 (1983) 93-110, lll-131. R.J. Evans, T.A. Milne and M.N Soltys, J. Anal Appl. Pyrol., 9 0986) 207-237. 3 A.P. Snyder, J. Anal. Appl. Pyrol, 17 (1990) 127-143 4 R.B. Prime and B. Shusan, Anal. Chem., 61 (1989) 1195-1201. 5 J.J. Morelli, J. Anal. Appl. Pyrol., 18 (1990) 1-18. 6 Interfacing of Curie point heating with quadrupole MS was achieved using a gold-coated heatable expansion chamber to moderate the pressure pulses. H.L.C. Meuzelaar, M A. Posthumus, P.G. Klstemaker and J. Klstemaker, Anal. Chem., 45 (1973) 1546-1549. 7 C.S. Gutteridge and J.R. Norris, J. Appl. Bacterlol., 47 (1979) 5-43. 8 H.L.C. Meuzelaar and S.M. Huff, J Anal. Appl. Pyrol, 3 (1981) 111-129. 9 R.E. Winans, Mass spectrometric studies of coals and coal macerals, in H.L C. Meuzelaar (Ed.), Advances in Coal Spectroscopy, Plenum Press, New York, 1991, pp. 255-274. l0 H.R. Schulten, N. Simmlelt and R. Mueller, Anal. Chem., 59 (1987) 2903 ll This heating rate is used on our JEOL DX-303 machine at FOM-AMOLF when Pt/Rh filaments (100/tin diameter) are used. 12 B. Plage and H.R. Schulten, J. Anal. Appl. Pyrol., 19 (1991) 285-299. 13 G. Montaudo, C. Pughsi, C Berti, E. Marlanucci and F. Pilati, J. Polym Sci, 27 (1989) 2277-2290, 2657-2672. 14 A.D. Pouwels, G.B. Eijkel, P.W Arisz and J.J. Boon, J. Anal. Appl. Pyrol., 15 (1989) 71-84. 15 A.C. Tas, J. van der Greef, J. de Waart, J Bouwman and M.C. ten Oever de Brauw, J. Anal. Appl. Pyrol., 7 (1985) 249-255. 16 An excellent summary of a number of studies in this respect has been published by A.C. Tas, Mass spectrometric fingerprinting: soft ionisation and pattern recognition, Ph.D. Thesis, University of Leiden, 1991. 17 E. Van der Heijden, J J. Boon and M.A. Scheijen, Pyrolysis mass spectrometric mapping of selected peats and peatified plant tissues, in R. Sopo (Ed.), Peat 90---Versatile Peat, The Association of Finnish Peat Industries, Jyskae, Finland, 1990, pp 148-163. 18 D.G. Van Smeerdijk and J.J. Boon, J. Anal. Appl. Pyrol., 11 (1987) 377--402. 19 B. Odhlya, G.B. Eijkel, J. Pureveen and J.J. Boon, Unpublished report (1990). 20 J. de Waart, A.C. Tas, G.F. La Vos and J. van der Greef, J. Anal. Appl. Pyrol., 18 (1991) 245. 21 P.W. Arisz, J.A. Lomax and J.J. Boon, Anal. Chem., 62 (1990) 1519-1522. 22 R.J. Helleur and P. Jackman, Can J. Chem, 68 (1990) 1038-1044 23 J.A. Lomax, J.M. Commandeur, P.W. Arisz and J.J. Boon, J. Anal. Appl. Pyrol., 19 (1991) 65-79. 24 A. Balhstreri, G. Montaudo, D. Garozzo, M. Giuffrida and M.S. Montaudo, Macromolecules, 24 (1991) 1231-1236. 25 R.E. Winans, J. Anal. Appl. Pyrol., 20 (1991) 1-13. 26 J.M. Challinor, J. Anal. Appl. Pyrol., 16 (1989) 323-333. 27 J.P Dworzanski, L. Berwald, W.H. McClennen and H.L.C. Meuzelaar, J. Anal. Appl. Pyrol., 21 (1991) 221-232. 28 J.M. Richards, W.H. McClennen, H.L.C. Meuzelaar, J.P. Schockcor and R.P. Lattimer, J. Appl. Polym. SOl., 34 (1987) 1967-1975. 29 J.J Boon, A.D. Pouwels and G.B. Eijkel, Blochem. Soc. Trans., 15 (1987) 170-174.

784

J.J. Boon/Int J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

30 T.P. Wampler and E.J. Levy, J. Anal. Appl. Pyrol., 12 (1987) 75-87. 31 Commumcated at the Gordon Research Conference on Pyrolysis, Holderness, 1983 by R.J. Lloyd. Also. R.J. Lloyd, J. Chromatogr. 284 (1984) 357. 32 D.R. Budgell, E.R. Hayes and R.J. Helleur, Anal. Chim. Acta, 192 (1987) 243-253. 33 R J. Helleur, J. Anal. Appl. Pyrol., 11 (1987) 297-311. 34 G. Montaudo and C Puglisi, Thermal degradation mechanisms in condensation polymers, in N. Grassie (Ed.), Developments in Polymer Degradation, Applied Science, London, 1987, p. 35. 35 G. Montaudo and C. Puglisl, Thermal degradation of condensation polymers, m Comprehensive Polymers Science, 1992, in press. 36 A. Guarine, M. Vlncenti, P. Guarda and G. Marchionm, Abstracts 12th International Mass Spectrometry Conference, Amsterdam, 1991, p. 392. 37 R. Luijk, H.A.J. Govers, G.B. Eljkel and J.J. Boon, J. Anal. Appl Pyrol., 20 (1991) 303-319. 38 Older literature is reviewed by: W.J. Irwin, J. Anal. Appl. Pyrol., 1 (1979) 3-25, 89-122. H.R. Schulten and R.P. Lattlmer, Mass Spectrom. Rev., 3 (1984) 231-315. 39 J.J. Boon and J.W de Leeuw, J. Anal. Appl. Pyrol., 11 (1987) 313-327. 40 T.O. Munson and D.D. Fetterolf, J. Anal. Appl. Pyrol., 11 (1987) 15. 41 M. Langhammer, I. Luederwald and A. Slmons, Fresenius' Z. Anal. Chem., 324 (1986) 5-8. 42 P.F. Crain, Mass Spectrom Rev., 9 (1990) 505. 43 A Van der Kaaden, J. Haverkamp and J.J. Boon, Blomed. Mass Spectrom., 11 (1984) 486-492. 44 H.R. Schulten and W. G6rtz, Anal. Chem., 50 (1978) 428. 45 J R. Obst, J Wood Chem. Technol., 3 (1983) 377-397. 46 C. Saiz-Jlmenez and J.W. de Leeuw, Org. Geochem., 6 (1984) 417--422. 47 A D. Pouwels and J.J. Boon, J. Anal. Appl. Pyrol., 17 (1990) 97-127. 48 M M. Mulder, J.B.M. Pureveen, J.J. Boon and A.T. Martinez, J. Anal. Appl. Pyrol., 19 (1991) 175-191. 49 R. Hempflmg and H R. Schulten, Org. Geochem., 15 (1990) 131-145. 50 E.R.E. Van der Hage, J.M. Commandeur, J.B.M. Pureveen, M.M. Mulder, N. Terashima and J.J. Boon, Abstracts 12th International Mass Spectrometry Conference, Amsterdam, 1991, p. 396 51 C. Largeau, S. Derenne, E. Casadevall, A. Kadoun and N. Sellier, Org. Geochem., 10 (1986) 1023-1032. 52 M. Nip, E W. Tegelaar, J.W. de Leeuw, P.A. Schenck and P J. Holloway, Org. Geochem., 10 (1986) 769-778. 53 P Metzger, C Largeau and E. Casadevall, Llpids and macromolecular lipids of the hydrocarbon rich microalga Botryococcus brauntt. Chemical structure, biosynthesis, geochemical and blotechnologlcal importance, in W. Herz, H. Grisebach, G.W. Kirby and C. Tamm (Eds.), Progress m the Chemistry of Orgamc Natural Products, Vol. 57, 1991, p. 1-70. 54 E.W Tegelaar, J.W. de Leeuw, C Largeau, S. Derenne, H.R. Schulten, R Mueller, J.J Boon, M. Nip and J.C M Sprenkeis, J. Anal. Appl. Pyrol., 15 (1989) 29, 55 E.W. Tegelaar, J.W. de Leeuw and P.J. Holloway, J. Anal. Appl. Pyrol., 15 (1989) 289-295. 56 T.L. Bluhm, G.K. Hamer, R H. Marchessault, C.A. Fyfe and R.P. Veregin, Macromolecules, 19 (1986) 2871. 57 H. Monkawa and R.H. Marchessault, Can J. Chem., 59 (1981) 2306

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

785

58 R.J. Helleur, Polym. Prepr., Am. Chem. Soc., Div. Polym. Chem., 29 (1988) 609. 59 B.E. Watt, S.L. Morgan and A. Fox, J. Anal. Appl. Pyrol., 19 (1991) 237-251 60 J.J. Boon, B. Brandt-de Boer, W. Genult, J. Dallinga and E Turkstra, Characterisation of particulate organic matter from sediments in the estuary of the Rhine and from off-shore dump sites of dredging spoils, in M.L. Sohn (Ed.), Organic Marine Geochemistry, ACS Symp. Set. 305, American Chemical Society, Washington, DC, 1986, pp. 76-90. 61 A. Ballistreri, D. Garozzo, M. Giuffrida, G. Impallomeni and G. Montaudo, J Anal. Appl. Pyrol. 16 (1989) 239-255. 62 A. Ballistren, G. Montaudo, D. Garozzo, M. Giuffrida and M.S. Montaudo, Macromolecules, 24 (1991) 1231-1236. 63 G. Montaudo, Rapid Commun. Mass Spectrom., 5 (1991) 95-100. 64 H Preustlng, A. Nijenhuis and B. Witholt, Macromolecules, 23 (1990) 4220-4224. 65 D Radlein, J. Piskorz and D.S Scott, J. Anal. Appl. Pyrol, 19 (1991) 41-63. 66 A.D. Pouwels, G.B. Eijkel and J.J Boon, J. Anal. Appl. Pyrol, 14 (1989) 237. 67 D. Radlein, A. Gnnshpun, J. Plskorz and D.S. Scott, J. Anal. Appl Pyrol., 12 (1987) 39. 68 G.N. Richards, J. Anal. Appl. Pyrol., l0 (1987) 251. 69 A.D. Pouwels, G.B. Eijkel, P.W Ansz and J J Boon, J. Anal. Appl. Pyrol., 15 (1989) 71-84. 70 J.J. Boon, Gordon Research Conference on Pyrolysis, New Hampton, 1989 (see also ref. 76) 71 P.W. Arisz, personal communicahon, 1990. 72 F. Shafizadeh, R.H. Furneaux, T G. Cochran, J.P. Scholl and Y. Sakal, J. Appl Polym. Scl., 23 (1978) 3525. 73 A.C. Tas, A. Kerkenaar, G.F. LaVos and J. van Der Greef, J. Anal. Appl Pyrol., 15 0989) 55-70. 74 R.J. Helleur and R. Guevremont, J. Anal. Appl. Pyrol., 15 (1989) 85-94. 75 J.A. Lomax and J.J. Boon, in preparation. 76 J.A. Lomax, R.A. Hoffmann and J.J. Boon, Carbohydr. Res, 221 (1991) 219-233. 77 J.A. Lomax, J.M. Commandeur and J.J. Boon, Biochem Soc. Trans., 19 (1991) 935-940. 78 M L. Coates and C.L. Wilkins, Anal. Chem., 59 (1987) 197-200. 79 M.A. Scheijen and J.J. Boon, Rapid Commun. Mass Spectrom., 7 (1989) 238-240 80 M.A. Scheljen, Analytical Pyrolysis Studies on Tobacco, Ph.D. Thesis, University of Amsterdam, 1991. 81 J.J. Boon, M. Nip, G.B. E1jkel, B. Brandt-de Boer and M. van Amsterdam, Characterisatlon of citrus and apple pectins by pyrolysis mass spectrometry in PY-EIMS, DCI-MS and PYGCMS modes, in P. Longevlalle (Ed.), Advances in Mass Spectrometry, Vol. 1l, Heyden and Sons, London, 1988, pp 1100-1102. 82 The papers selected give access to the relevant literature. 83 H.L.C. Meuzelaar, F Hlleman and J. Haverkamp, Pyrolysis Mass Spectrometry of Recent and Fossil Blomatenals" Compendium and Atlas, Elsevier, New York, 1982. 84 A.C. Tas, J. de Waart, J. Bouwman, M.C. ten Oever de Brauw and J. van der Greef, J Anal. Appl. Pyrol, l l (1987) 329-340. 85 J.J. Boon, B. Brandt-de Boer, G.B Eijkel, E. Vlegels, L Sijtsma and J.T.M. Wouters, Differentiation of phage sensltwe and phage resistant Streptococcus cremorts strains by pyrolysis mass spectrometry and dlscrimmant analysis of the cell walls, in E. Heinzle and M. Ruess (Eds.), Mass Spectrometry in Biotechnologlcal Process Analysis and Control, Plenum Press, New York, 1987, pp. 187-208. 86 J.G de Nobel, Permeabihty of the Cell Wall in Saccharomyces cerevistae, Ph.D Thesis, University of Amsterdam, 1991.

786

J.J. Boon~Int. J Mass Spectrom. Ion Processes 118/119 (1992) 755-787

87 A.C Tas, H.B. Bastiaanse, J. van der Greef and A Kerkenaar, J. Anal. Appl. Pyrol., 14 (1989) 309-321. 88 S.M. Huff, J.M. Matsen, W. Windig and H.L.C. Meuzelaar, Blomed. Environ. Mass Spectrom., 13 (1986) 277-286. 89 J.J. Boon, Tracing the origin of chemical fossils in microbial mats: Biogeochem~cal investigations of Solar Lake cyanobacterlal mats using analytical pyrolysis methods, in Y. Cohen, R.W. Castenholz and H.O. Halverson (Eds.), Microbial Mats, M.B.L. Lectures in Biology Vol. 3, Alan Liss, New York, 1984, pp. 313-342. 90 J.J. Boon, J.W. de Leeuw and W.E. Krumbem, Biogeochemistry of Gavish Sabkha sediments II. Pyrolysis mass spectrometry of the laminated microbial mat m the permanently covered zone before and after the desert sheet flood of 1979, in G.M. Friedman and W.E. Krumbein (Eds.), Hypersaline Ecosystems, Springer-Verlag, Berlin, 1985, pp. 368-380. 91 S.L. Morgan, B.E. Watt, K. Ueda and A. Fox, Pyrolysis GCMS profihng of chemical markers for microorganisms, in A. Fox, S.L. Morgan, L. Larsson and G. Odham (Eds.), Analytical Microbiology Methods, Plenum Press, New York, 1990, pp. 179-200. 92 J.J. Boon, An introduction to pyrolysis mass spectrometry of lignocellulosic material: case studies on barley straw, corn stem and Agropyron, in A. Chesson and E.R. Orskov (Eds.), Physico Chemical Characterisation of Plant Residues for Industrial and Feed Use, Elsevier Applied Science, London, 1989, pp. 25-50. 93 G.J. Niemann, R.P. Baayen and J.J. Boon, Ann. Bot. (London), 65 (1990) 461. 94 M.M. Mulder, O. Dolstra and J J Boon, PYMS as a scanning tool for plant breeders: a study of two public inbred lines of Zea Mays, in G.C Galett~ (Ed.), Production and Utilization of Lignocelluloslcs, Elsevier Applied Science, London, 1991, pp. 291-307. 95 G.J. Nlemann, J J. Boon, J.B.M. Pureveen, G.B. Eijkel and E. van der Heijden, J. Anal. Appl. Pyrol., 19 (1991) 213-236. 96 M.J.M. Hagedoorn, T.P. Traas, J.J. Boon and L. van der Plas, Plant Physiol., 137 (1990) 72-80. 97 M. Scheijen and J.J. Boon, J. Anal. Appl. Pyrol., 19 (1991) 153-175. 98 C. Salz-Jlmenez, J.J. Boon, J. Hedges, J.K.C. Hessels and J.W. de Leeuw, J. Anal. Appl. Pyrol., 11 (1987)437. 99 S.A. Stout, J.J. Boon and W. Spackman, Geochim. Cosmoch~m. Acta, 52 (1988) 405-414. 100 E. Van der Heijden, J.J. Boon, F. Bouman and M.M. Mulder, Anatomy and pyrolysm mass spectrometry of peat forming and peatified plant tissues, in D.A.C Manning (Ed.), Organic Geochemistry, Advances and Apphcations in the Natural Environment, Manchester Umversity Press, 1991, pp. 460-464. 101 R. Hempfling, W. Zech and H.R. Schulten, Soil Sci., 146 (1988) 262-276. 102 H.R. Schulten, R. Hempfling and W. Zech, Geoderma, 41 (1988)211-222. 103 N. Slmmlelt and H.R. Schulten, Chemosphere, 18 (1989) 1855-1869. 104 H.R. Schulten, N. Simmleit and H.H. Rump, Int. J. Environ. Anal. Chem., 27 (1986) 241-264. 105 M. Schnltzer, Soil Scl., 151 (1991) 41-58. 106 W.G M. van Loon, A.D Pouwels, P. Veenendaal and J.J. Boon, Int. J. Environ. Anal. Chem., 38 (1990) 255. 107 W.M.G. van Loon, J.J. Boon and B. de Groot, J. Anal. Appl. Pyrol., 20 (1991) 275 108 A.M. Shedrinsky, T.P. Wampler, N. Indictor and N.S. Bear, J. Anal. Appl. Pyrol., 15 (1989) 393. 109 T F.M. Oudemans and J.J. Boon, J. Anal. Appl. Pyrol., 20 (1991) 197. 110 S.R. Larter and J.T. Senftle, Nature, 318 (1985) 277.

J.J. Boon~Int. J. Mass Spectrom. Ion Processes 118/119 (1992) 755-787

787

111 B. Horsfield, Pyrolysis studies and petroleum exploration, in J Brooks and D.H Welte (Eds.), Advances in Petroleum Geochemistry, Vol. 1, Academic Press, London, 1984, p. 247. 112 E.W Tegelaar, J.W. de Leeuw, S Derenne and C. Largeau, Geochlm Cosmochlm. Acta, 53 (1989) 3103. 113 G.R. Alken, D McKnight, R.L. Wershaw and P. McCarthy (Eds.), Humlc Substances in Soil, Sediment and Water, Wiley, New York, 1985. 114 B Horsfield, U. Disko and F Lelstner, Geol Rundsch, 78 (1989) 361. 115 T.I. Eglinton, R.P. Phllp and S.J. Rowland, Org. Geochem., 12 (1988) 33. 116 J S. Slnnlghe Damste, T.I. Eghnton and J.W. de Leeuw, The presence of abundant macromolecularly bound tetrapyrrole ptgments m kerogens as determined by alkylpyrrole distrabuUons in flash pyrolysates, m D.A.C Manning (Ed.), Organic Geochemistry, Advances and Applications m the Natural Environment, Manchester University Press, 1991, pp. 452-455. 117 J.S. Smmghe Damste, T I Eglinton, W.I.C. Rijpstra and J.W. de Leeuw, Characterisation of organically bound sulphur in high molecular weight sedimentary orgamc matter using flash pyrolysis and Raney nickel desulphurisation, in W.L. Orr and C.M White (Eds.), Geochemistry of Fossil Fuels, ACS Symp. Ser. 420, American Chemical Society, Washington, DC, 1990, p. 486. 118 T.I. Eglinton, J S. Sinmghe Damste, W. Pool, J W de Leeuw, G.B. Eijkei and J.J. Boon, Geochlm. Cosmochim. Acta, (1992) m press. 119 J W de Leeuw, P F. van Bergen, B G.K van Aarssen, J.-P. L.A. Gatelher, J.S. Slnmghe Damste and M E. Collinson, Proc. R. Soc London, Ser. B, 333 (1991) 54. 120 T.I. Eglinton, S.R. Larter and J.J. Boon, J. Anal. Appl. Pyrol., 20 (1991) 25-45. 121 H.L.C. Meuzelaar, A.M. Harper, R.J. Pugmlre and J. Karas, Int. J. Coal Geol., 4 (1984) 143. 122 P J J. Tromp, J.A Mouhjn and J.J Boon, Characterlsatlon of coal by means of Curiepoint pyrolysis techniques, in Y. Yuerum (Ed.), New Trends m Coal Science, Kluwer, Dordrecht, 1984, pp. 241-269. 123 R.E. Winans, R.G. Scott, P.H. Nelll, G R. Dyrkacz and R. Hayatsu, Fuel Proc Techn., 12 (1986) 77. 124 Y. Yun, H.L.C. Meuzelaar, N. Slmmlelt and H.R. Schulten, Energy and Fuels, 5 (1991) 22-29