Bobov Dol subbituminous coal from Bulgaria was studied by chloroform extraction and subsequent TMAH ther-mally assisted hydrolysis andmethylation. Themolecular composition of the “free” bitumenwas comparedwiththe products of residue pyrolysis. GC/MS separation registered lipids, i.e. n-alkanes and terpenoids, mainlysesqui- and diterpenoids, predominantly aromatized. Pyrolysis released additional portions of n-alkanes, regularisoprenoids, n-fatty acids, hopanes, etc. Direct extraction of “free” bitumenwith chloroform and TMAH thermallyassistedhydrolysis andmethylation of the residue attained somewhat different lipid pools of coal organicmatter.The results of TMAH thermally assisted hydrolysis and methylation demonstrated that, despite the relativelyadvanced geological age of the sample (Late Oligocene) some lignin remnants were still preserved. The followingspecies were registered: 3-methoxy-4-hydroxybenzaldehyde, 3-methoxy-4-hydroxyacetophenon and 3-methoxy-4-hydroxy benzoic acid. By analogywith the lignin structure ofmodern trees, the abundance of vanillicstructures indicated conifers as the predominant coal-forming vegetation in the Bobov Dol peat bog.Fatty acids in “free” bitumen and in the products of thermochemolysis were compared and certain differenceswere depicted. The presence in the pyrolysate of linear mono- and dicarboxylic fatty acids, mainly of higherplant origin, revealed the role of ester linkages in coal organicmatter structuring at anRo = 0.42–0.43%maturity.The experimental data denoted that lipid components and conifer lignin had considerable contribution to BobovDol coal organic matter.
Brown coal deposits in Bulgaria are mainly located in the westernpart of the country i.e. the Bobov Dol, Pernik and Pirin coalfields, andnear the Black Sea, i.e. Černo More coalfield. In 2010, the total produc-tion of brown coal, extracted from both underground and opencastmines, amounted to 3 million tonnes. The Bobov dol coalfield is thelargest deposit of brown coal in the country. There are significant coalreserves and resources, amounting to some 100 million tonnes. Miningis carried out at one opencast and two underground mines. A total of1 million tonnes of brown coal was produced by the three mines inthe cited year. The coal is mainly supplied to the nearby thermalpower plant, while about 10–12% is used by households (http://www.euracoal.be).
The basin, as one of the primary sources of energy in the country, hasbeen well studied: mineralogical and geochemical characterizations, aswell as a retrospective on previously performed investigations can befound in Vassilev et al. (1994 and references therein); petrology and de-position environment are described by Zdravkov and Kortenski (2004
ghts reserved.
and references therein); fossil flora and their biostratigraphic significancein comparison with available paleobotanical data was published byPalamarev et al. (1998). However systematic study of the organic mat-ter composition is missing.
Molecular organic geochemistry in coal research seeks to findrelationships between organic compounds in coal and specific taxa.Currently, limited studies try to draw systematic and phylogenetic rela-tionships (Otto andWilde, 2001). Once sufficient and unequivocal infor-mation on fossil biomarkers is available for source-specific compounds,additional palaeoenvironmental reconstructions will be possible andfloral diversities during geological ages can be proposed (Stefanovaand Simoneit, 2008). Such data will enhance knowledge of coal precur-sors and their diagenetic transformations during coalification. A draw-back of extractable portion (bitumen) molecular analysis is the factthat only a small part of the organic matter is considered. To overcomethis problem and with the aim of generating supplemental informationon organic matter composition, the insoluble portion remaining afterbitumen extraction can be pyrolysed.
Analytical pyrolysis coupled with gas chromatography-mass spec-trometry (GC/MS) is a powerful technique for characterization of naturalbiopolymers and geomolecules. Polymeric material undergoes thermal-ly induced covalent bond cleavage to produce lower molecular weight
2 M. Stefanova / International Journal of Coal Geology 118 (2013) 1–7
compounds that reflect the composition of the sample. Conventional py-rolysis is improved via flash heating of the sample in the presenceof tetramethylammonium hydroxide (TMAH), a technique knownas thermally assisted hydrolysis with methylation or, more briefly,thermochemolysis. In contrast to conventional pyrolysis, this approachavoids decarboxylation and produces esters, and can operate “off line”on a preparative scale (Grasset and Amblès, 1998). Application of thetechnique to analyse bio- and geological materials using different alkyl-ation reagents was recently reviewed by Shadkami and Helleur (2010).
Currently, TMAH thermally assisted hydrolysis and methylation hasreceived its worthy place as an attractive approach for coal organic mat-ter study. On the other hand, not many papers dealt with TMAH coaltreatment, asmost applications described results frompyrolysis of lignitehumic substances (Válková et al., 2009). Del Rio et al. (1996) wereamong the first who applied thermally assisted hydrolysis and alkylationfor the structural study of natural biopolymers and geomacromolecules,i.e. humic substances, kerogen, asphaltenes, peat, lignite, etc. They havetreated xylitic and humic brown coals and, in addition to lignin buildingblocks, linear fatty acids and benzene polycarboxylic acids were identi-fied (as methyl esters). A study closely related to the present one waspublished by Dutta et al. (2012). The authors analysed preserved ligninstructures from Early Eocene Surat Lignites, India produced duringthermochemolysis. The abundant syringic structures in the pyrolysates,by analogy with the lignin structure of modern taxa, were interpretedby the authors as corresponding to angiosperms in the coal palaeofloralcommunity.
The aimof the present study is to analyse the organicmatter compo-sition of Bobov dol subbituminous coal using the biomarkers present inthe solvent extractable portion, so-called “free” bitumen. Additional in-formation will be obtained by TMAH thermally assisted pyrolysis andmethylation of the coal residue remaining after bitumen extraction.The research should be considered as preliminary, because of limitationin sample number, and comparative, inasmuch as the data are discussedin relation with the palaeoenvironments of other Bulgaria coals alreadystudied. Some assumptions about coal parent vegetation and transfor-mations with coalification will be offered.
2. Material and methods
2.1. Study area and sample material
TheBobovdol coal basin is situated in SWBulgaria. It belongs to Pernikprovince. The coal-bearing Paleogene sediments were deposited in NW-SE graben structure over denudated crystalline shists, Triassic and Jurassicsediments. Bobovdol subbituminous coal is dark brown to black in colour,bright and banded. According to Valčeva (1990) the main Bobov dol coallithotypes are clarain, vitrain, with rare semifusain and fusain. Some of itscharacteristics are as follows: Wa = 6 ÷ 12%; VM = 43 ÷ 51%; Ad =15 ÷ 65%; Cdaf = 71 ÷ 75%; Hdaf = 5.3 ÷ 5.8%; Sdaf = 1.8 ÷ 3.7; andRo = 0.42–0.43%. Šiškov et al. (1988) proposed the late Oligocene agefor the coal formationwhile Palamarev et al. (1998) based onpaleobotan-ical data argued for the Latest Oligocene–Early Miocene.
The present study was done on material prepared from the vacantamounts after microscopic investigations of 21 samples of Bobov dolcoal (Zdravkov and Kortenski, 2004). First, coal samples were crushedto b1 mm and small portions were mounted with epoxy resin, groundand polished for maceral analysis. Each sample's coal rank was deter-mined by measuring huminite reflectance. The Ro values were in theranges from 0.34 to 0.45%. According to the Bulgarian classification sys-tem this magnitude corresponded to the O2–O3 coalification stage. Themean reflectance determined by Zdravkov and Kortenski (2004) was0.4%. This value was in agreement with data published by Šiškov et al.(1988) and Valčeva (1990) for Bobov dol coal.
The rests of 21 samples after maceral analysis were additionallyground, mixed and sieved to b0.2 mm to prepare “average” sample. It
was kept in a container under N2. The sequence of analysis is illustratedin Fig. 1.
2.2. Bitumen extraction
An aliquot of 30-gramme of the “average” coal sample was placedin a Soxhlet thimble and inserted into the extractor. Chloroform(~300 ml) was used for extraction. The heating mantle was turned onand the reflux process continued for at least two days or until thesolvent ran clears from the thimble (about 30 h). The yield of “free”bitumen was determined to be the mean value of two extractions. Thetotal chloroform extract was concentrated under reduced pressure.First, asphaltenes were precipitated by pouring a toluene solution ofbitumen extract into cold n-hexane (1:100 v/v). The soluble portionwas concentrated and subsequently separated into neutral (n-hexaneeluent), aromatic (toluene eluent), and polar (acetone eluent) fractionsaccording to the procedure described in Stefanova et al. (2005a).
2.3. Isolation of “free” fatty acids
A portion of the “free” chloroform bitumen (~40 mg) was dividedinto neutral and acidic parts by column chromatography separation onKOH-impregnated SiO2. The procedure developed by McCarthy andDuthie (1962) for isolation of “free” fatty acids from other lipids wasused. The neutral fraction was eluted with 10 ml diethyl ether, andthe acidic one with 10 ml of a mixture of diethyl ether/formic acid(9:1, v/v). The acidic portionwasmethylated by CH2N2 and GC/MS sub-sequently studied. Separation of fatty acid methyl esters (FAMEs) wastracked by specific ions as described in Stefanova and Disnar (2000).
The residue after bitumen extraction was subjected to “off line”TMAH thermally assisted hydrolysis/methylation treatment. It was car-ried out according to the procedure developed by Grasset and Amblès(1998). About one gramme of the residue after bitumen extractionwas placed in a ceramic boat and 2 ml of TMAH, 50% v/v in MeOH,were added drop wise. After that the soaked sample was left overnight.The next day it was inserted into a Pyrex tube (60 cm × 3 cm) andheated at 400 °C for 1 h. Volatile products were swept by a stream ofN2 (100 ml/min) to two subsequently connected traps of ice-cooledchloroform. At the end of the treatment the product was concentratedto ~ 20 ml on a rotary evaporator. Subsequently, it was separated intofour fractions on a SiO2 mini-column using mixtures of diethyl ether inpetroleum ether with increasing polarity (Grasset and Amblès, 1998;Stefanova et al., 2008). Afterwards the third sub-fraction was acetylatedwith acetic anhydride in pyridine at 50 °C for 20 min. The acetates werestable derivatives of alcohols, phenols and polar diterpenoids. MS detec-tion was done bym/zM+. + 42n, where n corresponded to the numberof OH groups in the molecule.
2.5. Instrumental analysis
2.5.1. Rock-Eval pyrolysisA Rock-Eval study was carried out with a “Turbo” model RE6 pyro-
lyzer (Vinci Technologies, France). This instrument determined “free”volatile hydrocarbons at 300 °C (S1), hydrocarbons formed during py-rolysis at 600 °C (S2), CO2 released during pyrolysis at 390 °C (S3), andtotal organic carbon (TOC), Tmax (the temperature of S2 peak maxi-mum). The hydrogen index, HI, is defined as the amount of pyrolyzableorganicmatter (S2) per unitweight of organic carbon; the oxygen index,OI, is the amount of CO2 (S3) per unit weight of organic carbon. Param-eter definitions and their interpretations were described by Espitaliéet al. (1985) and applied by Vandenbroucke et al. (1988), Disnar et al.(2003) and references therein.
Catechols,rests oflignins
SiO2 column
CHCl3
extraction
Residue
COAL
TMAHthermochemolysis
I II III IV
Hydrocarbons Fatty acids MEsPhenols
Acetylation
Polars
GC/MSGC/MS
GC/MS
Neutrals Aromatics
GC/MS GC/MS
InsolubleSoluble
KOH/SiO2
Neutrals
“Free”Fatty acids
GC/MS
Asphaltene precipitation
“Free” bitumen
Fig. 1. Scheme of separation and analysis.
3M. Stefanova / International Journal of Coal Geology 118 (2013) 1–7
2.5.2. GC/MS analysisBitumen fractions and products of TMAH thermally assisted hydro-
lysis and methylation were analysed with a Hewlett-Packard 5-MS in-strument equipped with a DB-5 capillary column (0.22 mm × 30 m;0.25 μm film thickness). A FID at 300 °C and a split/splitless capillary in-jectormaintained at 300 °C in the splitlessmode (valve reopened 1 minafter injection) were used. After a 0.5 min hold at 85 °C the oven tem-perature was increased from 85 °C to 200 °C at 20 °C/min and from200 °C to 320 °C at 5 °C/min. Peak assignments were done by compar-isonwith theNIST library or literature data (Philp, 1985). Products werealso analysed using GC and GC/MS. For GC a Hewlett Packard HP 6890chromatograph [split injector, 250 °C; flame ionization Detector (FID)at 300 °C] and fused silica column (SGE BPX 5%, 30 m × 0.25 mm i.d.,0.25 film) were used with He as carrier gas. The GC oven was tempera-ture programmed from 60 °C to 300 °C at 5 °C min−1 (hold 20 min).GC/MS was performed with a Thermo Finnigan Trace GC coupled to aThermoFinniganAutomass (sameGC conditions). TheMSwasoperatedin the EI mode at 70 eVwith ion separation in a quadrupole filter. Prod-ucts were identified on the basis of GC retention time and MS spectra(commercial standards) and literature data (Philp, 1985). Componentsof the “free” bitumen were quantified by the application of 4-Decene(C10H20, M+.140) as an inner standard. For quantification of “free”fatty acids in the bitumen acidic portion, p-heptyl benzoic acid, methylester (C15H24O2, M+. 234) was applied.
The following fragments were track: m/z 85 for n-alkanes; m/z 202and m/z 206 for sesquiterpenoids; m/z 123 for diterpenoids; m/z 74 forn-fatty acidmethyl esters (FAMEs) in “free” bitumen and formed duringTMAH thermally assisted hydrolysis/methylation;m/z 191 andm/z 217for hopanes and steranes;.
3. Results and discussion
3.1. Bulk characteristics
The bulk characteristics of the coal were summarised in Table 1.They indicated that the sample was representative of Bobov dol coal,as the parameters were in the ranges defined by Valčeva (1990). Addi-tional characteristics were recovered by Rock-Eval pyrolysis. The dataassigned a terrestrial character to the sample. In the HI (mg hydrocar-bon/g TOC) vs. OI (mg CO, CO2/g TOC) diagram for classification of fossilmaterial, disseminated organic matter, petroleum source rocks, etc.,Bobov dol coal was ascribed as a Type III kerogen (Baudin et al., 2007;Disnar et al., 2003; Espitalié et al., 1985). The position of the sample inthe HI/Tmax diagram corresponded to immature coal and was in agree-ment with the reflectance index value Ro from 0.34 to 0.45%. The mag-nitude of Tmax in Table 1 was located below the threshold of 0.5% ofvitrinite reflectance.
Table 1Bulk characteristics of Bobov dol “average” coal sample.
Total sulfur (St) 1.54i.e. sulfatic sulfur (Sso4) 0.05pyritic sulfur (Spyr) 0.27organic sulfur by diff, (Sorg) 1.22
Rock Eval dataTotal organic carbon (TOC), in wt.% 63.11Hydrogen index (HI), in mg HC/g TOC 119Oxygen index (OI), in mgCO, CO2/g TOC 23Tmax, in °C 410S1 in mg HC/g coal 3.38S2 in mg HC/g coal 75.18S3 in mg CO2/g coal 14.54
a analytical base;.d dry base;.daf dry, ash free base;.
0
5
10
15
20
25
23 24 25 26 27 28 29 30 31 32 33 34
Carbon number
n-Alkanes
Con
tent
, mic
rog/
g C
org
.102
Fig. 2. Distribution of long chain n-alkanes in “free” chloroform bitumen, in μg/gCorg.
4 M. Stefanova / International Journal of Coal Geology 118 (2013) 1–7
3.2. “Free” bitumen composition
The modern concept of coal organic matter structuring considers itas a two-phase model, composed of a three-dimensional cross-linkedportion (macromolecular phase) and a mobile (molecular) phase. Themacromolecular network did not dissolve at low temperature unlessdegradationswere applied. It was assumed that components of themo-lecular phase were trapped in the macromolecular phase by means ofelectron donor-acceptor interactions. Some portions of the molecularphase might not be solvent-extractable, owing to restricted orificesizes of the pores in the macromolecular network. All treatments thatdecreased the degree of cross-linking raised the extract yield as a resultof the increased ability of the solvent to penetrate into the porous coalstructure. Data on coal extractability and the influence of different fac-tors on extract yields can be found in Stefanova et al. (1995) andFurmann et al. (2013).
The yield of the chloroform “free” bitumen was 2.1 wt.%(33.3 mg/g Corg). This value was comparable with the yields deter-mined by Fabianska (2004) for bitumen contents of brown coals. Overa mini-column the soluble part of the extract was separated into ali-phatics (50.6%) and aromatics (9.7%). Bitumen components irreversiblyadsorbed on the column were attributed to NSO polar compounds. Nodoubt their study would supply valuable data on the composition ofthe bitumen but it was not done at the present state of the study. Infor-mation about the fatty acids in the “free” bitumen was obtained byacidic portion analysis (see later in the text).
GC/MS separation of the first fraction revealed the presence of thefollowing series:
3.2.1. n-Alkanes in “free” chloroform bitumenThe n-alkane distribution was shown in Fig. 2 and was confirmed
by single ion monitoring (SIM) of m/z 85. It was characterized bynC29 dominance. The sum of homologues shorter than nC25 wasnegligible, so only the distribution of the long chain members wasillustrated.
The carbon preference index (CPI) was calculated for the nC24 tonC34 homologues:
CPI ¼ 1=2X
C25 � C33ð Þ=X
C26 C34ð Þ þX
C25 C33ð Þ=X
C24 C32ð Þh i
¼ 3:75
This value assumed a contribution of epicuticular wax from higherplants as a possible source for the n-alkanes in the “free” chloroformbitumen.
3.2.2. Diterpenoids in “free” chloroform bitumenDiterpenoids are common constituents of coal resins. Their composi-
tions and importance have been discussed in previous papers (Stefanovaet al., 2005a,b, 2008, 2013). The diterpenoid composition of the sampleunder study was presented in Table 2. It was rather simple. The strongpreponderance of aromatized diterpenoids over saturated diterpenoidhydrocarbonswas evident. Simonellite, 57.19 μg/gCorg strongly dominat-ed all other components. The diterpenoid distribution pattern illustratedthe more advanced geological age of the Bobov dol coal formation incomparison with other Bulgarian Neogene lignites (Stefanova et al.,2013 and references therein). The prevalence of products of abietanediagenetical transformation, i.e. simonellite and retene, was in agree-ment with the expressed assumption of advanced maturation.
3.2.3. n-Fatty acids in “free” chloroform bitumenThe acidic portion amounted up to 13% of the total “free” chloroform
bitumen. Its GC/MS separation (Fig. 3) revealed that it was mainlycomposed of n-fatty acids in the range of nC14–nC30. The total contentof n-fatty acids in the “free” chloroform bitumen was 89.8 μg/gCorg.The distribution, followed by SIM m/z 74 and expressed in μg/gCorg,was bimodal (Fig. 3). A strong even/odd carbon number predominancewas obvious. A noticeable maximum of a ubiquitous fatty acid nC16(27.4 rel. %) was apparent. The short chain homologues, i.e. nC14–nC20,represented 43.6 rel.% of the total sum of linear fatty acids. Branchedchain fatty acids amounted up to to 5.12 μg/gCorg, i.e. i-, ai-C15 and i-,ai-C17. They exclusively attested to bacterial activity in the peat bog(Stefanova and Disnar, 2000). Monounsaturated fatty acids nC16:1 andnC18:1 and hydroxyl fatty acid methyl esters were not detected.
Fig. 3. Distribution of fatty acids in “free” chloroform bitumen, in μg/gCorg.
Table 3GC/MS registered compounds in the first fraction of TMAH thermally assisted hydrolysisand methylation product.
The yield of the product from preparative “off line” TMAH thermallyassisted hydrolysis and methylation of Bobov dol sample residue was10.2% calculated on a “dry ash free basis” or 138 mg/g TOC. By SiO2 col-umn chromatography pyrolysate was fractionated into: (i) linear/cyclichydrocarbons (10.6%); (ii) n-fatty acidmethyl esters (FAMEs) + phenols(28.7%); (iii) catechols + lignin remnants (20.1%); (iv) and polars (8.0%).The fractions with phenols/catechols presence were acetylated (Fig. 1).
GC-MS separation of the first pyrolysate fraction was illustrated inFig. 4 and compounds identificationswere listed in Table 3. The compo-sition strongly resembled the chloroform “free” bitumen assemblage, asthe same diterpenoids and their aromatized counterparts dominatedthe distribution. Some sesquiterpenoids, i.e. cadalene, and substitutednaphthalenes/tetralines were additionally presented. Pairs of alkenes/alkanes (nC14–nC30) with a monomodal distribution configurationwere depicted by SIM m/z 85 tracking (not shown). The profile wassmooth, maximal in the range nC16–nC20, and no “odd” over “even”
Fig. 4. TIC of TMAH thermally assisted hydrolysis/methylation product (first fraction).(o–n-Alkanes; numbers correspond to carbons in linear chain).
member preponderance was recognized. The calculated CPI = 1.03was close to unity and indicated a different source contribution com-pared to the “free” hydrocarbons in the bitumen. The distribution pat-tern gave a hint of the occurrence of resistant aliphatic biopolymers asstructural elements, probably originating from cutin and suberin (deLeeuw and Largeau, 1993).
Regular isoprenoids, i.e. prist-1-ene, M+. 266 (C19H38,m/z 57, 100%)and prist-2-ene m/z 69, (100%) were detected as well (Fig. 4). Theyproved the existence of bound isoprenoid chains in the coal matrix atthe coalification level Ro = 0.42–0.43%. Their origin could be relatedto phytol or tocopherols (Goossens et al., 1984). Hopanes were anotherspecies attached by covalent bonds to themacromolecular phase of coalorganic matter and released during thermally assisted hydrolysis andmethylation. Hopene/hopane pairs in the range of C27–C30 (C28 absent)were tracked by SIM m/z 191. Steroid composition was simple, as only5α-Stigmastane (20R), C29H52, M+. 400was detected. The other stereo-isomer, 20S, was visible as well.
The second pyrolysate fractionwas composed of phenol, its alkylatedhomologues, and series of fatty acids methyl esters (FAMEs), (Fig. 5).FAMEs tracking by SIM m/z 74 was demonstrated in Fig. 6. FAMEs werethe main aliphatic products of TMAH thermally assisted hydrolysis andmethylation. The most likely they originated from transesterification ofesters present in the macromolecular phase of the sample under study(Grasset and Amblès, 1998).
In Fig. 6, three pools of FAMEs were distinguished:
- The shortest-chain FAMEs with 9.5 rel. % content were representedby nC6 ÷ nC11, with nC9 somewhat prevalent. They probably origi-nated from oxidation of the double bonds of Δ9-fatty acids (Grassetand Amblès, 1998).
- Shorter-chain members (nC12 ÷ nC20) were represented by theubiquitous nC14, nC16 and nC18 acids (17.3 rel. %). There were no in-dicators of bacterial activity (absence of i-, aiC15 and i-, aiC17 acids).
- The longer-chain region (nC21 ÷ nC34) was dominated by nC24 andnC26 acids. The total sum of the longer FAMEs was 70.8 rel. %.These acids were associated with ester hydrolysis and methylation.
In a study on FAs from Polish brown coals, Fabianska (2004) hasremarked that thedominance of long chain evennumbered homologuesdisappeared with a depth at the expense of the shorter members. In our
Fig. 5. TIC of TMAH thermally assisted hydrolysis/methylation product (second fraction).(o-Fatty acids, methyl esters, FAMEs; numbers correspond to carbons in linear chain).
6 M. Stefanova / International Journal of Coal Geology 118 (2013) 1–7
experiment only one “average” sample from Bobov dol subbituminouscoal was analysed. No correlations with depth were done. In the samestudy, Fabianska (2004) has observed that detritic coals were character-ized by a dominance of longer FA homologues, while in xylitic coals theshorter-chain fatty acids prevailed.
In the present study the comparison of FA distribution patterns of“free” and “bound” FAs demonstrated certain differences. Proof of mi-crobial activities associated with the presence of — i, and ai-FAs weredistinguishable only in the “free” FAMEs set. In “free” acids the distribu-tion patternwas dominated by nC16 fatty acids and the relative contentsof shorter and longer homologues were comparable. In the “bound”FAMEs set generated during TMAH thermochemolysis the distributionwas governed by the long-chain homologues (70.8 rel.%) where nC24and nC26 were maximal. FAMEs distributions gave hints that differentpools of organic matter were attained during extraction and pyrolysis.Namely: (i) short-chain fatty acids in “free” bitumen could originatedfrom ester hydrolysis of diagenetically reduced cutins, suberins or glyc-erides from cell membranes; (ii) while hydrolysis of epicuticular waxeswas the presumed source of the longer homologues in “bound” FAMEs.
0
2
4
6
8
10
12
14
16
6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
%
Carbon number
Fig. 6. Fatty acids, methyl esters (FAMEs) distribution in products of TMAH thermallyassisted hydrolysis and methylation, in rel. %. (numbers correspond to carbons in linearchain).
Even numbered α,ω-di-FAMEs were registered by m/z 98 tracking,maximal at nC14 and nC26 with a smooth distribution of the longer ho-mologues. Their occurrence was explained by the higher plants inputto the palaeomire. Diacids were explained by ω-oxidatioin of FAs toω-OH-FAs and subsequently to α,ω − di FAs. In addition, according tode Leeuw and Largeau (1993), they could originate from suberin andcutin contributions, inasmuch as di-FAs in the nC14–nC20 range weretheir structural elements. α,ω-di-FAs played an important role in coalorganic matter structural organization, as they could assure entangle-ment of the linear chains by covalent bonds.
In the products of thermochemolysis apart from the linear fatty acids,some diterpenoid acids (as methyl esters) were detected (Fig. 5).Podocarpatic acid, methyl ester (C21H36O2, M+.320, m/z 163) anddehydroabietic acid, methyl ester (C21H30O2, M+. 314, m/z 239) wereregistered. Diterpenoid acids, in combination with 3-ketoferruginol,methyl ether (C21H30O2, M+.314, m/z 299) were considered as charac-teristic biomarkers for conifer contribution to coal-forming vegetation.Side chain methylation of phenylpropane units in lignin yieldedbutanedioic acid, dimethyl ester (C6H10O4, M+.146, m/z 115).
Acetylation of the third pyrolysate fraction permitted detection ofbenzenediols and their alkylated homologues, i.e. predominantly cate-chols, C6Cn(OH)m, n = 1 ÷ 4;m = 1 ÷ 2, someof themweremethoxyderivatives, i.e. 1,2-dimethoxybenzene, 3,4-dimethoxytoluene. Werethey naturally occurring in coal or produced during TMAH treatment?There was no convincing answer in this study. According to HatcherandClifford (1997), the contemporary viewof peatification/coalificationpresumed cleavage ofβ-O-4 bonds in lignin and subsequent realkylationof the aromatic ring. After that, demethylation of methoxy side chainfunctionalities took place to form catechol-like structures. Catecholstructures were considered, respectively, as a clue for lignin diagenetictransformation.
Despite the fact that the Bobov dol coal formation was assigned tothe Late Oligocene age some lignin building blocks still could bedepicted. In the polar fraction accompanied by phenols/catecholsdominance, some aromatic components structurally related to ligninwere identified, i.e. 4-hydroxy-3-methoxybenzaldehyde (vanillin),M+.152, C8H8O3; 4-hydroxy-3-methoxyacetophenon (acetovanillonе),and M+.166, C9H10O3; as well as methyl esters of 4-hydroxy-3-methoxybenzoic acid (vanillic acid) M+.168, C8H8O4 and 4-hydroxybenzoic acid, M+.138, C7H6O3.
4. General discussion
Based on aromatic nuclei substituents Hedges and Mann (1979) di-vided aldehydic, ketonic, and acidic phenolic compounds inheritedfrom lignin and produced during oxidation of geological samples intothree groups — vanillic (V), syringic (S), and cinamic (C). All theywere considered as remnants of lignin building blocks. The position ofthe studied sample in the C/V vs. S/V diagram allowed to distinguishnon-vascular plants, gymnospermwoods (non-flowering plants includ-ing conifers), non-woody gymnosperm tissues, angiosperms (floweringplants including hardwood trees) and non-woody angiosperms (herbs,grasses, etc.). In this way, the general vascular plant sources in thepalaeoenvironment could be recognized and related to plant variety,lignin concentration, tissue type and degree of fungal alteration duringdeposition.
In the batch pyrolysate of the present residue treatment, all phenolicderivatives possessed a vanillic (V) structural arrangement andwere un-equivocally explained byGymnosperm input to the coal-formingvegeta-tion. The presence of diterpenoid acids, methyl esters, i.e. podocarpaticand dehydroabietic, as well as ketophenol argued for Gymnosperms'prevalence in the palaeomire as well.
A high preponderance of vanillyl structures (indicative of Gymno-sperm contributions) and a total lack of syringyl units (proof of Angio-sperms) were registered in the products of TMAH thermolysis of themajority of Bulgarian Neogene coals (Stefanova et al., 2013 and
7M. Stefanova / International Journal of Coal Geology 118 (2013) 1–7
references therein). Respective of this, it was surmised that the fossilplant taxa of all the deposits, i.e. Maritza-East, Chukurovo, Balsha, etc.were strongly dominated by coniferous vegetation in the coal formingorganic matter. Pliocene-aged lignite in the Dacian coal-bearing prov-ince was an exception to the total picture, as appreciable quantities ofsyringic (S) structures, indicative of Angiosperms contributing to coalorganic matter structuring, were depicted, and flowering higher plantswere assumed to be important coal progenitors (Stefanova et al., 2008).
According to the scheme offered by Stefanova et al. (2005b), thepossible diagenetic pathways of Pinacea and Taxodiaceae tricyclicditerpenoid transformation supposed aromatization of abietane tocadalene/retene with concomitant simonellite formation. Strong evi-dence of the predominance of conifers in the Bobov dol palaeomirewas the biomarker assemblage of the “free” extractable bitumen,complemented and enriched by the TMAH pyrolysate composition.The experimental data attested that conifer lignin and lipid componentshave contributed to Bobov dol coal organic matter formation.
5. Conclusions
A sequence of chromatographic and pyrolytic techniques provided re-liable information on the molecular composition of coal organic matter.Direct extraction with chloroform and TMAH thermally assisted hydroly-sis and methylation isolated different lipid pools. Namely, n-alkanes of“free” bitumen probably originated from epicuticular waxes while, inthe pyrolysate, contributions from cutin and suberin were denoted.Short-chain fatty acids in the “free” bitumen were related to ester hydro-lysis of diagenetically reduced cutins, suberins or glycerides from cellmembranes.
TMAH thermally assisted hydrolysis and methylation, besides theclassical products of coal pyrolysis, i.e. alkene/alkane doublets, alkylatedphenols/benzenediols, etc., released a variety of carboxylic moieties inthe geological material, i.e. linear, benzene and terpenoid fatty acidsall as methyl esters. Enriched information about coal structural unitsand their alteration with deposition was obtained. In addition TMAHthermochemolysis liberated regular isoprenoids and hopanoids initially“bound” to the coal macromolecular phase.
Linear mono- and dicarboxylic fatty acids, predominantly of higherplant origin, designated ester linkages presence in the organic matterat a coalification level of Ro = 0.42–0.43%. Aliphatic carboxylic acidswere designed as molecular entities attached to the coal matrix. Theyimplied esterification as a possible reaction proceeding during coaldiagenesis.
Lignin-derived phenolic derivatives with COOH groups depictedcoalification as a gradual oxidation through aldehyde/ketone to ben-zene carboxylic acid formation. The results demonstrated that despitethe relatively advanced geological age of the sample under study,some lignin remnants were still available. Their assignment to vanillicstructures definitively assumed conifers as the predominant coal pro-genitors in the peat bog of Bobov dol coal. The biomarker assemblageof the “free” bitumen, the presence of diterpenoid acids, methyl estersas well as ketophenol in the pyrolysate, all argued for Gymnosperm'sprevalence in the palaeomire.
Acknowledgements
The author would like to express her gratitude to the authorities ofthe University of Poitiers, France and personally to Prof. A. Amblès andDr. L. Grasset for the possibility to run TMAH experiments and for valu-able discussions. Special thanks to the anonymous reviewers and toProf. Dr. R. Littke, they helped me a lot with their remarks and recom-mendations to improve the text.
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