The in situ analytical pyrolysis of two dierent organic
components of a synthetic environmental matrix doped
with [4,9-
13
C] pyrene
Paul F. Greenwood
a,*, Elizabeth A. Guthrie
b,1, Patrick G. Hatcher
b aCSIRO, Division of Petroleum Resources, PO Box 136, North Ryde, NSW 1670, AustraliabDepartment of Chemistry, Ohio State University, Columbus, OH 43210, USA
Abstract
Laser micropyrolysis GC±MS was used for in situ analysis of the coal and lignin components of a synthetic mixture. Designed to mimic environmental matrices such as soils and sediments, the mix comprised several possible soil pre-cursors and was also amended with [4,9-13C]pyrene as part of concurrent research on the interaction of PAH pollutants
and sedimentary organic matter. The labeled spike was consistently detected as the major pyrolysate in the in situ analyses of both lignin and coal components of the synthetic mix, indicating its eective sorption by these moieties of the mix. The remaining hydrocarbon distribution detected from the lignin was dominated by guiaicyl (i.e. methox-yphenol) compounds, whereas high abundances of aromatic (e.g. benzene, naphthalene, phenol and alkyl derivatives thereof) and aliphatic (e.g.n-alkene/alkane, prist-1-ene, hopanes) products were detected in the coal. Apart from the high concentrations of the 13C-spike, these data were very similar to molecular data obtained from the respective
pyroprobe pyrolysis GC±MS analysis of pure lignin and coal samples. The untainted (i.e. apart from the13C-spike)
molecular signatures detected from the in situ analysis of the coal and lignin constituents indicates minimal organic contamination from the other constituents of the synthetic mix, successfully demonstrating the capability of the laser micropyrolysis GC±MS technique to selectively analyse the discrete organic entities within complex and heterogeneous mixtures.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Laser pyrolysis; GC±MS; Soil; Polycyclic aromatic hydrocarbon; Sorption;13C-labeling
1. Introduction
Soils and sediments, by their very nature of forma-tion, represent a highly complex mixture of organic and mineral matter. The major organic precursors are degraded plant matter that may comprise a wide array of autochthonous compounds due to variable plant type and composition and allochthonous components transported by aquatic or aeolian processes. Analytical pyrolysis has
provided useful molecular information from soils (Saiz Jimenez and de Leeuw, 1984, 1986; Saiz Jimenez, 1992, 1994). Traditionally, these analyses have been applied to bulk samples or chemically derived fractions of the bulk soil. This approach can not account for the hetero-geneous morphology of the organic matter that exists prior to chemical manipulation and disturbance.
Distinct organic entities within heterogeneous materials such as soils may now be analyzed with the recently developed technique of laser micropyrolysis gas chromato-graphy mass spectrometry (GC±MS) (Stout, 1993; Greenwood et al., 1993, 1996, 1998). This technique has emerged from the need to analyze micro-sample quan-tities of organic matter in organic geochemistry research. Recently, laser microprobes have been used in combination with various traditional analytical methods to analyse microscale portions of soils and soil fractions.
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* Corresponding author. Present address: Isotope and Organic Geochemistry Laboratory, Australian Geological Sur-vey Organisation, PO Box 378, Canberra, ACT 2601, Aus-tralia. Fax: +61-6249-9961.
For example, the use of a laser microprobe with mass spectrometric detection revealed the heterogeneous binding pattern of PAHs to geosorbents at a lateral resolution of 40mm (Gillette et al., 1999).
To test whether the laser micropyrolysis technique can be used to identify and characterize organic con-stituent microparticles, it is used here to analyze two morphologically distinct components of a synthetically prepared mixture. Puri®ed lignin, coal, cuticle, peat, humin, and humic acids were mixed together to produce an environmental matrix typical of the complex mixture of chemical compounds found in soils and other organic sediments. Lignin, coal, and cuticle are representative of composite phases often recognisable in soils and sedi-ments. Sapropelic material from an algal-dominated sediment (its humin fraction) and humic acids are representative of the types of biopolymeric organic matrices present in sediments. The in situ analysis focused on the lignin and coal components because these components are easily recognised by microscopic observation and by their molecular composition. The lignin was from a sample of brown-rot infected wood collected from Mount Rainier, Washington state (Hatcher et al., 1988) and the coal was collected from an outcrop of the Wilcox Formation near San Antonio, Texas. Lignin, is a resistant component of wood cell walls that is preferentially preserved during biological degradation. For this reason, it is an important terres-trial marker in soils and sediments (Hedges et al., 1985; Shevchenko and Bailey, 1995). Coals usually represent a more modi®ed version of the plant remains found in soils and sediments (Wilson, 1987; Schmidt et al., 1996, 1999).
Conventional pyrolysis data obtained from the pyro-probe pyrolysis GC±MS of pure lignin and coal samples was used to assess the purity of the laser pyrolysis data. A 13C-labeled, four ring polycyclic aromatic
hydro-carbon, [4,9-13C]pyrene, was added to the synthetic mix
so that the capabilities of this technique for detecting PAH contaminants in analogous environmental settings could also be assessed. The use of 13C labeled organic
contaminants in conjunction with pyrolysis GC±MS detection to measure the extent of [4,9-13C]pyrene
sequestration by the refractory humin fraction of a sediment was recently demonstrated (Guthrie et al., 1999). Complementary data concerning the molecular fate of the 13C label following the interaction of 13
C-labeled compounds with sedimentary organic matter has also been previously obtained by other analytical methods including chemolysis (Richnow et al., 1997, 1998), thermo-chemolysis GC±MS (Knicker and Hatcher, 1997), 13C
NMR (Hatcher et al., 1993; Bortiatynski et al., 1994, 1997; Castro et al., 1995; Guthrie et al., 1999) and C-isotopic analysis (Richnow et al., 1998).
Here, we assess the sorptive capacity of a13C-probe
to dierent matrix constituents (i.e. lignin and coal) by
laser micropyrolysis GC±MS. Sorptive interactions between dierent soil biomacromolecules and organic pollutants may dier dramatically (Luthy et al., 1997). However, it should be stressed that monitoring the13
C-probe by laser micropyrolysis GC±MS alone, will only provide a qualitative analysis. Additional factors (e.g., relative lability/bonding strengths) may also in¯uence the detection of such pollutants. The consideration of GC±MS data in association with complementary data from analytical techniques such as13C NMR and/or13C
isotopic analysis may provide a more de®nitive quanti-tation of the degree of sorption. However, analysis of the lignin and coal entities of the synthetic mix by these techniques was beyond the scope of the present paper.
2. Experimental
2.1. Sample composition and preparation
The composition of the synthetic mix is shown in Table 1. The dierent components were simply mixed together in a glass beaker and stirred with a steel spatula. None of the components were ground before or during the mixing process to maintain the morphology of the coal, lignin and cuticle constituents. 2 mg of [4,9-13C]pyrene (synthesized as reported by Guthrie et
al., 1999) was added to 2.22 g of the synthetic mix via 50
ml of methanol carrier. The mixture was air-dried for four hours to allow for the evaporation of the methanol. An even distribution of the 13C-probe throughout the
mixture cannot be assumed since it consists of granules of irregular size and shape. The synthetic mix did not appear to be physically altered by the spiking process. A photomicrograph of the synthetic mix in which the lig-nin and the coal components are readily identi®able is shown in Fig. 1. (NB: This photo was taken with an Olympus PM-10ADS photomicrographic camera system interfaced to a Zeiss Universal microscope equipped with Epiplan HD objectives, in epi-dark®eld mode using Kodak Elite II ED135-36 transparency ®lm.)
Table 1
Composition of the synthetic mixture
Component Source Mass
(mg)
Humin Warwick pond, Bermuda 1000 Coal and resin Wilcox lignite, TX, USA 430 Peat Everglades peat, FL, USA 330 Lignin Brown-rotted wood from Mt Rainier,
WA, USA
250
Humic acid Everglades peat, FL, USA 170 cuticle Tomato cuticle (provided by
Dr. Erik Tegelaar)
2.2. Laser micropyrolysis GC±MS
Details of the hardware and experimental protocol associated with the laser technique have been outlined
previously (Greenwood et al., 1996, 1998) and only a brief description will be included here. The laser microprobe comprises a Laser Applications continuous wave Nd:YAG laser and an Olympus re¯ected light/
Fig. 1. A photomicrograph of the synthetic mixture. L = lignin and C = coal components. Scale bar = 1 mm.
¯uorescence microscope. A small portion of the syn-thetic sample was dispersed on a 5.9 mm DeckglaserTM
glass cover-slip and located in the sample pyrolysis chamber. The pyrolysis chamber is interfaced to the GC via an intricate gas inlet system designed for ecient product transfer. A window at the top of the pyrolysis chamber provides the laser microprobe with access to the sample inside.
The samples were typically pyrolysed with a laser energy of 19 watts/s for a period of 10 s. Individual
lignin and coal grains were of sucient size (0.1±1 mm) to accommodate the relatively large (200mm diameter)
laser craters obtained with these parameters when focusing the laser beam through the10 microscope objective.
Where possible, large craters are preferred as they give rise to higher pyrolysate concentrations. The pyrolysis chamber was manually positioned under the microscope objective such that the particular granule (or part thereof) to be analysed was located in the target area of the laser.
GC±MS detection of the laser pyrolysates was per-formed with a HP 5890/Series II gas chromatograph interfaced to a Micromass-Autospec UltimaQ mass spectrometer. Chromatography was carried out on a 25 m DB-5 capillary column (5% phenyl polysiloxane, 0.32 mm i.d., 0.52mm ®lm thickness). The GC was typically temperature-programmed for an initial 40C, held for 2
min, then increased at 4C/min to a ®nal temperature of
300C, held for 25 min. Full scan mass spectra (m/z50±
550) were obtained using standard detection parameters (electron energy = 70 eV; ®lament current = 200 mA; source temperature = 250C; electron multiplier = 200
V; mass resolution = 1000).
2.3. Pyroprobe pyrolysis GC±MS
Conventional analytical pyrolysis was performed with a Chemical Data Systems 160 pyroprobe inter-faced to a HP 6890/5973 GC±MS. A small powdered portion (1±2 mg) of the pure lignin, pure coal and bulk
synthetic mix were separately pyrolysed at a tempera-ture of 650C which was applied for 10 s. The
pyr-olysates were swept from the pyroprobe interface onto the GC-column with a helium carrier (1.3 ml/min). Chromatography was carried out on a 30 m, AT-5 capillary column (5% phenylmethyl silicone, 0.25 mm i.d., 0.25mm ®lm thickness). The pyrolysates were cryo-genically focussed onto the GC column by immersing a short section of the column in a liquid nitrogen bath for 1 min. A GC temperature program of an initial 40C (2 mins hold) increased at 2C/min to 300C
(25 min hold) was started on removal of the column from the cold trap. A HP 5973 mass spectrometer was used for product detection with relatively standard MS parameters (70 eV; m/z 50±550; 250C source
temperature).
3. Results and discussion
3.1. Analysis of the Mt Rainier lignin moiety of the synthetic mixture
The total ion chromatogram (TIC) from the in situ laser micropyrolysis GC±MS analysis of a lignin com-ponent of the synthetic mix is shown in Fig. 2. Product assignments are listed in Table 2 and were based on both mass spectral and GC retention time data. The major peak eluting at a retention time of42 min can be assigned as [4,9-13C]pyrene, indicating eective sorption
of the13C-labeled probe onto the lignin component of
the synthetic mix. It was consistently the most abundant product detected from several repeat analyses of lignin constituents of the synthetic mix.
Table 2
Pyrolysates detected from either the synthetic mixture and/or the lignin and coal components of the mixture
The remaining product distribution is dominated by guaiacyl units re¯ecting the gymnosperm origins of the Mt Rainier lignin (Hatcher et al., 1988). The major peaks are phenol (8), guaiacol (13), 4-methylguaiacol (17), vinyl-guaiacol (21), eugenols (22,23,29) and coniferylaldehyde (33). Subordinate peaks were assigned as alkylphenols (11,12,15), other side-chain oxidized products (e.g.
31,32) and polycyclic hydrocarbons (e.g.10,16,28). The phenolic products are indicative of a biodegraded wood (Sigleo, 1978; Obst, 1983), whereas the signi®cant PAH abundances are probably a consequence of the pyrolysis mechanism.
In a recent study in which we compared the laser micropyrolysis and pyroprobe pyrolysis GC±MS ana-lyses of (pure) Mt Ranier lignin (unpublished data), the applicability of laser energy as a source of pyrolysis for the study of lignin was established. Except for the major [4,9-13C]pyrene peak, the hydrocarbon distribution from
the in situ analyses of lignin components of the synthetic mix (e.g. Fig. 2) was almost identical to the guiaicyl dominated pyrolysis distribution from the laser pyr-olysis GC±MS of pure Mt Ranier lignin observed in our recent study. The laser result was also in good agreement with previous data obtained from Mt Rainier lignin (Hatcher et al., 1988). The consistency between the laser pyrolysis GC±MS data from the in situ analysis of a lignin component in the heterogeneous matrix and cor-responding molecular data obtained from pure Mt Rainier lignin indicates minimal contamination from the other precursors of the synthetic matrix. Thus, the laser
microprobe has been successfully used to selectively pyrolyse the lignin moiety of a complex environmental matrix without prior chemical or physical isolation.
3.2. Analysis of the Wilcox coal moiety of the synthetic mixture
The total ion chromatogram (TIC) from the in situ laser micropyrolysis GC±MS analysis of a coal component of the synthetic mixture is shown in Fig. 3. Product assignments are listed in Table 2. [4,9-13C]pyrene was
again the major pyrolysate consistently detected from the in situ analysis of several coal components, indicating that this moiety of the synthetic mixture has also eciently sorbed the13C-probe. The TIC was deliberately set o
scale (3) in Fig. 3 so that the complete pyrolysate
dis-tribution can be more readily observed. A high abundance of aromatic pyrolysates including alkylaromatic, alkyl-phenol and polycyclic aromatic hydrocarbons was detected. A signi®cant distribution of aliphatic pyrolysates con-sisting of prist-1-ene in high abundance, a wide MW distribution ofn-alkene/alkanes and a number of high MW hopane/hopenes was also detected.
Apart from the large peak due to the 13C-probe, the
pyrolysate distribution detected from the in situ analysis of the coal component of the synthetic mix is generally consistent with molecular data obtained previously from py-GC±MS analysis of low-rank coals (Larter and Hors®eld, 1993). The Wilcox coal was additionally ana-lysed here by pyroprobe pyrolysis GC±MS to provide a
Fig. 4. (a)m/z57 and (c) them/z191 mass chromatograms from the in situ laser micropyrolysis GC±MS analysis of a coal component of the synthetic mixture; (b)m/z57 and (d) them/z191 mass chromatograms from the pyroprobe pyrolysis of Wilcox coal. Pr=prist-1-ene;
comparative data set. The respectivem/z57 andm/z191 mass chromatograms obtained from the in situ analysis of a coal component of the synthetic mixture by laser micropyrolysis GC±MS and the analysis of pure Wilcox coal by pyroprobe pyrolysis GC±MS are shown in Fig. 4. The aliphatic products consist of a bimodal distribution of n-alkene/alkanes from C10out to C31, with maxima
at C11 and C27, and a very prominent prist-1-ene. The
hopanoid distributions consist of C27, C29 and C30
hopanes and hopenes. The respective acyclic aliphatic and hopanoid data sets re¯ect little qualitative dierence. The small variance observed in the relative abundance of some products may be due to the dierent heating regimes of the pyrolyis methods (laser>>pyroprobe) or simply may re¯ect the reproducibility limitations of these methods. A slight intermolecular chemical hetero-geneity within the coal macromolecule (averaged out by the bulk pyroprobe analysis) might also be evident.
3.3. Pyroprobe pyrolysis of the synthetic mixture
Consistent with the multi-precursor composition of the synthetic mixture, the pyrolysate distribution detected from the bulk mix is considerably more complex than
the corresponding data from its coal and lignin moieties. The total ion chromatogram (TIC) andm/z57 chromato-gram from the pyroprobe pyrolysis of the synthetic mixture is shown in Fig. 5. The bulk pyrolysates essen-tially constitute a mean of the molecular composition of each of the precursors of the synthetic mixture. Several molecular features due to the lignin and coal precursors can be recognised. Vinylguiaicol (21) and trans-isoeugenol (29), two of the major pyrolysates detected from the lignin moiety of the mix (Fig. 2), are also detected in high abundance in the synthetic mixture (Fig. 5a).
In contrast to the direct guiaicyl product/lignin pre-cursor link, aliphatic hydrocarbons are not so source speci®c. Nevertheless, the bimodaln-alkene/alkane dis-tribution and prominent prist-1-ene observed from the
m/z57 chromatogram detected from the synthetic mixture (Fig. 5b) is largely consistent with the aliphatic distribution measured from the Wilcox coal (Figs 4a and b). The Wilcox coal is probably the major source of aliphatic hydro-carbons in the synthetic mixture, inundating the alipha-tic contribution of the other precursors.
Many other products detected from the bulk pyrolysis of the synthetic mix cannot be attributed to either the coal or lignin precursors. Products which may have derived from
other precursors of the synthetic mixture include alkylated pyrolles, pyridines and several other low MW nitrogen containing compounds (NB: pyroprobe pyrolysates detected from the Everglades humic acid) and low MW branched aliphatic products (NB: pyroprobe pyrolsates detected from the tomato cuticle). The complexity of the pyrolysate distribution detected from the synthetic mixture is typical of the complex chemical material which accu-mulates in soils and sediments.
4. Summary and conclusions
Selective in situ analyses of the lignin and coal moieties of a synthetic mixture was achieved by laser micropyrolysis GC±MS analysis. Apart from a high abundance of [4,9-13C]pyrene, the13C-labeled compound
with which the mixture was amended, the remaining hydrocarbon distributions detected from moieties proved consistent with conventional pyrolysis data obtained from pure lignin and coal samples. Guiaicyl pyrolysates were dominant in the lignin analysis whilst the coal was characterised by a more complex distribu-tion of C10±C30n-alkene/alkane doublets, an abundant
prist-1-ene, alkylated benzenes, naphthalenes and phe-nols. No chemical contamination from other precursors in the mixture was evident in the in situ analysis of these two components. As expected, input from the lignin, coal and other precursors was evident in the complex pyrogram obtained from the bulk analysis of the syn-thetic mix.
The consistent detection of [4,9-13C]pyrene as the
major product in these analyses was interpreted as indi-cating strong sorption of this PAH to the surface of the coal and lignin moieties of the synthetic mixture. These results demonstrate the potential of this approach for qualitatively monitoring the sorption capacity of PAH pollutants to the distinctly recognisable phases of naturally occurring organic materials.
The encouraging results reported here from this very basic synthetic mixture spiked with [4,9-13C]pyrene gives
con®dence that more detailed information concerning the interaction between pollutants and biomacromolecules may be obtained with more expansive experiments. For example, the particular fate of a 13C-label compound
following its sorption by a speci®c entity of a simulated or real organic setting may be subsequently monitored by repeat laser micropyrolysis GC±MS analyses of the entity at advanced time intervals. For example, is the pollutant labile and lost over time or, alternatively, is it incorporated in to the organic environment through various reactive or physically protective processes. A variety of signi®cant environmental issues (e.g. reme-diation of heavily contaminated sites) may be addressed if the consequences of organic pollution can be better de®ned at the molecular level.
Acknowledgements
We thank the Oce of Naval Research through Grants N-00149510209 and N-00149910073 for ®nancial assistance. We also thank Dr. Erik Tegelaar for providing a sample of tomato cuticle. The reviews by Drs. Claude Largeau and Pim van Bergen were appreciated and their comments signi®cantly improved the manuscript.
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