Structure elucidation of soil macromolecular lipids by
preparative pyrolysis and thermochemolysis
V. GobeÂ, L. LemeÂe, A. AmbleÁs *
Laboratoire de SyntheÁse et ReÂactivite des Substances Naturelles, UMR 6514, Faculte des Sciences, 40 Avenue du Recteur-Pineau, 86022 Poitiers, France
Abstract
The structure of macromolecular lipids from an acid anmoor soil was investigated using preparative pyrolysis and thermochemolysis. The matrix is formed by cross-linked aliphatic chains. Alkanols, triterpenoid alcohols e.g.a-amyrin, sterols, stanols can be bound to the matrix via ether or ester groups. Fatty acids can be incorporated through ester-i®cation. Dicarboxylic acids and other compounds such as hydroxyacids represent alkyl bridges between the poly-methylene chains. Various components are trapped in the macromolecular network. The two techniques give complementary results. Despite higher yields, thermochemolysis does not permit one to distinguish between methyl ester groups present in the structure and methyl ester groups formed on degradation.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Macromolecular lipids; Pyrolysis; Thermochemolysis; Hydrocarbons; Fatty acids; Alcohols; Aldehydes; Ketones
1. Introduction
Lipids play an important role in soil processes (Ste-venson, 1982) because they aect plants or soil micro¯ora and in¯uence soil physical properties (Jambu et al., 1983). Previous studies concerning lipids present in soils have shown that this soluble fraction includes simple lipids with a plant or a microbial origin but also complex, macromolecular lipids, corresponding to the polar lipid fraction when using the McCarthy and Duthie (1962) procedure for lipid fractionation. This latter fraction has been studied using chemical degradation methods such as alkaline hydrolysis under phase catalysis conditions, ether bond cleavage with boron tribromide or alkaline potassium permanganate oxidation (AmbleÁs et al. 1991, 1993a). It was shown that soil macromolecular lipids contains preserved biomacromolecules of plant or microbial origin (AmbleÁs et al., 1991). The (rever-sible) incorporation of exogenic organic molecules in
macromolecular lipid fraction, for instance by ester groups, has then been clearly demonstrated (AmbleÁs et al., 1994).
In hydromorphous environments, these macro-molecular lipids can partly escape biodegradation (AmbleÁs et al., 1991) and become part of the sedimen-tary organic matter. As a consequence, some analogy between these soil macromolecular lipids and the organic matter present in ancient sediments may exist and macromolecular lipids can be compared with a proto-kerogen in some soils (Shioya and Ishiwatari, 1983; AmbleÁs et al., 1991).
In this paper, the structure of macromolecular lipids from an acidic anmoor soil was investigated using pre-parative o-line pyrolysis (Behar and Pelet, 1985; Lar-geau et al., 1986; Metzger and LarLar-geau, 1994) and a new preparative thermochemolysis technique which permits the treatment of more than one gram of product (Grasset and AmbleÁs, 1998b). In the latter technique, an alkylating agent is used for the thermally assisted hydrolysis and alkylation, as developed for analytical ¯ash on-line pyrolysis (del Rio et al., 1996). The soil samples originated from a peaty soil (Plateau de Mill-evaches, France) where organic matter is accumulating and as such the structural study of macromolecular
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* Corresponding author. Tel.: 5-4945-3866; fax: +33-5-4945-3501.
lipids from these soils may give information about early diagenetic processes.
2. Experimental
2.1. Samples
Samples were collected from a peaty soil (Sphagnum peat), an acidic anmoor located on the Plateau de Mill-evaches (North CorreÁze). The macromolecular lipids (ML) from the surface 0±20 cm horizon were previously described by AmbleÁs et al. (1991; 1993a). The samples used for the present work originated from the 30±50 cm horizon which is waterlogged, organic-rich, with a higher degree of decomposition of plant debris (saprist) than the surface horizon (®brist).
2.2. Separation of the macromolecular lipid fraction
The procedure is shown in Fig. 1. Representative soil samples were dried and sieved to 2 mm. Total lipids were extracted with chloroform in a Soxhlet (324 h).
The original soil sample contained 56% of total organic
matter (TOM) (determined by combustion). Lipids con-tributed 5% to the TOM. The total lipid extract was fractionated on a silica-potassium hydroxide column. In order to avoid problems of separation between the acid and the polar fractions in the classical McCarthy±Duthie procedure (AmbleÁs et al.,1991) on the one hand, and to release lipids possibly trapped by the macromolecular network (such as HCT, Fig. 1) (AmbleÁs et al., 1991) on the other hand, the procedure of separation was slightly modi®ed: the ``classical'' acid and the polar fractions were mixed and then methylated using trimethylsi-lyldiazomethane (Hashimoto et al., 1981). The most polar fraction (corresponding to the polar fraction of the McCarthy±Duthie procedure) obtained after liquid chromatography (MLA) contains macromolecular lipids. Other macromolecular lipids (MLN) were obtained by liquid chromatography of the neutral frac-tion (Fig. 1). The total macromolecular lipid fracfrac-tion (ML) was obtained after addition of MLA and MLN. It gave no elution in thin layer chromatography using polar eluents. The other fractions (in italics in Fig. 1) were not studied in this work.
Macromolecular lipids contributed 15% to the total lipid extract (0.7% of TOM). The elemental composi-tion of the ML is : C 72.39%, H 10.16%, O 16.96% (by dierence), N 0.27%. The value of the atomic H/C ratio is 1.68.
2.3. Preparative o-line pyrolysis
The same device as that described by Behar and Pelet (1985) was used. A quartz tube containing the complex lipids (60±70 mg) was placed in a stainless steel tube and put in an oven, at 425C for 1 h under helium (¯ow rate
30 ml minÿ1). Pyrolysis products were collected in two
successive traps containing solvent cooled toÿ30C. After
evaporation of the solvent, the pyrolysate from seven experiments was separated on a SiO2/KOH column
(McCarthy and Duthie, 1962) in a neutral fraction and an acid + polar fraction. The later was methylated. Each fraction was chromatographed on a silica column, the various components were eluted with diethyl ether/pet-roleum ether mixtures of increasing polarity (Fig. 1). Three chromatographic fractions were obtained for hydrocarbons: HC I:12 mg, HC II:1 mg; HC III:1.5 mg.
2.4. Preparative o-line thermochemolysis
ML (349 mg) was placed in a ceramic boat and moistened with 3 ml of a methanol 50% w/w solution of tetramethylammonium hydroxide (TMAH) (Aldrich). The ceramic boat was then transferred into a 603 cm
i.d. Pyrex1 tube. The tube was maintained at 425C
(oven) for 1 h under helium (¯ow rate 100 ml minÿ1). The pyrolysate was collected in two successive traps, as described above. The pyrolysis products were directly Fig. 1. Procedure of fractionation of total lipid extract and of
separated by liquid chromatography on SiO2 (diethyl
ether/petroleum ether elution).
2.5. Analyses
The products were analyzed by capillary GC and GC±MS. GC separations were carried out with a Shi-madzu GC-14A gas chromatograph in splitless injection mode, using a CPSil 8-CB (Chrompack) capillary column (50 m0.25 mm i.d., 0.12mm ®lm thickness) and a FID.
The temperature of the column was programed from 50C
(2 min isothermally) to 200C (2 min isothermally) at 5C
minÿ1then to 300C (30 min isothermally) at 3C minÿ1.
The temperature of the injector and the detector was 300C.
The quantities of each kind of products are given as apparent concentrations; they were determined on the basis of liquid chromatography for the various famil-ies and on the basis of GC response (peak area) for a determined family. No correction was made for derivatization.
GC±MS were performed on a FINNIGAN MAT-INCOS 500 mass spectrometer coupled with a VAR-IAN 3400 gas chromatograph. The GC conditions were the same as for GC analysis. The mass spectrometer was operated in the electron impact mode (70 eV). Transfer line and ion source were 290 and 180C,
respectively. Mass spectra were acquired by scanning the region m/z50±600 at a rate of 1 s per decade and recorded by a Data General 20 computer. The various products were identi®ed on the basis of their GC reten-tion times and their mass spectra (comparison with standards). Alcohols were identi®ed as acetates (acet-ylation with acetic anhydride/pyridine). Acids were methylated with trimethylsilyldiazomethane (Hashi-moto et al., 1981). Fourier transform infra-red (FT-IR) spectra were obtained in CCl4using a Nicolet 750
spec-trometer. NMR spectra were recorded in CDCl3
solu-tion on a BRUKER AVANCE DPX 300 (300 MHz for
1H and 75 MHz for13C). Tetramethylsilane was used as
internal reference.
3. Results and discussion
3.1. Bulk properties
The results of the elemental analysis and the value of the atomic H/C ratio (see experimental section) indi-cated a rather aliphatic nature for the macromolecular lipids of the studied soil. The point was corroborated by the FT-IR spectrum (Fig. 2) which exhibited strong bands centered at 2926, 2853, 1464 and 1377 cmÿ1which
can be attributed to CH3 and/or CH2 groups (GobeÂ,
1998). NMR measurements gave con®rmation of this predominantly aliphatic nature: 1H NMR spectrum
presented bands in the 0.5±1.7 ppm region (Fig. 3a) and the13C NMR spectrum (Fig. 3b) showed signals in the
aliphatic 9±35 ppm region. The FT-IR spectrum revealed also the presence of hydroxyl groups (broad band centered at 3413 cmÿ1) and broad bands at 1737
cmÿ1(carbonyl), 1252 (nCsp3-O), 1174, 1132 and 1032
cmÿ1 (nCsp3-O) which can be attributed to ester and
ether groups. In the 1H NMR spectrum (Fig. 3a),
sig-nals at 3.6 ppm (H±C±O), 2.3 ppm (H±C±CO) and 1.5
ppm (very broad) (H-C-C-O) can con®rm the presence of ester groups, as well as the signal at 169.5 ppm (CO) on the13C NMR spectrum.
3.2. Pyrolysis and thermochemolysis
ML were investigated using o-line preparative pyr-olysis (Behar and Pelet, 1985 ; Largeau et al., 1986) and o-line thermochemolysis as described by Grasset and AmbleÁs (1998b). The results are given in Table 1. Seven elemental o-line pyrolysis on 415 mg of material yiel-ded 98 mg of pyrolysis products corresponding to 23.6% of the original macromolecular lipids. The remaining residue represented 64.8% of initial ML. The loss of weight was probably due to volatile compounds. Preliminary experiments (GobeÂ, 1998) clearly showed that the pre-pyrolysis used for the study of sedimentary organic matter (Largeau et al., 1986) is not useful for the study of soil organic matter, pre-pyrolysis and pyr-olysis yielding the same products.
Preparative thermochemolysis aorded degradation products with a much higher yield (234 mg; 67% of ML) but TMAH was present among the pyrolysis pro-ducts. The loss of products was 23 mg (6.6% of the initial ML).
Fig. 2. FT-IR spectrum of macromolecular lipid fraction (CCl4
The composition of the pyrolysates from both experiments was determined by liquid chromatography. The main series of compounds are listed in Table 2. Polar products corresponded to the remaining material and/or to more or less transformed macromolecular lipids. The yield of GC amenable components was much higher for thermochemolysis (Table 2).
The hydrocarbon fraction formed in pyrolysis (HC I, Fig. 1) showed the classical series of n-alkane/n -alk-1-ene doublets, ranging from C16 to C36 (Fig. 4a). The
same distribution was obtained upon thermochemolysis.
Alkenes can be produced byb-scission of radicals (Larter and Hors®eld, 1993). The origin of n-alkane/n-alkene doublets on pyrolysis is diverse. One origin can be the thermal degradation of esters which is known to produce chie¯yn-alkanes from an acid moiety andn-alk-1-enes from the alcohol moiety (Van de Meent et al., 1980). These esters correspond to preserved protective layers of higher plants, as suberin, cutin (de Leeuw and Largeau, 1993 ; Macko et al., 1993) from above-ground parts as well as from roots (Nierop, 1998). The presence of alkene/alkane doublets in pyrolysates is indicative of the Fig. 3. 1H NMR (a) and 13C NMR (b) spectra of macromolecular lipids (CDCl
presence of resistant aliphatic macromolecules (cutan, suberan...) in soils (Tegelaar et al., 1989b; van Bergen et al., 1997a; Augris et al., 1998). Various terpenoids, nor-hopanes, norhopenes, hopadienes, hopenes and hopanes (derived from bacteriohopanetetrol or related com-pounds) and a sterene, 24-ethyl 5a(H) cholest-2-ene were also identi®ed, eluting between nC29 andnC36 alkanes
(Fig. 4a; Table 3). These triterpenoid compounds were not identi®ed on hydrolysis (AmbleÁs et al., 1991; 1993a) which indicates a possible ether linkage to the macro-molecular matrix on carbon 3 of the A ring of the terpe-noid/steroid. Despite a higher quantity of released hydrocarbons (Table 2), the number of polycyclic struc-tures were less numerous on thermochemolysis. Only 5 components were present 22,29,30-trisnorhop-17 (21) ene, 17a(H),22,29,30-trisnorhopane, 30-norhop-17(21) ene, 17b(H), 21 b(H)-30-norhopane, 17a(H),21 a (H)-hop-22(29)-ene. The presence of the two isoprenoids prist-1-ene and prist-2-ene, indicated the occurrence of chemically-bound phytol (Larter et al., 1979) and/or tocopherols (Goossens et al., 1984). Among the pyrolysis
products, two minor chromatographic fractions were also obtained (Fig. 1). The HC II fraction (1 mg) con-tained onlyn-alkanes with abundant C27and C29
mem-bers, superimposed on the Gaussian C21±C37
distribution. The HC III fraction (1.5 mg) contained dominant even C18, C20, C22and C24saturated members
(Fig. 4b). The origin of these even hydrocarbons is very probably fatty acid or alcohol reduction. These hydro-carbons were obviously trapped in the macromolecular network and released after alteration of the matrix. OkomeÂ-Mintsa (1991) reported the presence of suchn -alkanes in the hydrolysis products of macromolecular lipids from the same soil. The same phenomenon was observed after chemical degradation of kerogens from ancient sediments (Baudet et al., 1991; Halim et al., 1997).
The alcohol fraction from both experiments presented the same composition.n-Alkanols were present in the range C14±C32with dominant even members and a clear
max-imum at C22, accompanied with neolupenol [identi®ed in
some roots (Ageta et al., 1981)], a-amyrin, sterols also Table 1
Results (mg) of pyrolysis and thermochemolysis of macro-molecular lipids
Mass of sample
Pyrolysate Residue Loss of weight
Pyrolysis 415 98a 270 47
Thermochemolysis 349 234b 922 23
a Seven elemental pyrolysis.
b Presence of TMAH.
Table 2
Components produced by thermal degradation of macro-molecular lipids from Plateau de Millevaches (% of pyrolysate)
Pyrolysis Thermochemolysis Range
Hydrocarbons 14.5 26 C16±C36
Fatty acids (methl esters)a
4.1 C14±C34
Fatty acid methylesters 6.1 19 C14±C19;
C20±C32
Aliphatic diacids (dimethylesters)a
7.2 15 C11±C28
Alcohols (acetates)a 2.6 5 C
14±C32
Ketones 1.7 5 C17±C33
Aldehydes 0.7 0.1 C19±C31
a-Hydroxymethylesters 1 2.1 C22±C27
o-Hydroxymethylesters 2.1 2.8 C16±C28
(o-1)-Ketomethylesters 2.1 ND C22±C29
(o-1)-Ketoalcohols 1 ND C17±C29
Fatty acid octylesters 1.9 ND C14±C22
Polar products 55 25
a After derivatization.
mainly of plant origin (i.e. stigmasterol, methyl- and 24-ethylcholesterol) and their corresponding stanols. The sterol/stanol ratio ranged from 0.5 (cholesterol/cholesta-nol) to 0.9 (24-ethylcholesterol/24-ethylcholesta(cholesterol/cholesta-nol). The sterol distribution with a dominant 24-ethylcholesterol is the same as that from the simple lipids from the same soil providing evidence that simple lipids can become incorporated into a macromolecular lipid fraction (AmbleÁs et al., 1991). As the same structures (including stanols) were found after alkaline hydrolysis (AmbleÁs et al., 1991; 1993a), incorporation was most probably via ester groups. The presence of stanols is a consequence of anaerobic conditions in this hydromorphic soil, as in marine or lacustrine sediments (Gaskell and Eglinton, 1975). Microbial reduction of sterols occurs when the OH at position 3 is not bound, the attachment to the matrix occurring later probably via a transesteri®cation process (de Leeuw and van Bergen, personal communication).
The distribution of n-alkanols formed by thermal degradation of macromolecular lipids does not resemble that of alcohols present either in the above-ground parts of plant in the studied sample or in the soil simple lipid fraction [highly dominant C28 member (AmbleÁs et al.
1991)]. They can partly be inherited from plant wax esters (Jambu et al., 1993; AmbleÁs et al., 1993a), or from highly resistant biopolymer in root material as suberin (Nierop, 1998), or result from the enzymatic reduction
of fatty acids (Kolattukudy, 1976). No close correlation with the distribution of fatty acids can be expected inasmuch as the occurring processes are biological pro-cesses (with discrimination with chain length) and maybe competitive processes.
The distribution of aliphatic monocarboxylic acids formed on pyrolysis of macromolecular lipids (Fig. 5a) showed a strong even over odd carbon number predomi-nance. These acids could originate from plant protective polyesters (Tegelaar et al., 1989a,b). Unsaturated C15:1,
C16:1, C18:1, C18:2 components, and branched iso- and
anteisoC15, C16and C17acids of bacterial origin (Boon
et al., 1977 ; Perry et al., 1979), C31and C321721b(H),
21b(H) hopanoic acids were also present. Fatty acid methyl esters, corresponding to 6.1% of the pyrolysate (Table 2) which were also identi®ed amongst the pro-ducts (Fig. 5b), showed a similar distribution to that of fatty acids present in simple lipids. Thermochemolysis aorded only methyl esters arising mainly from the transesteri®cation of esters (Martin et al.,1995; Gonza-lez-Vila et al.,1996). The relative yield of products was much higher than in pyrolysis. Conversely, thermo-chemolysis does not permit to distinguish fatty acids bound by ester linkage from fatty acid methyl esters, if any, trapped in the macromolecular matrix. The dis-tribution of the fatty acid methyl esters corresponded to the combined distributions of Fig. 5.
Table 3
Triterpenoid hydrocarbons detected in Fig. 4
Number Name Structure Number Name Structure
1 17a(H)-22,29,30-trisnorhopane 8 17b(H),21a(H)-hop-22(29)-ene
2 24-ethyl-5a(H)-cholest-2-ene 9 17a(H),21b(H)-hopane
3 C29 17b(H),21a(H)-hopadiene 10 C32 17b(H),21a(H)-hopane
4 C30 17b(H),21a(H)-hopadiene 11 17b(H),21b(H)-homohopane
5 17a(H),21b(H)-30-nor-neohop-13(18)-ene 12 C32 17b(H),21b(H)-hopane
6 17b(H),21b(H)-norhopane 13 C32 17b(H),21b(H)-hopene
Dicarboxylic acids in pyrolysis or dimethylesters in thermochemolysis were identi®ed in the C11±C28range
with an even predominance and a dominance of the C22
member (and an important C16component). They were
bound as diesters in the macromolecular matrix. Both experiments exhibited the same distributions.
a- ando-hydroxyacid methyl esters were observed in both experiments, respectively in the C20±C29 range
(max. C24) (Fig. 6) and C16to C28with a maximum at
C22.a- hydroxyacid methyl esters give a prominentm/z
90 ion on mass spectrometry (McLaerty rearrange-ment) while the o- isomers led to the classicalm/z74. Although they were produced (as methyl esters, without derivatization) in pyrolysis with a lower yield, this method indicated that they were bound to the matrix via the hydroxyl groups only, the methyl ester group being
pre-existant in the macromolecular structure. Otherwise, if the COOH groups were esteri®ed to the matrix, hydroxyacids would have been produced upon pyr-olysis. o-Hydroxyacids are present in cuticular waxes, cutins and suberin from plants (Eglinton et al., 1968; Ketola et al., 1987) whilea-hydroxyacids have a micro-bial origin, resulting from the oxidation of fatty acids (Eglinton et al.,1968; Cranwell, 1984). The presence of
a- and o-hydroxyacids in the degradation products of macromolecular lipids con®rms that this fraction con-tains partly preserved biopolymers of plant or microbial origin (AmbleÁs et al., 1991).
Ketones (Fig. 7) were essentially methylketones in the C13-C29 range accompanied with the isoprenoidal
6,10,14-trimethyl-pentadecan-2-one and steroidal 24-ethylcholest-4-en-3-one, 24-methyl- and 24-ethylcho-lesta-3,5-dien-7-one (or stigmastadienone). The iso-prenoid ketone originates probably from the microbial degradation of phytol, as reported by Brooks and Maxwell (1974). The origin of methylketones in thermal degradation products is not well established. Among soil (simple) lipids, they arise exclusively from oxidation of plant hydrocarbons or b-oxydation-decarboxylation of fatty acids (AmbleÁs et al., 1993b) and are pre-dominantly odd-carbon numbered components. The distribution of Fig. 7 could indicate that these ketones were trapped in the macromolecular matrix. Methylk-etones could also have been produced from the scission of an ether linkage on carbon 2 or the scission of an alkyl chain on thea position of a hydroxyl group. An other mechanism involving a radical scission at the b
position of a free hydroxyl group was postulated by Hartgers et al. (1995). Several mechanisms contribute probably to the formation of methylketones on thermal degradation of macromolecular material. At present, this point remains unclear. Steroidal ketones could also have been trapped compounds or bound to the network by an ether bond at the carbon 3 position. The occur-rence of ester bond can also be postulated insofar as the presence of esteri®ed cholesterol, cholestanol, 24-ethyl-cholesterol and 24-ethylcholestanol in the macro-molecular lipids of the surface 0±20 cm horizon was previously reported (AmbleÁs et al., 1991; 1993a).
Linear C19±C31 aldehydes with maxima at C23 and
C29 were present in minor amount, (Table 2). They
Fig. 5. Fatty acids produced upon pyrolysis [m/z74 fragmen-togram of the methyl ester derivatives (i :iso; a :anteiso)].(a) Aliphatic monocarboxylic acids (as methyl esters); (b) aliphatic monocarboxylic acid methyl esters produced on pyrolysis.
Fig. 6. Distribution of a-hydroxyacids obtained by pyrolysis (m/z90 fragmentogram).
could have been formed from chains bearing an OH group (Gelin et al., 1994; Hartgers et al., 1995) or could arise from the cleavage of an ether group (Grasset, 1997; GobeÂ, 1998). Series of (o-1)-ketoacid methyl esters in the C22±C29range with a Gaussian distribution centered
on C25, and (o-1)-ketoalcohols in the range C17±C29with
a regular distribution also centered on C25 were also
produced upon pyrolysis. Several tentative hypotheses
could explain the presence of these products. For example, (o-1)-ketoalcohols could have been trapped in the macromolecular matrix, or were covalently bound to the matrix with one linkage (acting as a mono-substituent of the matrix) or covalently bound with two linkages (acting as bridges in the matrix). Keto groups could also partly originate from dehydrogenation of hydroxyl groups.
Octyl esters of C14, C16 (abundant), C18(dominant)
and C20fatty acids were only detected upon pyrolysis.
They were also found in the pyrolysates of other soil samples and of kerogen from phosphate rocks (unpub-lished results). They are never found in the products from thermochemolysis whatever material is degraded. Their origin is maybe related to a transesteri®cation process occurring only under the pyrolysis conditions.
4. Conclusions
In this work, a detailed study of the structure of complex, macromolecular lipids present in soil was conducted using preparative o-line pyrolysis and pre-parative thermochemolysis. The two techniques pro-vided evidence that the studied soil organic fraction is aliphatic in nature (AmbleÁs et al., 1991; 1993a). The identi®cation of degradation products permits one to advance a schematic representation of the modes of occurrence of structural building blocks (Fig. 8). The matrix of soil macromolecular lipids is formed by cross-linked aliphatic chains, a part of which are partly pre-served biopolyesters. Ether and ester groups are promi-nently involved in the cross-linking of the chains. Some aliphatic moieties are acting as monosubstituents of the matrix, re¯ected as aliphatic monocarboxylic acids (or methyl esters), alcohols, methylketones and aldehydes upon pyrolysis or thermochemolysis. The components with two functional groups (aliphatic dicarboxylic acids, (o-1)-ketoalcohols, etc.) provide evidence of the occur-rence of alkyl bridges in the network. Nevertheless, some of these components are bound to the matrix by only one linkage releasinga- ando-hydroxyacid methyl esters and (o-1)-ketoacid methyl esters on thermal degradation. As in kerogens from ancient sediments after chemical degradation (Halim et al., 1997), aliphatic compounds such as some alkanes, fatty acid methyl esters, possibly methylketones etc. are trapped in the macromolecular structure and are released when the structure of the matrix is altered. Direct ``thermo-evaporation'' of the trapped compounds could also occur on pyrolysis and thermochemolysis. The use of more ecient extraction methods, such as supercritical ¯uid extraction could free the trapped compounds as evidenced from a study of the released triterpenoids. Taking into account hydrolysis products (AmbleÁs et al., 1991; 1993a), it can be established that sterols, stanols, neolupenol,a-amyrin are bound to the matrix by ester (and maybe ether) linkages through the 3 carbon atom, hopanoic acids are bound by ester groups. The loss of the alcoholic group resulted in the formation of the corresponding unsaturated compounds. Contrary to other reported results using ¯ash Curie point pyrolysis (van Bergen et al., 1997b), the loss of the OH group is not total, probably as a consequence of a lower
tem-perature. Hopanes are present as trapped molecules. Stigmastadienone, and related compounds could also be present as trapped molecules. Alternatively, they are linked to the matrix as etheri®ed 7-ketosterols (hydro-lysis did not aord any 7-ketosterol), the stable con-jugated dienone being formed on thermal degradation.
It is interesting to note that, as reported for ¯ash on-line pyrolysis, preparative thermochemolysis in the pre-sence of tetramethylammonium hydroxide is a very use-ful and poweruse-ful technique for the study of natural macromolecules. Preparative thermochemolysis aords higher yields of products than conventional preparative pyrolysis, which is useful for representative structure determination. Heating in the presence of an alkylating agent causes additional chemolysis of the resistant ali-phatic parts of the matrix, allowing, as an example, transalkylation of polyesters resistant under classical pyrolysis conditions. As a consequence, the importance of structural parts can be largely underestimated when using the classical method (del Rio et al., 1996). Never-theless, pyrolysis only permits one to distinguish the groups present as methyl esters from the acid groups linked as esters to the matrix. Conversely, the separation procedure is much more time consuming than for pre-parative thermochemolysis.
From the results presented in this work, it can be concluded that there is, on the one hand an analogy between soil macromolecular lipids and the aliphatic part of humin from the same soil (Grasset and AmbleÁs, 1998a) and, on the other hand similarities with the structure of kerogen present in immature ancient sedi-ments (Tegelaar et al., 1989a,b; AmbleÁs et al., 1991, 1993a; AmbleÁs et al., 1996; Kribii et al., 1996).
Acknowledgements
The authors gratefully acknowledge the authorities of the French ReÂgion Poitou-Charentes for a doctoral grant (V.GobeÂ.) and C.N.R.S. for ®nancial support. The authors thank Dr. P.F. van Bergen and an anonymous reviewer for very helpful comments and suggestions.
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