MS and pyrolysis-field ionization mass spectrometry

Org. Geochem. Vol. 15, No. 2, pp. 131-145, 1990 Printed in Great Britain

0146-6380/90 $3.00 + 0.00 Pergamon Press plc

Chemical characterization of the organic matter in forest soils by Curie point pyrolysis-GC/MS and pyrolysis-field ionization mass spectrometry R. HEMPFLINGand H.-R. SCHULTEN Fachhochschule Fresenius, Department of Trace Analysis, Dambachtal 20, 6200 Wiesbaden, Federal Republic of Germany (Received 29 March 1989; accepted 29 July 1989)

Abstract--The chemical composition of three different forest humus layers (mull, moder, mor) was studied by flash pyrolysis-gas chromatography/electron impact mass spectrometry (Py-GC/EIMS) and by temperature-programmed, direct pyrolysis-field ionization mass spectrometry (Py-FIMS) in order to evaluate the relevance of individual humus constituents for geoecological and geochemical processes. Pyrolysis products with a molecular weight up to 430 Dalton that derive from carbohydrates, intact and degraded lignins, proteins, lipids, polyphenols, and aliphatic polymers were identified by accurate mass measurements in the FI mode and by GC/EIMS. However, the pyrolysis products that caused the main differences between the Py-FI mass spectra of the three humus types in the mass range above m/z 300 were not recorded by Py-GC/EIMS. Their assignments are based on in-source pyrolysis and high resolution FIMS. They derive mainly from biphenyl-, diarylpropane-, phenylcoumaran-, and resinol-type subunits from lignin, and phytosterols and their dehydration products. Furthermore, free and bound lipids such as homologous series of n-fatty acids (C:z-C32), alkyl monoesters (C40-Cs2), and aromatic esters (C27 C~4) are found. Key words--analytical pyrolysis, accurate mass measurements, chemical composition, forest humus, time-resolved pyrolysis mass spectometry

INTRODUCTION Forest humus comprises the organic layers of the forest floor (SSSA, 1987) that consists mainly of plant litter (primary resources) and humified plant remains or humic compounds. In addition, residues from animals and microorganisms (secondary resources) also contribute to the organic matter in forest soils. Besides various effects on soil fertility, plant nutrition and the dynamic of inorganic and organic pollutants, the organic material on the forest floor constitutes a major parent material for biogeochemical cycles. The various intermediate products of these cycles has been the object of numerous pyrolysis studies by organic geochemists (van Smeerdijk and Boon, 1987; Philp and Gilbert, 1987; Nip et al., 1988; Hatcher and Lerch, 1989, and others). Also parent plant materials such as wood, roots, and leaves have been studied by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and direct Py-MS (Schulten and Simmleit, 1986; van Smeerdijk and Boon, 1987; Simmleit and Schulten, 1989a, b) but there is insufficient chemical information, on a molecular basis, on the heterogenous mixture of primary and secondary resources and humified material on the forest floor. Some remains of plants and animals disappear during the decomposition process, but others are stabilized in humic substances (Hatcher and Spiker, 1988; Hempfling and Schulten, 1989). In particular, 131

lignin enters the geological cycle and survives early diagenetic reactions because of its high chemical stability and resistance to rapid microbial degradation (Saiz-Jimenez and de Leeuw, 1986; Hatcher, 1988). In addition, lipids in free or bound form are particularly useful as biogenic indicators. They partly show source-specificity, and certain structural types persist over long periods of time with only limited modifications. Thus, the structures of biopolymers present in plant tissue and their chemically altered counterparts generated upon humification can be used to find missing links between bio- and geomaterials. As concluded by Philp and Oung (1988), the larger and more complex these biomarkers, the more useful they are for providing geochemical information. Depending on the local environmental conditions such as chemical composition of the parent material, climate, acidity, and biological activity, the genetically different forest humus types mull, moder, and mor develop that are characterized by different chemical properties (Swift et al., 1979). These chemical properties effect geoecological and geochemical processes. Thus, bulk samples from sites that represent the characteristic forest humus types mull, moder, and mor have been examined. The complementary use of Curie point Py-GC/EIMS and temperature-programmed, direct Py-field ionization (FI) MS should provide more detailed information

132

R. HEMPFLINGand H.-R. SCHULTEN

about low- and high-molecular weight building blocks of forest humus. The main objectives of the present study were: (1) to identify pyrolysis products from different forest humus types by Curie point Py-GC/EIMS; (2) to propose structures for high-molecular weight pyrolysis products that are not GCamenable by time-/temperature-resolved and high mass resolution Py-FIMS; (3) to evaluate correlations between generated pyrolysis products and original structural subunits of the organic matter in forest soils. EXPERIMENTAL

Samples The bulk samples investigated derived from the three humus types mull (395 m above sea level), moder (365m), and mor (1010m) located in the surroundings of Bayreuth (F.R.G.). The humus samples with carbon contents of 16% for mull, 32% for moder, and 33% for mor were typical examples of the three types. The main parent material for mull was ash litter, for moder, beech litter, and for mor, spruce litter with a humus layer thickness of 16 cm for mull, 17.5cm for moder, and 17.5cm for mor. Besides plant litter at different stages of decomposition, the humus samples also contain significant amounts of fine material which represents the product of the humification process. Detailed morphological and chemical descriptions of the individual horizons from these forest humus layers, as well as a chemical characterization of extracted humic substances, have been published previously (K6gel et al., 1988; Hempfling et al., 1988; K6gel-Knabner et al., 1988; Hempfling and Schulten, 1989). As pretreatment for pyrolysis, the samples were only freeze-dried and ground. In order to distinguish between free and bound aliphatic components, some fresh material from mull, moder, and mor was extracted for 16 h with a chloroform/methanol mixture (2:1) in a Soxhlet apparatus. The extracts were reduced to a small volume on a rotary evaporator, transferred to vials, taken to dryness under a stream of N2, and dried under high vacuum.

Curie Point Pyrolysis-Gas Chromatography/Electron Impact Mass Spectrometry For Py-GC/MS, the on-line combination of Curie point (Cp) pyrolyzer (Fischer 0316, Meckenheim) and gas chromatograph (Varian 3700, Darmstadt) with the mass spectrometer (Finnigan MAT 212, Bremen) was used. Samples of about 5 mg were pyrolysed for 10 s total heating time at 500°C: a split of about 1:50 was used. In addition to volatile components, pyrolysis generates high-boiling products that partly condense in the pyrolysis chamber.

To minimize this condensation process, the pyrolysis reactor was surrounded by heating tape that maintained a constant temperature of 300°C in the pyrolysis chamber. Gas chromatographic separation was achieved by separate runs on 30 m capillary columns (0.25pm film thickness) coated with polyphenylmethylsiloxane (DB-17) and polydimethylsiloxane (DB-I). The starting temperature for gas chromatographic separation was 40°C and the final temperature 300°C, with heating rates of 4, 6, 10 and 20°C per min for the separate runs. For the open coupling of the gas chromatograph to the mass spectrometer an uncoated fused silica capillary column was employed (Schulten and Halket, 1986). For mass spectrometric detection with the Finnigan MAT 212, a combined EI/FI/FD ion source was used in the E1 mode with 3 kV accelerating voltage, 70 eV ionizing energy, 2.2 kV multiplier, 1.1 s/mass decade scan speed, and m/z 50-500 mass range. Signals that were not resolved in the ion chromatograms of the total ion current could mostly be separated by examining single ion chromatograms, and this allowed the assignment of appropriate E1 mass spectra. The evaluation of the mass spectra obtained by Py-GC/EIMS was carried out using a library search program (expanded NBS library), interpretation of mass spectra, comparison with data from the literature, GC-retention times, and comparison with the proposed elemental compositions from accurate mass measurements.

Time-~Temperature-Resolved Pyrolysis-Field Ionization Mass Spectrometry During temperature-programmed heating of nonextracted humus samples both evaporation products and thermal degradation products are generated. As both processes cannot be separated exactly the term pyrolysis is used for both processes in the following, because the resulting thermograms (see Fig. 2) indicate that thermal decomposition dominates. For time-/temperature-resolved Py-FIMS about 200 pg of the freeze-dried and ground samples were placed in quartz crucibles of the direct inlet system of a double focusing Finnigan MAT 731 mass spectrometer. The samples were thermally degraded under high vacuum between 50 and 750°C at a heating rate of 1.2°C s- 1, The resulting thermal degradation products were ionized using high-temperature activated tungsten wire emitters (Schulten and Beckey, 1972) in the high electric field. When the sample molecules approach the emitter from the gas-phase (field ionization), only few tenths of an eV are transferred during this soft ionization procedure, and the consecutive mass spectrometric fragmentation due to excess ionization energy is minimized. The resulting ions are separated according to their m/z values and recorded electrically, or on photoplates. The FI-technique has been described in detail by Beckey (1977) and Schulten et al. (1987a, 1989). Within the temperature range used, about 35 single

Organic matter in forest soils spectra were recorded electrically by repetitive magnetic scans. These single spectra were integrated by the Spectro-System SS 200 to give a summed spectrum. The resulting thermograms describe the changes of total ion intensity (TII) and total ion current (TIC) with increasing temperature. This description is also possible for single ions or groups of ions. In addition, selected single spectra that represent distinct temperature ranges can be evaluated. For high-resolution (HR) FIMS, the ions were recorded on photoplates (Ilford, Q2, U.K.). The registered mass range on the photoplate was between m/z 18 and m/z 502 for the forest humus samples investigated. The determination of accurate mass numbers was achieved by calibration with a mixture of standards such as perfluorokerosene, perfluorotributylamine, and tris(perfluoroheptyl)-s-triazine. Evaluation of the photoplates was performed using a comparator and usually the maximum of blackening was taken as the signal center. As incompletely resolved multiplets were often obtained for the humus samples, it was necessary to examine the raw data on the screen, and to determine the positions of multiplet components optically using cursor and special software (Schulten et al., 1987a). The resolution obtained was up to 50,000 (10% valley definition) with an average mass accuracy of approximately 0.002 Dalton. On the basis of these accurate masses elemental compositions were proposed.

RESULTS

AND

DISCUSSION

Curie Point Py-GC/EIMS As an example for the forest humus layers examined by Py-GC/MS the total ion chromatogram of one mor sample GC-trace is shown in Fig. 1. The tentatively identified compounds (see Table 1) represent pyrolysis products of plant materials and their decomposition products, respectively, such as polysaccharides (4-7, 10-12, 14, 20, 23, 24, 28, 31, 32, 36, 38, 41, 44, 48, 50, 58, 70), lignin (11, 21, 26, 27, 30, 41, 49, 51, 52, 54, 60, 61, 63, 64, 68-72, 75, 77, 79, 80, 85, 86, 88, 90, 94, 95),proteins (11, 17-19, 21, 26, 27, 34, 37, 41, 54, 56, 78), lipids (91, 98, 99, 105, 106, 113, 116, 124-136), polyphenols (51, 54, 60, 61, 70, 71, 95), and aliphaticpolymers (13, 15, 22, 33, 35, 42, 43, 55, 57, 65, 67, 74, 76, 81, 82, 87, 89, 93, 96, 97, 100-103, 107-112, 114, 115, 117-120, 122, 123). Similar pyrolysis products have been reported for soil organic matter fractions by Saiz-Jimenez and de Leeuw (1986) and for peat by van Smeerdijk and Boon (1987). Most of these can also be identified in the mull and moder samples. In addition, monomeric pyrolysis products from syringyl subunits such as syringol, methylsyringol, syringylethene, syringaldehyde, syringylethane, syringylpropene, syringylethenone, syringylpropanealdehyde, and sinapyl alcohol were obtained from angiosperm-derived humus samples. The ion chromatogram shown in

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Low and High Resolution Py-FIMS The summed Py-FI mass spectra and related thermograms of the three different forest humus types, mull, moder and mor, are shown in Fig. 2. Clearly, different thermal behavior is obtained for the angiosperm- and gymnosperm-derived humus samples, as the maximum and the whole temperature range of evaporation and thermal degradation is broader for the mor sample. This may be due to a greater heterogeneity of existing types of chemical linkages and physical interactions in the mor sample. Even in the low resolution mode, the signal pattern of the FI mass spectra presented in Fig. 2 is rather complex indicating the great variety of subunit types that are released and recorded by Py-FIMS. For the mass range m/z 50-220 (low-molecular weight pyrolysis products) the signal pattern of the three spectra is similar. These signals are mainly due to polysaccharides (rn/z 72, 74, 82, 84, 96, 98, 110, 112, 114, 126, 128, 132, 144, 162), proteins (m/z 67, 79, 81, 93, 117, 131), and monomeric pyrolysis products from lignin (m/z 124, 138, 140, 150, 152, 154, 164, 166, 168, 178, 180, 182, 194, 196, 208, 210, 212) as identified by Py-GC/MS. An interesting aspect is the ratio of the

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Table 3. Pyrolysis products identified by GC/MS that correspond to elemental compositions from accurate mass measurements of the mull, moder and mor samples Accurate mass

Proposed elemental composition

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Assignment, proposed structure

56, 55 56 58 58 60 67 68 67, 68, 53 55, 70 72 72, 57 57, 72 74 78 79, 52, 51 81, 80, 53 82, 54, 53 82, 81, 53 67, 54, 82 67, 82, 81 84, 54 55, 84 69, 84 56, 55, 69 86 70, 69 91, 92 93, 66 94, 66, 65 79, 94, 77 94, 95 96, 95 96, 95, 53 55, 81, 96 98, 97, 81 98, 55 55 56, 70, 55 100 102 103, 76 104, 103, 78 77, 105, 106, 51 91, 106 91, 106, 105 79, 108, 107, 77 108, 107, 79 108, 109, 80 110 110, 109 95, 110 109, 110 I10, 109, 67, 95 54, 67 112 55, 56, 57, 70 114, 58 115, 116, 89, 63 117, 90, 116 117, 90, 89, 63 117, 118, 115, 91 119, 91, 64 118, 119, 117, 91, 90 105, 120, 77, 51 91, 120, 119, 65 120 105, 120 122, 121, 65, 93 107, 122 122, 121, 77, 107, 79 122, 121 124, 109, 81

Propenal Butene Acetone Ps Butane Acetic acid Ps Pyrrole Pr Furan Ps Methylbutadiene Pentene Pyruvaldehyde Ps Butanone Ps Pentane Hydroxypropanal Ps Benzene Lg, Pr, Ps Pyridine Pr N-Methylpyrrole Pr Cyclopentenone Methylfuran Ps Cyclohexene Methylcyclopentene Furanone Ps Pentenone Methylbutenone Hexene Butandione Ps Hydroxybutanal Toluene Lg, Pr Aniline Pr Phenol Lg, Pr Methycyclohexadien Dimethylpyrrole Pr Furaldehyde Ps Dimethylfuran Ps Methylhexadiene Hydroxymethylfuran Ps Methylfuranone Ps Methylpentenone Heptene Pentanedione Ps Hydroxyketobutanal Ps Benzonitrile Styrene Benzaldehyde Ethylbenzene Lg Xylene Benzyl alcohol Lg Cresol Pr Formylmethylpyrrole Dihydroxybenzene Pp Methylfuraldehyde Ps Ethylmethylfuran Ps Propylfuran Ps Trimethylfuran Octadiene Hydroxylmethycyclopentenone Ps Octene Hydroxydihydropyranone Ps lndene Benzyl cyanide Pr lndole Methylstyrene Hydroxybenzonitrile lndoline Acetophenone Lg Tolualdehyde Vinylphenol Lg Ethylmethylbenzene Lg Hydroxybenzaldehyde Lg Ethylphenol Pp Methoxymethylbenzene C3-Alkylpyrazine Guaiacol Lg

Origin

[Continued--

136

R. HEMPFLINGand H.-R. SCHULTEN Table 3--continued] Accurate mass

Proposed elemental composition

126.0317

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128.0473 129.0591 130.0790 131.0736 132.0605

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Major El-signals (m/z 50-500; > 25% base peak) 98, 68, 53, 96 126, 55 97, 126, 69 128 129, 102, 128, 76, 51 130, 115, 129 130, 131 132, 104, 103 131, 132, 51, 77 117, 132 105, 77, 134, 51 134, 119, 91 134, 133 119, 134, 91, 105 121, 136 138, 123, 95 140, 125, 97 142, 141, 115 69 146, 131, 145 131, 146, 117, 77 147, 77, 146 77, 105, 131, 91 107, 150 150, 135 151, 152 137, 152 154, 139, 93 164, 149 151, 166 137, 166 168, 153 178 137, 180, 162 137, 180 165, 180 137, 182 182, 181 167, 182 186 192, 177 194 181, 196 181, 210, 85, 93 181 121, 214, 51, 77 149 60, 73, 57, 129, 228 73, 60, 57, 129, 256 57, 73, 60, 129, 340 73, 60, 57, 129 394, 275, 253 396, 255, 213 398, 215, 344 174, 410 124, 229, 412 414, 396, 213, 303 95, 69, 125 165, 164, 430

Assignment, proposed structure Levoglucosenone Hydroxymethylpyranone Hydroxymethylfuraldehyde Hydroxymethyldihydropyranone Isoquinoline Methylindene Methylindole Indanone Methylbenzofuran Ethylstyrene Benzofuranone Methyldihydrobenzofuran Phenylpropene C4-Alkylbenzene C3-Alkylphenol Guaiacylmethane Hydroxyguaiacol Methylnaphthalene Dianhydrohexose C2-Alkylbenzofuran Methylindanone Methoxybenzylcyanide Methoxyphenylpropene p-Coumaryl alcohol Guaiacylethene Vanillin Guaiacylethane Syringol Eugenol Acetovanillone Guaiacylpropane Methylsyringol Coniferyl aldehyde Coniferyl alcohol Guaiacylpropan-2-one Syringylethene Guaiacylacetic acid Syringaldehyde Syringylethane Biphenol Syringyl isomer Syringylpropene Syringylethenone Sinapyl alcohol Syringylpropanealdehyde Phenoxybenzoic acid Diethylphthalate Myristic acid Palmitic acid Behenic acid Lignoceric acid Ethylcholestatriene Ethylcholestadiene Ethylcholestene Stigmastadienone Stigmastenone fl-Sitosterol D:A-Friedooleanan-3-one :t-Tocopherol

Origin Ps Ps Ps Ps Pr

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Abbret~iations: Lg = lignin; Lp = lipid; Pp = polyphenol; Pr = protein; Ps = polysaccharides.

signal intensities of coniferyl alcohol (m/z 180) and sinapyl alcohol (m/z 210) for the angiosperm-litter derived humus samples. This ratio is much lower for the mull sample, indicating a higher proportion of syringyl subunits as lignin building blocks there. This may be one reason for the rapid decomposition process occuring in the mull profile. Distinct qualitative and quantitative differences are obvious in the mass range m/z > 300 (high-molecular weight pyrolysis products). These signals relate to dimeric pyrolysis

products from lignin and noncarbohydrate aliphatic structures. Their chemical characterization constitutes a major objective in this study. For the mull, moder, and mor samples, elemental compositions could be assigned for a number of nominal mass signals within the mass range m/z 50 to 502. Figure 3 shows examples of high resolution data for the mass range m/z 100-117. Accurate masses and proposed elemental compositions for these signals are listed in Table 2. Already this small

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section of the high resolution data demonstrates that several nominal mass signals are occupied by various elemental compositions and thus by different chemical structures. Moreover, the pyrolysis products identified by GC/MS demonstrate that in addition to isomers, one elemental composition may represent different compounds (Table 3). The data shown in Table 3 confirm that a significant number of similar pyrolysis products are generated by Curie point and micro-oven pyrolysis although different pyrolysis conditions are used.

Assignment of High-Molecular Weight Pyrolysis Products Recorded by FIMS Lignin Among the major components of primary resources it is difficult to find a relationship between pyrolysis products identified and the mechanisms of thermal degradation for lignin. The reasons are the complex structure of the lignin macromolecule and the lack of adequate methods to separate lignin from other plant constituents without chemical changes.

138

R. HEMPFLINGand H.-R. SCHULTEN

compounds, and lignin preparations, in combination with high resolution measurments. Evans et al. (1986) have proposed three plausible structures for a parent 20ion at m / z 272 with the formula Cl6Hl604 that 10, incorporate the fl-O-4-alkyl-arylether bond, phenyl/ coumaran and diarylpropane linkage. Knowing that the fl-arylether linkage is broken at lower tem., i,J .,,1 Li .... L~ . . . . l l J . , , . . ,L i peratures than C-C linkages, we suppose pyrolysis 10~:6 ...... ..... io~:6 ..... i0:,:6 ..... 10816..... i6'1.6..... io~:6 i~ products derived from phenylcoumaran- and diaryl~ 1 O0 propane-type subunits to be the most likely for 60] thermally stable lignin building blocks of this group ~ 20(Table 4). Other lignin subunits that are known to be lOrelatively thermally stable are biphenyl- and resinoltype structures. These four types of subunits are the 2 " skeletons for structural proposals of dimeric pyrolysis products from lignin that are summarized in Table 4. 106.8 107.8 108.8 109.8 110.8 >. 105,8 The substituents proposed for these thermally stable co lignin skeletons correspond to the substitution pat~ 100: ~ 60tern of monomeric pyrolysis products from lignin identified by GC/MS. 20In addition, some elemental compositions indicate 10~ dimeric pyrolysis products from lignin that have been 6.thermally modified by loss of hydroxyl, methoxyl, and methyl groups, but the intensities of mass signals 1 ..... ,, ~ loik ILl, Ii, d i l i,dlL t i L ~ l ,i.I related to thermally modified lignin subunits are 114.7 115.7 116.7 111.7 112.7 113.7 > mjz small compared to intensities of the pyrolysis prodFig. 3. High resolution data for the mass range m/z 100 to ucts listed in Table 4. Modified dimeric pyrolysis m/z 117 after pyrolysis-field ionization of the mor sample products from lignin assigned by H R - F I M S cover with photographic detection. the mass range rn/z 154 to 288 (Hempfling, 1988). Besides data from high resolution MS, and investigations of synthetic lignins and lignin extracts Therefore, until now mainly monomeric pyrolysis (Haider and Schulten, 1985), the assignment of products from lignin have been discussed (Obst, 1983; dimeric lignin subunits can also be confirmed by Saiz-Jimenez and de Leeuw, 1984). Some of these Py-FI mass spectra of wood samples. This is shown lignin subunits identified by Py-GC/MS of the for beech and spruce wood in Fig. 4. The wood of humus samples have been listed already in Table 3. beech and spruce trees consists of 25-30% lignin. The According to Obst (1983) and Saiz-Jimenez and de relations between the monomeric building blocks Leeuw (1984) monomeric subunits from lignin are coniferyl, sinapyl, and p-coumaryl alcohol are 94: 1: 5 released during Curie point pyrolysis by the break- for spruce and 56:40:4 for beech lignin. These subdown of C-O-C and C-C linkages within the lignin units are bound in the macromolecular structure of skeleton. At a heating rate of 12°C min-x the thermal lignin by arylether (spruce: 54-56%, beech: 65%), cleavage of alkyl-arylether linkages occurs between diarylpropane (7%, 15%), biphenyl (9.5-11%, 3%), 150 and 320°C (Domburg et al., 1974). This depends phenylcoumaran (9-12%, 6%), diphenylether on the kind and position of functionalities on the (3.5-4%, 1.5%), and resinol (2%, 5%) linkages aromatic ring and the propane chain. Biphenyl-, (Fengel and Wegener, 1984). All nominal mass sigphenylcoumaran-, and pinoresinol-type subunits are nals listed in Table 4 yield intensities between 0.1 and more stable and the thermal cleavage of links within 4.5% TII in the Py-FI mass spectra from spruce and these subunits occurs between 325 and 400°C beech wood. The softwood lignin can be clearly (Nguyen et al., 1981). Based on the binding energies distinguished from hardwood lignin by the intensities Evans et al. (t986) consider alkyl-arylether linkages of monomeric and dimeric syringyl units (m/z 154, in the lignin macromolecule to be most important in 168, 194, 196, 208, 210, 332, 386, 388, 418). These determining the major primary pyrolysis reactions. In mass signals should be the most suitable ones to this way also dimeric pyrolysis products of lignin are distinguish angiosperm- and gymnosperm-derived generated. Some dimeric chemical degradation prod- humus layers on the basis of different structural ucts of lignin have been identified by Mycke and subunits of lignin. The intense mass signals at m / z Michaelis (1986). However, so far no GC/MS data 420, 450, 476, and 504 in the spectrum from spruce for authentic dimeric pyrolysis products from lignin wood can be plausibly assigned to trimeric lignin have been reported in the literature. Structural as- subunits by theoretical calculations, although this has signments are based on the identified chemical degra- not yet been confirmed by high resolution mass dation products and thermal investigations of model spectrometry. 100-

60-

L.., I ,.i.,,,.......,t,,,d.

Table 4. Assignment of dimeric pyrolysis products from lignin with phenylcoumaran (a)-, biphenyl (b)-, diarylpropane (c)-, or resinol (d)-ty~ structures Proposed elemental Skeleton m/z composition R~ R2 (a) R1 C . ~ H

OCH~

270 284 296 298 300

Ct6H 1404 CI7H,604 C~s H ~60~ CisH~sO,~ CnHI605

-H --CH3 --CH=CH2 42H~-CH~ --CH2OH

310 312 314

CI9HI80,I C~8H ~6O5 C~sH~sO5

326

C~oH~sO~

-CH---CH-CH 3 --CH--CHOH -CH2~H2OH --CH3 -CH--CH~H2OH

-H -H -OCH 3 -H

330 340 342

C~sH~sO6 C:~oH2oO~ C~gHI~O6

-CH--CH2 ~HzOH ~H--CH-CH~ ~H--CHOH

-OCH3 ~CH~ ~:)CH~ -OCH3

356

C~0H2006

-CH--CH-CHzOH

-OCH 3

272 286 298 300 302

CI6HI604 CI7H,804 C is H is04 CI8H2oO4 CI7HI80s

-H

-H

~H 3 -CH--CH2 -CH--CH 3 --CH2OH3

312 314 316

C~9H2oO4 CisHIsO 5 CI8H2oO5

-H -H -H -H -OCH 3 -H -H

-H

C

O

R 2 ~ .

OCH~ OH

I~1 C

~

H2 F

OCHs

HC

'O

~

\

OCH3

328

C,9H2005

OH

-CH--CH~H 3 --CH=CHOH -CH2~H2OH -CH; -CH=CH--CH2 OH

~H--CH~ 332 342 346 358

Skeleton (b)

-H

R

R

OH

OH

CH30

CIsH2006 C2oH 22O5 CI9H2206 C20H2206

~H2OH -CH=CH-CH~ 42H2--CH2OH

-CH--CH-CH2 OH

m/z

Proposed elemental composition

246 260 272 274

CI4HI404 CtsHI604 Ci6Hi604 CI6HIsO 4

286

ClTHtsO4

300

CisH2oO4

302

CI7HIsO 5

312 316 328

Ci9H2oO4 CisH2005 CI9H2005

-H (2) ~[?H3, -H ~H---CH z, -H ~2H 3, ~CH 3 ~ H 2 - C H 3, -H --CH--CH-CH 3, -H -CH=CH2, --CH3 -CH--CH-CH3, 42H 3 -CH--CH2, -CHz-CH 3 42H--CH~H2OH, -H 42H---CH2, -CH2OH --CH---CH~H~, ---CH---CH2 -CH--CH42H2 OH , --CH3 -CH=CH-CH2 OH, 42H=CH2

272 302 332

CI6HI604 CI7 HI805 CI8H2006

-H (2) 4Z)CH3, -H -OCH 3 (2)

298 328 358 418

CisHisO4 CI9H2005 C2oH2206 C22H260~

-H (4)

OCH3

R°

OCH3

(c)

HC

~R-OH

HC

R~OCH3 OH (d) OH

HC-----CH I

HC

I

CHa

OH "Number of substituents in brackets (). o~ Is~,--a

139

4)CH 3, -H (3) --OCH3 (2), -H (2) -OCH 3 (4)

-H

-H -H -H -H -OCH 3

-H

-H -OCH 3

-H ~Z)CH3 -OCH 3 --OCH 3 -OCH 3 -OCH3

140

R. HEMPFLINGand H.-R. SCXUt,TEN

o)

100 A

80 60 ,,,

40 I 6

85

.jo]

~8

210

332

20

c

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O 50 1 0 0 e0

2oo

3go

550

," 650

.....j 800

b) 18o

E X~

60

< -2

40

~

20

12e 144 114

I 226

2;'2 300 /

85 504

420 450 475

• 0 5O

,,,l .......

200

~ .... J,t~i.~.,

3go m

/z

s6o .

.

.

.

.

6~o

86o

>

Fig. 4. Pyrolysis-fieldionization mass spectra of beech (a) and spruce (b) wood.

Free and bound lipids The straightforward assignment of noncarbohydrate aliphatic compounds to proposed elemental compositions and nominal mass signals by Py-GC/ MS investigation was possible only for some signals. With increasing mass number the proportion of these signals decreases. Therefore, besides extrapolation of the Py-GC/MS results other approaches have to be used to investigate the chemical background of proposed elemental compositions in the highmolecular weight range. These approaches are (1) time-/temperature-resolved Py-FIMS that allows the examination of single ion thermograms and single and multiple spectra; (2) comparison of the FI fragmentation pattern with Py-FIMS investigations of reference substances; (3) comparison with Py-FIMS investigations of lipid extracts; (4) comparison with wet-chemical investigations of lipid extracts and products of saponification; and (5) comparison with GC/MS studies of plant waxes using high-temperature columns. Tentative assignments of noncarbohydrate aliphatic compounds based on these possibilities are presented in the following section. Fatty acids. Several fatty acids such as lauric (m/z 200), myristic (m/z 228), palmitic (m/z 256), eicosanoic (m/z 312), behenic (m/z 340), and lignoceric (mz 368) acids have been identified by Py-GC/MS. Elemental compositions calculated from high resolution data, such as C22H440:, C24H4802, C26H5202, C2sH5602, and C30H6002, indicate that this series of n-fatty acids is continued in the higher mass range with a predominance of even carbon-numbered species. The higher mass range recorded by direct Py-MS may be due to the suppression of long chain fatty acids in GC-studies. Besides carboxylic acids, series of monoesters, of monounsaturated diols, or of hydroxyketones would also be reasonable structures for these elemental compositions. Time-/tern-

perature-resolved Py-FIMS offers the possibility of selecting the correct solution from these various possibilities. The single spectrum in Fig. 5 demonstrates that the high-molecular weight compounds related to this series of FI-signals (m/z 340-452) are volatilized almost at the same pyrolysis temperature around 180°C and furthermore, no mass spectrometric fragmentation can be detected. This pattern is typical only for carboxylic acids (Schulten et al., 1987b). For alcohols, a loss of water is typically observed, monoesters are characterized by splitting and protonation of the fatty acid subunits, and for branched carboxylic acids, side-chain splitting is expected. For these reasons, it is assumed that the proposed elemental compositions relate to a homologous series of n-fatty acids. Wax esters. The typical fragmentation pattern of wax esters offers the possibility of identifying a homologous series of compounds volatilized in the mass range m/z 592-760 without knowledge of elemental composition. Figure 6(a) clearly demonstrates that these humus constituents are characterized by similar thermal behavior with a small increase in volatilization temperature from 190 to 230°C with increasing molecular weight. The summed FIspectrum of scans 19 and 20 from the moder sample [Fig. 6(b)] shows the typical fragmentation pattern of these alkyl-monoesters that range from C40 to C52. The splitting of esterified C22-C28 n-fatty acids that are recorded as protonated species (m/z 341,369, 397, 425) indicates the chain length of the fatty acid subunits of these monoesters. In addition, the calculation of the chain length of the esterified alcohols with C~2-C30 becomes possible. Besides wax esters some sterols such as ergosterol (m/z 396) and fl-sitosterol (m/z 414) are volatilized at temperatures around 200°C. A similar homologous series of monoesters from plant waxes has been identified by GC/MS studies

Organic matter in forest soils

141 424

1O0 A 396

80

~- 60

E

~E 4o <

368 a2

20 452 56 24o

0 50

II t,

,lit

, 500

260 -

m/z

-

>

Fig. 5. Pyrolysis-field ionization mass spectrum of scan 16 (about 180°C) of the moder sample.

O)

1500.

4000A

I I I I

5ooo.

I

3000.

c

5000.

c

--:' 3000. CC 1500.

O"

5O

100

150

200

300

250

Temperature

b)

-

350

500

-

414

100.

A

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6 80.

<.25 676

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444

40. 85

..0

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20.

ol

50

,I

.,,., l I 260

lIT'

tJ

m/z

l

704 620

760

592 1 500

732

650

860

>

Fig. 6. (a) Single ion thermograms of m/z 592, 620, 648, 676, 704, 732, and 760 of the moder sample• (b) Summed pyrolysis-field ionization mass spectrum of the scans 19 and 20 (around 200°C) of the moder sample•

142

R. HEMPFLINGand H.-R.

using high-temperature columns (Dielmann et al., 1979) and supercritical fluid chromatography/mass spectrometry (Hawthorne and Miller, 1987). However, the chain lengths of the esterified alcohols and fatty acids reported differ in the various studies; this may be due to source-specificity of these ester structures. Sterols. Various phytosterols (e.g. fl-sitosterol) have been identified by Py-GC/MS of the humus samples. These sterols are abundant in non-humified plant remains and the intensities of related signals decrease in the spectra of soil horizons that represent more advanced stages of litter decomposition and humification (Hempfling eta/., 1988; K6gel et al., 1988). Therefore, it is assumed that some of the unsaturated ethylcholestanes identified by Py-GC/MS, or assigned on the basis of high resolution measurements (C29H42, C29H44) or both, may represent the dehydration products of phytosterols. This would explain the various double bonds of assigned ethylcholestanes. A possible explanation for the loss of the hydroxyl group is an ester or ether linkage. It can be shown by time-/temperature-resolved Py-FIMS that unsaturated ethylcholestanes are volatilized at temperatures higher than 300°C. The splitting of proposed linkages combined with the loss of the OH-group during pyrolysis therefore may be the reason for the thermal release of unsaturated ethylcholestanes by pyrolysis. Aromatic esters. Another homologous series of soil constituents with the elemental composition CnH2n.loO 4 (r/= 28-34) is characterized by the mass signals at m/z 432, 446, 460, 474, 488, 502, 516, and 530. Intense signals of this series are visible especially in Py-FI mass spectra of humus horizons where bark or root material has accumulated, or in the spectra of twig and root material (Hempfling, 1988; Hempfling et al., 1988). This fact, and the proposed elemental compositions suggest that the signals arise from phenolic ester structures, with an increasing chain length of the alkyl chain, released from suberin (Hempfling et al., 1988). Several authors have reported on the products of saponification of barks and roots, including a phenolic fraction and long chain alcohols, fatty acids, (o-hydroxy acids and dicarboxylic acids (e.g. Kolattukudy, 1980). It seems likely that the homologous series represents aromatic esters of these products of chemical depolymerization. The mass spectrometric assignment of highmolecular weight aliphatic structures discussed herein can be confirmed by the Py-FI mass spectra of chloroform/methanol extracts from mull, moder, and mor (Fig. 7). All three spectra are similar to spectra of the related bulk samples integrated for the temperature range 50-250°C. The signals assigned to fl-sitosterol (m/z 414), n-fatty acids (m/z 340-452) and aromatic esters (m/z 432-530) have significant abundances whereas those for alkanes and alkenes [m/z 58 (56)-618 (616)] are minor. Molecular ions of linolenic acid, linoleic acid, and oleic acids (m/z 278,

SCHULTEN 414

60

0) 4O i

T

28

458

20-

~7

312

C, iJll . . 50

704

J, . . .,..,,o , , ~ r . ' " ~. J,I , 200

.

.

• 500

350

650

800

41 4,

N

~)

40-

o

C

311 426

"

i~llilJ o ~J!~IILLJ:I,..,,,~,!,-L, . .L!.

o ~o20-

•

85 leO I 127 I 210

50

34O I3

280

200

350

414 ,

13 60-

<

• ;1

6148 704 , . .. 650

.

500

. 800

454 ,

c)

"O40-

3gd 20-

297 3403(18i

157

I .LJ,. o ,J:~, . . . . .......... :. 350 •

50

.

,

•

.

200

m/z

,

.

•

502

L,. I .

•

,

650

50O

•

.

,

800

>

Fig. 7. Pyrolysis-field ionization mass spectra of the chloroform/methanol extracts from mull (a), moder (b), and more (c).

280, 282) and the homologous series of wax esters (m/z 592-732) are more intense in the spectra of extracts from mull and moder, whereas signals related to 10-nonacosanol (m/z 157, 297, 406, 424) and to epoxy-D:A-friedooleanandione (mz 454) are only abundant in the spectrum of the mor extract. Particularly 10-nonacosanol is known as a typical constituent of the epicuticular wax layer of conifers and epoxy-D: A-friedooleanandione as well as D: Afriedooleanan-3-one represent lipid constituents of suberin. Therefore, the intense signal at m/z 454 in the mor spectrum indicates a high contribution of spruce bark and root material to the humus layer. As unsaturated ethylcholestanes exist in bound form in the samples, no signals related to these humus subunits were found in the spectra of the extracts. The single ion thermograms of sterols, fatty acids, aromatic esters, and wax esters allow differentiation between free (extractable) and chemically bound molecules (Hempfling et al., 1988). The signals representing n-fatty acids and aromatic esters show two maxima characteristic of free and bound molecules, whereas the sterols and wax esters recorded as intact molecules exist in the free form. A probable origin of the bound fatty acids is the fatty acid component of the wax esters because the fatty acid subunits of wax esters and the bound fatty acids in the samples are characterized by similar chain lengths. Also fatty

Organic matter in forest soils acids linked by ester groups to polysaccharide or lignin structures (Bridson, 1985) are possible. In the mor sample, where wax esters are less abundant, bound fatty acids contribute only to a minor extent to the related mass signals. Pyrolytic release of fatty acids from esterified polymers has been described recently by Tegelaar et al. (1989). In addition, the extraction of bound fatty acids as well as unsaturated ethylcholestanes from whole soils and humic acids has been demonstrated using supercritical pentane (Schnitzer and Schulten, 1989; Schulten and Schnitzer, 1989). Besides fatty acids and esters, the comparison of the three forest humus layers (Fig. 2) indicated no significant differences for sterols and ethylcholestanes but did so for aromatic esters. Based on morphological profile descriptions the contribution of suberin-containing plant material such as barks and roots to the humus layer increases from mull to moder and mor. This is clearly reflected by the intensities of signals related to suberin lipids and aromatic esters from suberin. Thus, the correspondence of morphological description and chemical data further confirms the correct assignment of the related mass signals.

Comparison of the Molecular Range of Pyrolysis Products Recorded by Curie Point Py-GC/MS and Py-FIMS Results from pyrolysis are influenced by the pyrolysis conditions such as the design of the pyrolysis device, heating rate, and type of heating. In addition to different pyrolysis products generated during Py-GC/MS some pyrolysis products may condense in the injector or be too polar to pass into the column. For lignin, these facts possibly explain why no dimers have been reported in Py-GC/MS studies. Nevertheless, a comparison of pyrolysis products detected by Py-GC/MS and Py-FIMS is

,oo] -q

143

shown in Fig. 8. The number of pyrolysis products identified by GC/MS, as a percentage of the number of elemental compositions proposed from HR-FIMS, clearly decreases with increasing mass number. In the mass range m/z 50-150 between 38 and 78% of the elemental compositions can be assigned by the Py-GC/MS data. However, in the mass range above m/z 150 this proportion decreases to less than 25%. For mass signals above m/z 300 only isolated mass signals from non-polysaccharide aliphatic structures can be assigned by Py-GC/MS. This clearly demonstrates the advantage of Py-FIMS for the characterization of biomaterials if highmolecular weight pyrolysis products are of special interest. C

O

N

C

L

U

S

I

O

N

S

The characterization of organic constituents from different forest humus layers by the combination of Curie point Py-GC/MS and Py-FIMS has provided further fundamental information to extend current knowledge, on a molecular basis, of the chemical composition of forest humus. The advantage of Py-FIMS especially for the characterization of highmolecular weight pyrolysis products of biomaterials has become obvious by the comparison of both techniques. This may be due to the loss of pyrolysis products that are not amenable to GC and to different pyrolysis products generated by flash pyrolysis and time-/temperature programmed pyrolysis. Differences observed among the mull, moder and mor samples by Py-MS are mainly due to differing subunits of the parent plant material. This indicates that properties of plant litter, e.g. resource quality, influence to a great extent the chemical conditions of forest soils and, furthermore, that humification processes occurring in these profiles mainly constitute a

__

60

o

20

50

~00

I

150

m/z

.

200

250

•

.

,

.

300

>

Fig. 8. Pyrolysis products identified by GC/MS as a percentage of the number of elemental compositions proposed from HR-FIMS.

144

R. HEMPFLINGand H.-R. SCHULTEN

Evans R. J., Milne T. A. and Soltys M. N. (1986) Direct mass-spectrometric studies of the pyrolysis of carbonaceous fuels. III. Primary pyrolysis of lignin. J. Anal. Appl. Pyrolysis 9, 207-236. Fengel D. and Wegener G. (1984) Wood: Chemistry, Ultrastructure, Reactions. De Gruyter Berlin. Haider K. and Schulten H.-R. (1985) Pyrolysis-field ionization mass spectrometry of lignins, soil humic compounds and whole soil. J. AnaL Appl. Pyrolysis 8, 317-331. Hatcher P. G. (1988) Dipolar-dephazing ~3C NMR studies of decomposed wood and coalified xylem tissue: evidence for chemical structural changes associated with defunctionalization of lignin structural units during coalification. Energy & Fuels 2, 48-58. Hatcher P. G. and Spiker E. C. (1988) Selective degradation of plant biomolecules. In Humic Substances and their Role in the Environment (Edited by Frimmel F. H. and Christman R. F.), pp. 59-74. Wiley, Chichester. Hatcher P. G. and Lerch H. E. (1989) Molecular evidence for the presence of the lignin structural units in coalified tissue from Cretaceous lignites. Geochim. Cosmochim. Acta. In press. Hawthorne S. B. and Miller J. D. (1987) Analysis of commercial waxes using capillary supercritical fluid chromatography-mass spectrometry. J. Chromatogr. 388, 397-409. Hempfling R. (1988) Charakterisierung verschiedener Waldhumusformen und ihrer Dynamik durch analytische Pyrolyseverfahren. Bayreuther Bodenkundl. Ber. 6, 1-126. Hempfling R. and Schulten H.-R. (1988) Characterization and dynamics of organic compounds in forest humus studied by pyrolysis-gaschromatography/electron impact mass spectrometry and pyrolysis-(high resolution) field ionization mass spectrometry. J. Anal. Appl. Pyrolysis 13, 319-325. Hempfling R. and Schulten H.-R. (1989) Selective preservation of biomolecules during humification of forest litter studied by pyrolysis-field ionization mass spectrometry. Sci. Total Environ. 81/82, 31-40. Hempfling R., Zech W. and Schulten H.-R. (1988) Chemical composition of the organic matter in forest soils. 2. Moder profile. Soil Sci. 146, 262-276. Kaaden van der A., Boon J. J., Leeuw de J. W., Lange de F., Schuyl P. J. W., Schulten H.-R. and Bahr U. (1984) Comparison of analytical pyrolysis techniques in the characterization of chitin. Anal. Chem. 56, 2160-2164. Krgel I., Hempfling R., Zech W., Hatcher P. G. and Schulten H.-R. (1988) Chemical composition of the organic matter in forest soils. 1. Forest litter. Soil Sci. 146, 124-136. Acknowledgements--The authors thank the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, for Krgel-Knabner I., Zech W. and Hatcher P. G. (1988) Chemical composition of the organic matter in forest financial support (Schu 416/3-1, SFB 137) and Professor soils 3. Humus profile. Z. Pflanzenerniihr. Bodenk. 151, W. Zech, Bayreuth, for his encouragement. 331-340. Kolattukudy P. E. (1980) Biopolyester membranes of plants: cutin and suberin. Science 208, 990-1000. REFERENCES Mycke B. and Michaelis W. (1986) Molecular fossils from Blazso M. and Schulten H.-R. (1990) Pyrolysis-field ionizchemical degradation of macromolecular organic matter. ation mass spectrometry of low rank coals. Org. Geochem. Org. Geochem. 10, 847-858. 15(1), 87-99. Nguyen T., Zavarin E. and Barrall E. M. (1981) Thermal Bridson J. N. (1985) Lipid fractions in forest litter: early analysis of lignicellulosic material. Part I. Unmodified stages of decomposition. Soil Biol. Biochem. 17, 285-290. material. J. Macromol. Sci.-Rev. Macromol. Chem. C20, Beckey H. D. (1977) Field ionization mass spectrometry. In 1-65. International Series in Analytical Chemistry (Edited by Nip M., Leeuw de J. W. and Schenck P. A. (1988) The Belcher R. and Frieser H.), Vol 61. Pergamon Press, characterization of eight maceral concentrates by means Oxford. of Curie point pyrolysis-gas chromatography and Curie Dielmann C. S., Meier S. and Rapp U. (1979) Highpoint pyrolysis-gas chromatography/mass spectrometry. temperature (GC)2/MS investigations. HRC&CC 2, Geochim. Cosmochim. Acta 52, 637-648. 343-350. Obst J. R. (1983) Analytical pyrolysis of hardwood and Domburg G., Rossinskaya G. and Sergeeva V. (1974) Study softwood lignins and its use in lignin-type determination on thermal stability of fl-ether bonds in lignin and its of hardwood vessel elements. J. Wood Chem. Technol. 3, models. Therm. Anal. 2, 211-220. 377-397.

mechanical disintegration of plant remains, rather than their disppearance. Important effects on soil fertility are expected from the great number of hydrophobic substances in forest humus that occur in the free and in the bound form. These compounds may stabilize aggregates in soils and increase water repellency. Soil lipids enclose the surface of mineral soil constituents thus suppressing the mineralization of nutrients. The compounds identified in the forest humus layers offer a wide variety of potential biomarkers from plant material that can be selectively preserved. The suitability of using dimeric pyrolysis products from lignin to discriminate angiosperm from gymnosperm-derived plant input that is better established by the P y - F I M S data is of special interest for geomaterials. As shown by Hempfling et al. (1988) the breakdown of lignin in forest humus layers mainly reduces the relative amount of monomeric pyrolysis products representing intact lignin but not that of the dimers. These "condense" subunits in lignin therefore seem to become important biomarkers. Also the distribution pattern of the homologous series of n-fatty acids with a maximum in the C22-C32 range and a strong even/odd predominance indicates an origin from higher plants. These compounds have been shown to occur in free and bound form in low rank coals of Hungary (Blazso and Schulten, 1990). The similar distribution pattern reported, demonstrates the suitability of these structures as biomarkers for original plant constituents in coal. In addition, the recalcitrance of these subunits during geochemical processes becomes obvious for free as well as for bound forms. The assignment of signals related to suberin offers the possibility of estimating the contribution of bark and root material to geomaterials. In addition, the assignment of aromatic esters that represent both aromatic and aliphatic subunits of suberin allows one to differentiate between the plant polyesters suberin and cutin.

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