Journal of Analytical and Applied Pyrolysis 47 (1998) 1 – 12
Structural study of soil humic acids and humin using a new preparative thermochemolysis technique L. Grasset, A. Amble`s * Laboratoire de Synthe`se et Re´acti6ite´ des Substances Naturelles, UMR 6514, Faculte´ des Sciences, 86022 Poitiers, France Accepted 15 March 1998
Abstract Humic acids and humin from an acidic soil from Plateau de Millevaches in France were investigated in their ester and ether groups. A new preparative thermochemolysis technique was used, in comparison with a classical determination based on chemical degradation methods. This technique allows the treatment of a high quantity of product ( 2 g). Preparative TMAH thermochemolysis of humin and humic acids yields various hydrocarbons (alkene/alkane doublets, sterenes, hopenes and hopanes), methyl esters of linear and branched fatty acids, linear dicarboxylic acids, v-methoxy fatty acids and 1-methoxyalkanes. These components are found to derive mainly from ester- and ether-bond cleavage, as demonstrated by specific chemical degradations. The results confirms that important structural differences do exist between totally insoluble humin and humic acids. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Preparative thermochemolysis; Humic acids; Humin; Ester; Ether; Soil; Hydrocarbons; Fatty acids; Pyrolysis
1. Introduction Humic substances play an important role in soil processes [1]. Moreover, these insoluble biomacromolecules can partly escape biodegradation and fossilize after deposition in aquatic or hydromorphous environments. Despite their geochemical importance, their structure remains largely unknown. Their heterogeneity and * Corresponding author. 0165-2370/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0165-2370(98)00084-9
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extreme complexity render structural information often difficult and controversial. Different analytical techniques must be used complementarily to avoid misinterpretations in structural determination. In this work, the lipidic part (part becoming soluble after thermal or chemical degradation) of insoluble humin and humic acids was studied using a new preparative thermochemolysis technique (pyrolysis with an in situ TMAH methylation). For confirmation of the results, a treatment with hydroiodic acid/cesium propionate was used, reacting for specific ether bond cleavage. Pyrolysis with in situ methylation is a recent technique used to avoid decarboxylation of acids and deshydratation of alcohols moities initially present as esters and also to prevent the loss of important structural informations [2–5]. This method is now usually used for the structural investigation of humic substances [6–11], of clay-organic complexes [12], and of total organic matter using whole soil pyrolysis [13], but only as an analytical procedure on a small quantity of sample. An interesting batch thermolysis procedure was developed recently by McKinney et al. [14] using a sealed pyrex tube and allowing the treatment of 3 mg of product. Nevertheless, to obtain quickly representative structural information on heterogenous complex organic matter, it is necessary to obtain significant amounts of degradation products. In this way, a new preparative thermochemolysis method was performed for the analysis of humin and humic acids: thermochemolysis was realized in a preparative off-line pyrolysis device [15,16]. This method permits the treatment of large quantities (1 – 2 g) of product. As thermochemolysis suggested the presence in the studied humic substances of alkyl chains bound to the matrix by ether groups, a specific chemical reaction was applied to corroborate the hypothesis. Iodhydric acid, used in the well-known Zeisel method for OCH3 titration [17], is known to selectively cleave ether bonds. HI treatment was previously applied on kerogen [18] and on algaenans [19].
2. Samples and methods
2.1. Samples The studied humin and humic acids were obtained from an acidic anmoor soil (dystric histosol) from Plateau de Millevaches (France), previously described by Amble`s et al. [20]. The soil sample contained a 56% weight of total organic matter (TOM) determined by combustion, and had a total organic carbon (TOC) content of 23%, on a ash-free basis. After Soxhlet extraction (CHCl3) of free lipids from representative soil samples and washing of the residue with 1 N HCl, humic acids were extracted with 1 N NaOH (under a nitrogen atmosphere) and separated from fulvic acids by precipitation at pH 1 (6 N HCl). The alkaline-insoluble residue corresponded to a humin concentrate. Prior to characterization, humic acids and humin were treated again with chloroform in Soxhlet (2 × 24 h) to extract possible adsorbed residual lipids. The detailed procedure is given in Grasset [21] and Grasset and Amble`s [22]. The prepared humin concentrate contained 37% weight of humin
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(insoluble organic matter) and 63% weight of resistant mineral matter. Humin and humic acids corresponded to 57 and 17% of the total organic matter, respectively. The elemental composition of humin and humic acids is given in Table 1. Humin and humic acids contributed to 62.3 and 18.2% of TOC, respectively.
2.2. Thermochemolysis The sample was placed in a ceramic boat and moistened with 2 ml of a methanol 50% w/w solution of tetramethylammonium hydroxide (TMAH) (Aldrich). The sample was then transferred in a 60× 3 cm i.d. Pyrex® tube and heated from 300 to 400°C (20 min isothermal period) at 5°C min − 1. Thermochemolysis products were carried out by helium (flow rate: 100 ml min − 1) to two successive traps containing chloroform cooled at −20°C. After evaporation of the solvent, products were separated on a SiO2 column.
2.3. Ether bond clea6age with HI/cesium propionate Humin or humic acids were suspended in 25 ml of a 47% aqueous solution of HI. The mixture was stirred for 60 h at 70°C under a N2 atmosphere. The reaction mixture was neutralized with a saturated NaHCO3 solution. I2 formed during the reaction was eliminated with Na2S2O3. After centrifugation and filtration, the reaction products were extracted with diethyl ether. Iodides resulting from three successive reactions were separated on a SiO2 column, and then allowed to react with cesium propionate in dimethylformamide (DMF) for 24 h, at 40°C, under an inert atmosphere. DMF was eliminated after adding 1 N HCl and evaporation. The propionate derivatives were then directly analyzed by GC and GC-MS.
2.4. Gas chromatography-mass spectrometry The products were analyzed by capillary GC and GC-MS. GC separations were carried out with a Packard Model 427 gas chromatograph using a CP Sil 5 CB (Chrompack) capillary column (25 m length). The temperature of the column was increased from 60°C (10 mn isothermally) to 300°C (20 mn isothermally) at 3°C min − 1. GC-MS was performed on a Finnigan Incos 500 mass spectrometer. The GC conditions were the same as for GC analysis. The various products were identified on the basis of their GC retention times, their mass spectra (comparison with standards) and literature data [23]. Table 1 Elemental composition (%) of the studied humin and humic acids Sample
C
H
N
O*
S
Humin Humic acids
45.57 48.23
7.05 4.37
3.43 3.33
43.44 42.69
0.46 1.38
a
By difference.
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Table 2 Results (mg) of thermochemolysis of humin and humic acids Sample
Mass of sample in mg (% OM)
Loss of weight in mg (% IOM)b
Thermochemolysis (% IOM)
Humin Humic acids
1267 (37.0)a 1830 (95.7)a
280 (59.7)b n.d.
107b,c (n.d.) 82 (4.6)b
a
Organic matter (%). Initial organic matter (%). c Presence of TMAH, n.d.: not determined). b
3. Results and discussion Humin and humic acids contributed to 74% of TOM and to 80.5% of TOC of the studied soil. The results of thermochemolysis of humin and humic acids are presented in Table 2. The products are more abundant for humin. The products were separated by liquid chromatography in a SiO2 column. Indeed, the complexity of the thermochemolysis products with many coelutions, does not a detailed identification and a precise quantification of the various series of products. The results of the separation given in Table 3 show important differences between humic acids and humin. Humic acids led to a high quantity of polar components which cannot be analyzed. In both samples, hydrocarbon fractions (Fig. 1a, b) are dominated by classical pyrolytic series of n-alkane/n-alk-1-ene doublets from C15 to C37. Alkenes are produced by b-scission of radicals. Among other origins, the alkane/alkene doublets can arise from the thermal degradation of esters which is known to produce chiefly n-alkanes from the acid moiety and n-alk-1-enes from the alcohol one [24–30]. Even with TMAH, we can never be sure that secondary reactions such as decarboxylation are totally impeded. Various steroids and triterpenoids, C27, C29 D-2 sterenes, C29 stera-3,5-diene (indicating a vegetal input), C27 hopene and C29, C31, C32 17b(H),21b(H)-hopanes (derived from bacteriohopanetetrol) were also identified, eluting between nC28 and nC34 alkanes (see list on Fig. 1). The release upon pyrolysis of covalently bound polycyclic products from various sediments is now well documented [31]. Prist-1-ene and prist-2-ene indicate the occurrence of chemically-bound phytol [32] and tocopherols [33]. Alcohols present as esters in humic acids and humin of the very same sample were precedently studied [21,22]. Sterols and stanols were released on hydrolysis but no hopanoı¨c structures (as alcohols or acids) and no phytol. This results indicate that hopanoid and phytyl components were bound via ether linkages and released by cracking on thermochemolysis. The second fraction contains mainly fatty acid methyl esters (FAMEs). The distributions of FAMEs were close in both samples (Fig. 2). The distribution pattern shows a strong even-over-odd carbon number predominance and maxima at C16:0 and C24:0 with the presence of unsaturated C16:1, C18:1 components. Branched iso -and anteiso-C15 and C17 FAMEs, typical of bacterial activity [34–36] were detected in minor amounts. As distributions of fatty acids present as esters in humin and humic acids were very similar [21,22], it can be concluded that a great
b
a
4266 1093
3755 2286
FAMEs
In mg/kg of organic humin or humic acids. n.d., Not determined, presence of TMAH.
Humin Humic acids
Hydro carbons 511 –
Methoxy alkanes
Table 3 Amounts of components released by thermochemolysisa
3776 610
Methoxy esters
1653 873
a,v-Diesters
5655 9717
Aromatics
n.d.b 22 673
Polar products
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Fig. 1. Gas chromatograph of hydrocarbons obtained after thermochemolysis of (a) humin; (b) humic acids (n-alkanes/n-alk-1-enes doublets; a: cholest-2-ene; b: 24-ethylcholest-2-ene; c: 24-ethylcholesta-3,5diene; 1: 22,29,30-trisnorhop-17(21)-ene; 2: 22,29,30-bisnorhopane; 3: 30-norhop-17(21)-ene; 4: 30norhopane; 5: hopane; 6: homohopane; 7: bishomohopane).
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Fig. 2. Gas chromatography of the fraction from the thermochemolysis products of humin containing fatty acids methyl ester ( : linear, saturated; : linear, unsaturated; : iso; 2: anteiso) and 1-methoxyalkanes (").
part of FAMEs from thermochemolysis were obviously released after ester bond cleavage. The esterified fatty acids present in humin ans humic acids are of plant or bacterial origin [21,22]. a,v-Dimethylesters were identified in the range C8 –C26. In humin (Fig. 3a), the C9 member is dominant while, only the even carbon-numbered members are present in the ‘‘long’’ mode with abundant C16 and C22 components. In humic acids, the long mode is dominant (Fig. 3b). As for FAMEs and due to similar distributions with diacids arising from hydrolysis, they are the result of ester bond cleavage. Sources of long-chain members are higher plant cutin and suberin [37] or microbial v-oxidation of fatty acids. The C9 member originates probably from the oxidation of the double bonds of D-9 fatty acids [38]. Most of the identified aromatics bear oxygenated chemical groups, principally methyl ester and methoxy groups. There is an obvious relationship with monomeric subunits of lignin polymers found in extant plant tissues [39] like vanillic acid 1, syringic acid 2, p-coumaric acid 3 and ferulic acid 4 (as esters). Indeed, the presence of similar products has been reported in flash pyrolysates of isolated gymnosperm/ angiosperm lignins and degraded derivatives [40,41]. These observations are consistent with the inferred contribution of plants in such humic structures.
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Fig. 3. Relative abundances of a,v-dimethyl esters obtained from humin (a) and humic acids (b).
Three series of methylated hydroxyacids (as esters) were identified in the thermochemolysis products from humin: 2-methoxy FAMEs of C22 and C24 acids and (v-1)-methoxy FAME of C28 acid are present in a very small amount. The main series corresponds to v-methoxy FAMEs having exclusively even-carbon numbers with major C16 and C22 components (Fig. 4). The distributions of v-methoxy fatty acid methyl esters and 2-methoxy fatty acid methyl esters produced from humic acids are similar. The presence of C16-C24 v-methoxy FAMEs with an even/odd predominance has been reported in cuticular waxes, cutins and suberins [42,43]. The presence of 2-methoxy FAMEs gives evidence of a bacterial input [44]. No hydroxy acids were released on the previously reported hydrolysis of the same humin and humic acids. It probably indicates that the hydroxyl group of such molecules is not initially bound to the macromolecular network through an ester linkage but through an ether one. Another family of long-chain compounds observed in humin, coeluting with FAMes (Fig. 2), is a series of 1-methoxyalkanes, corresponding to methylated n-alkanols, ranging from C14 to C30, maximizing at C22, with exclusively even carbon numbers (Fig. 5). A similar distribution was found for esterified n-alkanols in this sample [21,22]. It indicates that 1-methoxyalkanes formed by thermochemol-
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Fig. 4. Distribution of v-methoxy fatty acid methyl esters obtained from humin.
ysis were mainly alkanols bound by ester groups in humin structure. Some of them can be possibly bound to the matrix by ether linkages. In order to confirm (or to invalidate) the presence of alkyl chains bound by ether groups, humin and humic acids were treated with iodhydric acid. Alkyl iodides were then transformed into propionate derivatives on reaction with cesium propionate, instead of the classical treatment with LiAlD4 which leads to deuteroalkanes [18,45]. Indeed, on LiAlD4 reduction, the intermediate radical can react with a proton from the solvent, as well as with a deuterium atom from LiAlD4 [46]. In addition alkylpropionates show a highly characteristic MS fragmentation ion m/z= 75 arising from a double hydrogen rearrangement of the molecular ion. The distributions of alkylpropionates obtained from humin and humic acids are presented in Fig. 6. A strong even over odd carbon predominance is observed. Branched iso and anteiso C15 members (iC15, aC15, Fig. 6) are also present. For humic acids, the distribution is very simple (Fig. 6b), the even predominance of
Fig. 5. Distribution of 1-methoxyalkanes obtained from humin.
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Fig. 6. Distribution of propionate derivatives (m/z =75 fragmentograms) obtained after HI/PrOCs treatment of humin (a) and humic acids (b) (i: iso; a: anteiso).
linear compounds is much more pronounced than for humin (Fig. 6a). This result confirms that some of the 1-methoxyalkanes formed on thermochemolysis were initially alkyl chains bound to humin and humin acids matrix by ether groups. The ability to perform preparative TMAH-thermochemolysis in an open dynamic system provides some significant advantages over classical flash pyrolysis or preparative pyrolysis apparatus. The system permits the treatment of relatively high amounts of product. The high quantity of thermolysis products thus obtained in one experiment enables chromatographic separation and quantitative determination of the thermolysis products. On the whole, the results indicate that lignin and lipidic biopolymers contribute highly to the formation of complex organic matter in soil. Ester and ether groups are noticeably involved in the structure of humin and humic acids. The reticulation of moieties originating from microbiological metabolism or inherited from higher plants is partly assumed by these chemical groups. Considering ester group determination, the results arise from one experiment, compared to two series of stepwise hydrolyses. Nevertheless, complementary chemical determination can be useful.
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Acknowledgements This work was supported by the Centre National de la Recherche Scientifique (C.N.R.S.).
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