Biomarker binding into kerogens: evidence from hydrous pyrolysis using heavy water (D2O)

Biomarker binding into kerogens: evidence from hydrous pyrolysis

using heavy water (D2O)

LINDA STALKER*1, STEPHEN R. LARTER1 and PAUL FARRIMOND1

1Fossil Fuels and Environmental Geochemistry, Postgraduate Institute: (NRG), Drummond Building,University of Newcastle upon Tyne, Newcastle upon Tyne, NE1 7RU, UK

(Received 29 November 1995; returned to author for revision 12 April 1996; accepted 30 September 1997)

AbstractÐSolvent-extracted Kimmeridge Clay Formation kerogen was heated to 3158C for 72 h in thepresence of excess water (H2O) or heavy water (D2O). Hydrocarbons generated from the kerogenduring the D2O experiments contained variable amounts of deuterium atoms [0 to as high as 12 deuter-ium atoms per molecule in the compounds we examined (mean of <0.5±4.2 atoms), varying with com-pound type and pyrolysis conditions]. The observed patterns of deuteration are not a function ofsimple homogeneous exchange, but must derive from the cleavage process in the presence of excessD2O. The diagnostic mass spectral fragment ions (electron impact ionisation) of selected regular acyclicisoprenoid alkanes, hopanes and steranes generated in the presence of D2O showed highly localisedareas of deuteration, at or near the positions or functionality in the original biological precursor mol-ecules. We interpret deuteration to have occurred at, or in close proximity to, the inferred linkages (e.g.ether or sulphur) by which the di�erent biomarker hydrocarbons were formerly attached to kerogen(i.e. the tail of the asymmetric acyclic isoprenoids, the A-ring of steriods, and the side chain of hopa-noids). # 1998 Elsevier Science Ltd. All rights reserved

Key wordsÐdeuterium-labelling, hydrous pyrolysis, kerogen, isotope-labelling, Kimmeridge ClayFormation

INTRODUCTION

This paper presents one part of the ®ndings from a

study of the extent to which water and organic mat-

ter interact during arti®cial maturation experiments

in the laboratory (Stalker, 1994). We applied hy-

drous pyrolysis, a technique which generates pro-

ducts which resemble, in some respects, naturally

occurring crude oils, with a suite of fully saturated

hydrocarbons (Lewan et al., 1979). The presence of

water during pyrolysis experiments is in accordance

with the ubiquitous occurrence of water in sedi-

ments, which may be a source of hydrogen for

transfer during hydrocarbon generation (Hoering,

1984). The method of hydrous pyrolysis with isoto-

pically labelled waters was initiated by Hoering

(1984), and more recently followed up by Stalker et

al. (1994), as isotopically labelled waters may act as

path®nders for mechanisms involved in the inter-

action of organic matter with water during arti®cial

maturation. In particular, deuterium substitution at

the point of hydrocarbon cleavage from the kerogen

structure can allow the determination of the sites of

binding of the molecules evolved during hydro-

carbon generation. In the present work, a series of

hopanes, steranes and regular acyclic isoprenoid

alkanes were studied because of their structural

complexity and geochemical signi®cance. Biomarker

compounds, the basic structure of which can be

(unambiguously) linked to naturally-occurring bio-

chemical precursor compounds, are ubiquitous in

crude oils and sedimentary organic matter (e.g.

Mackenzie et al., 1982; Goossens et al., 1984;

Ourisson et al., 1984), and are widely used for

maturity and source characterisations in petroleum

geochemistry (e.g. Peters and Moldowan, 1993, and

references therein). However, the signi®cance of

kerogen-bound biomarkers has often been over-

looked in petroleum geochemistry, largely due to

our relative lack of knowledge of how they behave

as kerogens mature (compared with free bio-

markers), and the general di�culty of analysis.

Until now the most common means of investi-

gating kerogen-bound biomarkers has been by

chemical degradation methods. Sulphur-linked bio-

markers may be cleaved from kerogens and macro-

molecular organic matter by a variety of reagents

(Sinninghe DamsteÂ and de Leeuw, 1990; Richnow

et al., 1992; Hofmann et al., 1992) as can ether-

linked species (Mycke and Michaelis, 1986; Mycke

et al., 1987). These studies have clearly shown that

biomarkers become bound to macromolecular or-

ganic matter by a variety of functional groups (e.g.

-OH groups in sterols, bacteriohopanetetrol, phytol

etc., or via vulcanization attached by sulphur links

and can be subsequently removed intact by chemi-

Org. Geochem. Vol. 28, No. 3/4, pp. 239±253, 1998# 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0146-6380/98 $19.00+0.00PII: S0146-6380(97)00103-4

*Current address: Statoil, N-4035 Stavanger, Norway.

239

cal degradation. Additional studies have shown thatin the presence of deuterium labelled reagents, clea-vage points on the released biomarkers have been

labelled by 1±2 (steroids) to 3±4 (hopanoids) deu-terium atoms (Mycke and Michaelis, 1987;Richnow et al., 1992, 1993). While showing themode of incorporation of biomolecular precursors,

these experiments tell us little of the natural releaseof biomarkers during maturation and the role ofhydrogen in the form of water and organic hydro-

gen.A few studies have performed arti®cial matu-

ration experiments using hydrous pyrolysis in D2O,

notably one experiment by Hoering (1984) and sub-sequently by Stalker (1994). It is assumed in theseexperiments that the substitution of geological time

by elevated temperature will not critically alter themechanism of hydrocarbon generation, so that wecan monitor the cleavage of biomarker species frommacromolecular organic matter in a more natural

way, and to some extent determine the role of bothorganic and water hydrogen in the saturation ofthese species.

With all this in mind, we performed a series ofhydrous pyrolysis experiments with immatureKimmeridge Clay Formation kerogens in the pre-

sence of H2O and D2O, in part to con®rm the workof Hoering (1984) (by building on his single exper-iment by using a di�erence source rock, and demi-neralising it ®rst to avoid possible catalytic

reactions caused by clays), and for comparison withthe many chemical degradation experiments per-formed (Mycke et al., 1987; Sinninghe DamsteÂ and

de Leeuw, 1990; Hofmann et al., 1992; Richnow etal., 1992).

EXPERIMENTAL METHODS

Samples used

Two specimens of immature Kimmeridge Clay(Dorset Coast, UK) were used in this study. The

results of the experiments for both samples werevery similar (Stalker, 1994), therefore only one setof data is reported here to avoid confusion (sample

collected at Clavell's Hard, Dorset).

Sample preparation: extraction and kerogen isolation

The rock sample collected from Kimmeridge Baywas sonicated in methanol to clean any contami-nated surfaces before being crushed in a Tema mill

and extracted by soxhlet (9:1 dichloromethane:-methanol; 72 h). Carbonates were ®rst dissolved bythe addition of concentrated HCl, stirred for 24 h,

followed by washing (�3) of the residue in distilledwater. Then conc. HF was added and the rockpowder heated (approx. 608C) and stirred for 3

days. The solution was neutralised by repeatedwashing with distilled water. Finally, the residuewas stirred in hot conc. HCl (608C) for a further24 h and neutralised by repeated washings with dis-

tilled water. The sample was then air-dried before afurther soxhlet extraction. XRD analysis of the sep-arated kerogen concentrate (TOC = 65%) showed

no clays, ¯uorides or oxide minerals to be present;only kerogen and pyrite remained. We consider itunlikely, therefore, that mineral±organic matter

reactions could have taken place during the hydrouspyrolysis experiments.

Hydrous pyrolysis conditions and product isolation

Hydrous pyrolysis experiments were conducted

using 0.5 g of kerogen, placed in a stainless steeltube (T-316 SS) or ``bomblet'', with 18 ml internalvolume. 5 ml of distilled water (H2O) or D2O (99.8atom % purity) was added, and the unit sealed

under nitrogen after extensive purging to removeair. Calculations using liquid water vapour densitycurves and the expression described by Lewan

(1993) showed that liquid water was maintained atthe pyrolysis temperatures used. The sealed bomb-lets (run in duplicate) were then placed in a 0.5 l

general purpose reactor (Parr Co.) and rapidlyheated to the experimental temperature (taking 45±60 min, depending on the ®nal temperature), and

heated isothermally for 72 h at various temperatures(300±3308C) (Table 1). Internal pressure of the 0.5 lreactor ranged from 1500 to 1800 psi (10340±12410kg mÿ2, however the internal pressure of the indi-

vidual bomblets could not be measured.After cooling, the bomblets were thoroughly

washed out, ®rst with water, then methanol and

®nally dichloromethane. The products and washingsfrom the kerogen experiments were passed througha Buchner ®lter. The ¯uids collected from the kero-

gen were then added to a separating funnel. Thedenser, organic layer (dichloromethane) wasremoved and the remaining solutions were washeda further three times with dichloromethane. The

combined DCM washings were dried over anhy-drous MgSO4 and concentrated by rotary evapor-ation before chromatography.

The recovered materials were fractionated byelution over a silica column (60±120 mesh) with®rst 100 ml petroleum ether (aliphatic hydrocarbon

fraction), then 100 ml petroleum ether:dichloro-

Table 1. Summary of the kerogen pyrolysis experiments per-formed, where data have been reported. The pyrolysis conditions(time and temperature), water and kerogen used are all summar-ised in the legend description in the ®nal column of these labelsare used throughout the tables and ®gures in this paper

CH = Clavell's Hard kerogen: Da and Db are duplicate runs

Temperatureandtime conditions

Type of waterused (5 ml)

Weight ofkerogenadded (g)

Legend used intables

and ®gures

3158C, 72 h H2O 0.5 CH315/72H3158C, 72 h D2O 0.5 CH315/72Da3158C, 72 h D2O 0.5 CH315/72Db

L. Stalker et al.240

methane (80:20), followed by 100 ml dichloro-methane and ®nally 50 ml methanol.

Optimising pyrolysis conditions

The ®rst set of experiments reported here were

performed to optimise the hydrous pyrolysis con-ditions. Experiments were performed using 0.5 g ali-quots of kerogen heated in bomblets with 5 ml

distilled water at 300, 315 and 3308C for 72 h, usingthe procedure described above. Internal standards(n-nonylcyclohexane and squalane) were added

during workup. The aliphatic hydrocarbon fractions

of the pyrolysates were analysed by GC, and peakareas used to calculate the abundance of n-alkanes

(summed from n-C16 to n-C25), the acyclic isopre-noid alkanes pristane and phytane, and C27aaaRsterane in m g gÿ1 TOC.

Final pyrolysis experiments

The experiments performed to study the incor-

poration of deuterium or hydrogen from water andthus the cleavage points in various biomarkers wereperformed in duplicate. Two experiments were per-

formed in H2O acted as control experiments for

Fig. 1. Example of summed mass spectra of C16 regular acyclic isoprenoid alkane generated duringhydrous pyrolysis of Clavell's Hard kerogen at 3158C for 72 h in (a) H2O and (b) D2O. Major ions are m/z141 and 183. Note that deuteration levels in (b) are much higher in the m/z 141 fragment than in the m/z

183 fragment.

Biomarker binding into kerogens 241

comparison with duplicate experiments performedin D2O at 3158C for 72 h (see above for details).

GC and GC±MS conditions

The aliphatic hydrocarbon fractions were ana-lysed by gas chromatography (GC) using a Carlo

Erba HRGC 5300, ®tted with an HP-5 fused silicacapillary column (30 m�0.25 mm; 0.25 mm ®lmthickness) and employing a temperature program of

508C for 2 min, then ramping at 48C minÿ1 to3008C and holding for 20 min. The carrier gas washydrogen.Further analyses were performed by gas chroma-

tography±mass spectrometry (GC±MS). The ali-phatic hydrocarbon fractions recovered fromKimmeridge Bay kerogen pyrolysis experiments

were analysed by a HP 5890 Series GC linked to aVG TS 250 mass spectrometer run in EI mode. Thecolumn used was an HP-12 (25 m�0.32 mm;

0.17 mm ®lm thickness) fused silica capillary col-umn. The aliphatic fractions isolated from theClavell's Hard kerogen pyrolysates were analysedusing a Fisons Instruments GC 8000 Series gas

chromatograph linked to a Fisons Instruments Trio1000 mass spectrometer. The column used was anHP-5 (30 m�0.25 mm; 0.25 mm ®lm thickness)

fused silica capillary column. The GC temperatureprogram used for both machines began at 408C,holding for 1 min, increasing at 108C minÿ1 to

1758C, holding for 1 min, then ramping again at

68C minÿ1 to 2258C and holding for 1 min, and®nally heating at 48C minÿ1 to 3008C and holdingfor 20 min. The ionization energy used was 70 eV,

and a limited mass spectrometer scan range (m/z140±270), at 3 scans/s was used to enhance signalintensity.

Data manipulation

Biomarker mass spectra were generated by aver-

aging a series of scans across the widest part of theappropriate peak on each ion chromatogramobtained from the aliphatic hydrocarbon fractions.The following acyclic isoprenoid alkanes, hopanes

and steranes were selected for analysis of their deu-terium content as they all comprised abundant,clearly resolved components: of 2,6,10-trimethyl tri-

decane and 2, 6, 10-trimethylpentadecane (hereafterreferred to as 16i and 18i regular acyclic isoprenoidalkanes respectively), 17a(H),22,29,30-trisnorho-

pane, 17a(H),21b(H)-30-norhopane, 17b(H),21a(H)-30-normoretane, 17a(H),21b(H)-bishomohopane(22R) (referred to throughout the text as Tm, C29abhopane, C29ba hopane and C32abR hopane respect-

ively), and 5a(H),14a(H),17a(H)-cholestane (20R),and 5b(H),14a(H),17a(H)-cholestane (referred tothroughout the text as C27aaaR and C27baa sterane

respectively). Comparison of an averaged massspectrum of, for example, non-deuterated 16i, withthe averaged mass spectrum of the deuterated 16i

shows that there is a high level of background sig-

Fig. 2. Abundance (mg gÿ1 TOC) of n-alkanes (expressed as 10% of the sum of n-alkanes ranging fromn-C16 to n-C25, pristane, phytane and C27aaa 20R sterane (labelled sterane) for a series of duplicate

Clavell's Hard kerogen pyrolysis experiments performed at 300, 315 and 3308C for 72 h.


nal present in the mass spectrum of the deuterated

compound (Fig. 1). This high background partly

re¯ects the broadened mass ranges of individual

ions due to multiple deuterium incorporation, but

may also partly represent coeluting deuterium-iso-mers of minor components also (a problem associ-

ated with the chromatographic broadening of peaks

made up of multiple deuterium isomers, i.e. the

same compound with di�erent numbers of D atoms

substituted for H atoms). This background had to

be removed from the data in order to get a better

representation of the abundance of the di�erent D-

isomers in the compounds of interest. A number of

possible methods were considered (Stalker, 1994).

While GC±MS computer software generally has the

facility to subtract baseline noise during data pro-

cessing, it is di�cult to obtain a section of back-

ground that is truly representative of the noisepresent throughout the generated ion chromato-

gram. Alternatively, the subtraction of the mass

spectrum of a non-deuterated product [e.g. 16i spec-

trum presented in Fig. 1(a)] from its deuterated

equivalent [16i spectrum in Fig. 1(b)] would clearly

result in the removal of the major fragment ions

(e.g. m/z 183 and 141) only. Instead it was decided

that an arti®cial baseline could be constructed as a

Fig. 3. The relative abundance of each D-isomer is shown for both diagnostic fragment ions of C16 iso-prenoid (16i) generated in the presence of H2O (i.e. the control) and in the presence of D2O from thepyrolysis of Clavell's Hard kerogen. Presented in the form of a deuteration summary diagram, duplicateexperiments using D2O may be compared simultaneously with the control, thus showing the distri-bution of deuterium incorporation in the di�erent fragment ions. (a) the m/z 141 fragment ion is gener-ally indicative of the ``tail'' end of the molecule and (b) the m/z 183 fragment ion is indicative of the

``head'' (see Fig. 4).


series of horizontal steps for each ion cluster (e.g.around m/z 141 and related D-isomers andaround m/z 183; Fig. 1).

Following background subtraction, some of thedata were subsequently corrected for the presenceof 13C (18i, and C29 ab and ba hopanes only) using

the method of Biemann (1962). Sterane data werenot corrected for the presence of 13C due to compli-cations resulting from alternative mass spectral

fragmentations in the steranes (discussed below).Although time consuming, baseline subtraction andcorrection for 13C content improved the data qual-

ity whilst tending to give a value for the absoluteminimum quantity of deuterium present in each ofthe diagnostic fragment ions of the biomarkers stu-died.

Data presentation

The treated mass spectral data were used to pro-duce a series of reconstructed ion intensity plots (in

which the relative abundance of the various deuter-ated fragments could be compared for di�erent ex-periments simultaneously). In these deuterationsummary diagrams (e.g. Figure 2) the partial mass

spectra (redrawn as line graphs) of products of thecontrol experiments (i.e. biomarkers generatedduring kerogen pyrolysis in H2O) are compared

with the equivalent deuterated fragments generatedduring kerogen pyrolysis with D2O.A series of parameters are also used to show rela-

tive di�erences in deuteration between di�erent

biomarker fragments and di�erent kerogen pyrol-ysis experiments: Dmean, Dmode, Dmax, and %D0.The abundance of deuterium-isomers (or D-isomers;

Ross, 1992) present in each fragment was averagedto give the mean number of deuterium atoms perfragment, or Dmean, while the most abundant D-iso-

mer (Dmode) shown in the deuteration summary dia-gram de®ned the modal value of deuteriumincorporation into a given fragment of the bio-

marker molecules. The heaviest D-isomer present ina fragment is reported as Dmax. The percentages ofbiomarker fragments generated during GC±MS (EI)

which were found to contain no deuterium (i.e.those molecules which did not appear to interactwith the deuterium labelled water) were also calcu-lated, and are expressed as %D0. The shorthand

notation for the sample names is explained inTable 1.

RESULTS AND DISCUSSION

Optimising pyrolysis conditions for the generation ofbiomarker hydrocarbons

Typical hydrous pyrolysis conditions range from290±3608C for 24±120 h (Winters et al., 1983),although much longer periods of time may be used

(Saxby and Riley, 1984; Rae, 1990). The tempera-ture and time conditions most commonly reportedin the literature are 3308C for 72 h (Hoering, 1984;

Eglinton et al., 1988; Lewan, 1993), which tend to

Fig. 4. Structures and fragmentation patterns of the molecules investigated by D2O hydrous pyrolysis.Arrows in bold indicate major fragmentation pathways, and dashed lines show minor alternative frag-

mentations which may occur during GC±MS (EI).


result in the highest total pyrolysate yield.However, initial experiments performed with the

Kimmeridge Bay kerogen showed that temperaturesof 3308C may have been too high, resulting in amarked loss of biomarker hydrocarbons (data not

shown here). Thus a series of trial experiments wereperformed at various temperatures so that hydrouspyrolysis conditions could be optimised.The duplicate results (Fig. 3) appear consistent

for each temperature used. With increasing pyrol-ysis temperature, the abundance of selected n-alkanes (n-C16±n-C25) increased markedly from an

average of 837 mg gÿ1 TOC at 3008C to 2692 mm gÿ1

TOC at 3308C, a trend which was also observed byEglinton et al. (1988). The abundance of pristane

decreased slightly with increasing temperature(Fig. 3), but both phytane and C27aaaR steranereached peak generation at 3158C and then

declined. A pyrolysis temperature of 3308C wasapparently too high for our purposes, resulting inincreased degradation processes versus generationof the biomarker hydrocarbons studied (Fig. 3).

Therefore, conditions of 3158C for 72 h were uti-lised for the experiments with H2O and D2O.

Final pyrolysis experiments

Regular acyclic isprenoid alkanes. Gas chromato-

grams of aliphatic hydrocarbon fractions separatedfrom pyrolysates produced in the presence of D2Oshow substantial coelution of deuterated pristaneand deuterated n-C17 alkane, due to the broad

peaks composed of multiple D-isomers. Similar coe-lution occurred between deuterated phytane anddeuterated n-C18 alkane. With this in mind, 2, 6,

10-trimethyltridecane (16i) and 2, 6, 10-trimethyl-pentadecane (18i) were chosen instead for study, asthey elute midway between the n-C14 and n-C15 or

n-C16 and n-C17 alkanes respectively, thus avoiding

major coelution e�ects. In addition, both these

compounds have asymmetric structures (unlike pris-tane, studied in the same manner by Hoering,

1984), and thus each diagnostic fragment ion pro-duced during GC±MS (EI) may be related to di�er-

ent ends of the precursor molecules.

The summed mass spectra of 16i clearly showdi�erences between generation in the presence of

H2O and D2O (Fig. 1; labels explained in Table 1).Summary diagrams of the major fragment ions of

16i (Fig. 2) indicate that deuteration is apparently

concentrated in the ``tail'' fragment of the molecule(Fig. 4) (in this case, m/z 141).

Similar data, indicating that deuteration is also

concentrated in the ``tail'' (m/z 169) of the related18i molecule are shown in the form of Dmean,

Dmode, Dmax and %D0 values presented in Tables 2,

3, 4 and 5. For example, the Dmean values of bothmajor fragments of 18i appear close to zero in the

non-deuterated control (Table 2). Higher values ofDmean are recorded for both fragments in the deut-

erated samples, but these are especially high for the

m/z 169 fragment, ranging from 1.0 to 1.3 deuter-ium atoms per fragment. Similarly the Dmode

(Table 3) and Dmax (Table 4) values con®rm thispattern of preferred deuteration in the m/z 169 frag-

ment over the m/z 183 fragment. It must be remem-

bered, however, that these major fragment ions arenot totally exclusive to particular parts of the mol-

ecules (Fig. 4). For the C16 isoprenoid it is possibleto generate both m/z 141 and 183 fragment ions by

alternative, but less favourable, single-step fragmen-

tation routes. However, for the C18 isoprenoidalkane it is only possible to generate the m/z 169

ion in a single fragmentation process from the``tail'' end of the molecule, although m/z 183 frag-

ments may come from both ends of the molecule

(Fig. 4). We therefore interpret the low levels of

Table 2. Average number of deuterium atoms per fragment ion (Dmean) for selected acyclic isoprenoid, hopane and sterane aliphatichydrocarbons generated during pyrolysis in H2O (control) and D2O

Dmean

16i isoprenoid 18i isoprenoid C29ab hopane C29ba hopane C27aaaR sterane C27abb sterane

m/z 141 m/z 183 m/z 169 m/z 183 m/z 177 m/z 191 m/z 177 m/z 191 m/z 149 m/z 217 m/z 149 m/z 217

Control 0.02 0.02 0.04 0.02 0.22 0.18 0.05 0.07 1.88 0.64 2.70 0.38CH315/72Da 2.77 0.55 1.3 0.5 3.2 0.8 2.8 0.6 3.5 4.2 3.2 2.8CH315/72Db 0.92 1.05 1.0 0.6 1.7 1.0 1.4 1.1 1.8 2.5 2.4 2.7

Table 3. Most abundant D-isomer per fragment ion (Dmode) for selected acyclic isoprenoid, hopane and sterane aliphatic hydrocarbonsgenerated during kerogen pyrolysis in H2O (control) and D2O. Dmode of the steranes is partly a�ected by interference with the other

major ions of m/z 151 and 218

Dmode



Control 0 0 0 0 0 0 0 0 0 0 0 0CH315/72Da 0 0 1 0 0 0 0 0 0 1 0 1CH315/72Db 0 0 0 0 0 0 0 0 0 0 0 0

ND = not determined.


deuteration observed in the m/z 183 fragments of

both molecules to be a result of these more minor(``tail'' end) fragmentations, and that deuterium in-

corporation in the ``head'' of the molecule is negli-gible or absent.

That deuteration is preferred in (or even

restricted to) the m/z 141 (16i) and m/z 169 (18i)fragments or ``tail'' ends of the isoprenoid mol-

ecules (Fig. 4) is not surprising, as these comprise

the part of the molecules associated with the func-tionality in the precursor compounds (e.g. -OH

group of phytol; Brooks and Maxwell, 1974; or thechroman ring of tocopherols, Goossens et al., 1978;

Li et al., 1995). This positive evidence of localised

incorporation of deuterium con®rms Hoering's(1984) hypothesis that pristane too was most likely

deuterated around the functionality in the inferredprecursor molecule. Although of a symmetrical

nature (the major fragment ion, m/z 183, coming

from both ends of the molecule) the relative abun-dance of D-isomers of deuterated pristane showed a

bimodal distribution, suggesting that deuterium wasconcentrated in one of the two m/z 183 fragments

(Hoering, 1984; also observed in this study), ratherthan randomly distributed throughout the molecule.

Analysis of 16i and 18i now shows that deuteration

does indeed occur in the area of the molecule wherethe isoprenoid was most likely to have been

attached to the kerogen matrix.

Although deuteration in the regular acyclic iso-prenoid alkanes is extensive (between 58 and 71%

of 18i molecules have taken up at least one deuter-ium atom; Table 5), a signi®cant proportion remain

undeuterated. Non-deuterated hopanes and steranes

are also reported (Table 5). As the kerogen hadbeen exhaustively extracted prior to the hydrous py-

rolysis experiments, eliminating the possibility offree hydrocarbons, it is assumed that all the hydro-

carbon products in the pyrolysates have been

cleaved from the kerogen; a proportion of thesehave incorporated 1H instead of deuterium atoms.

The successful labelling of the majority of the iso-

prenoid molecules studied in these experiments mayin part be due to the excessive amount of 99.8

atom% D2O used (ratio of 10:1), of water:organic

matter). Unfortunately, we were unable to performexperiments which could more closely mimic nature

(i.e. reducing the water:OM ratio) with the availableequipment (the minimum quantity of water for a

hydrous pyrolysis experiment was used throughout)

to establish whether the extent of deuteration waslinked to the D2O content. The remaining 20±50%

(Table 5) of isoprenoids which did not become deu-terium labelled on cleavage from the kerogen may

be explained by the presence of an alternative

source of H (i.e. in the kerogen itself). This may bein the form of early catagenetic generation of water

from the kerogen, as it has been demonstrated thatduring con®ned pyrolysis experiments (Landais et

al., 1994) 50±60 mg gÿ1 TOC water were generated

at between 300±4008C in the con®ned pyrolysis ex-periments, and partially consumed as kerogen pro-

ceeds through the stages of maturation. Thedecrease in water abundance approximately midway

through the maturation experiment of Landais et

al. (1994) is believed to be due to the utilisation ofthe water for saturation of released aliphatic hydro-

carbons. Alternatively, later stage aromatizationreactions may also be a source of H in these exper-

iments (Landais et al., 1994). In the water-domi-

nated environment of our hydrous pyrolysisexperiments (containing approx. 18.5 g water gÿ1

TOC), it may then appear surprising that such highquantities of non-labelled species are observed. It

would therefore suggest that the donation of 1H

from aromatization reactions still dominate over

Table 4. Heaviest D-isomer per fragment ion (Dmax) for selected acyclic isoprenoid hopane and sterane aliphatic hydrocarbons generatedduring kerogen pyrolysis in H2O (control) and D2O

Dmax



Control 1 1 1 1 2 2 1 1CH315/72Da 10 3 10 3 10 5 10 3 11 12 8 9CH315/72Db 6 5 5 2 7 6 7 6 7 9 9 9

Table 5. Percentage of non-deuterated isomers generated per fragment ion (%D0) for selected acyclic isoprenoid, hopane and sterane ali-phatic hydrocarbons generated during kerogen pyrolysis in H2O (control) and D2O

%D0



Control 97.9 98.0 96.4 97.9 86.6 85.1 95.2 92.6 ± ± ± ±CH315/72Da 22.4 59.5 28.8 64.8 21.5 62.3 20.2 66.6 17.1 10.6 17.2 16.0CH315/72Db 46.4 48.1 41.8 64.6 37.5 64.3 42.1 62.8 34.4 22.1 27.3 21.4

± = not determined.


donation of D from water during the saturation of

many aliphatic hydrocarbon components during

arti®cial maturation experiments in large excesses of

water.

Hopanes. m/z 191 ion chromatograms of the ali-

phatic hydrocarbon fractions of the pyrolysates

were used to select hopanes to be analysed for levels

and location of deuteration. These compounds con-

Fig. 5. Summed mass spectra of C29ba hopane generated during hydrous pyrolysis of Clavell's Hardkerogen at 3158C for 72 h in (a) H2O and (b) D2O. Major fragment ions are m/z 177 and 191. Noteagain that deuteration levels in (b) are much higher in the m/z 177 fragment than in the m/z 191 frag-

ment.


sisted of Tm, C29ab, C29ba and C32abR, and were

selected for their mass spectral characteristics, since

they have diagnostic fragment ions for the di�erent

ends of the molecule. Tm contains none of the side-

chain present in the known biological precursors

(e.g. diplopterol or bacteriohopanetetrol), and thus

tests for deuterium incorporation in the ring system.

The C29 hopanes possess short (ethyl) side chains,

while the C32 hopane contains a remnant of the

extended functionalised side-chain from the precur-

sor bacteriohopanetetrol (or related C35 com-

pounds; Rohmer et al., 1992).

While Tm present in the aliphatic fraction gener-

ated from kerogen in the presence of H2O was

found to elute cleanly, deuterated Tm was found to

partially coelute with the deuterated C28baa sterane

(due to peak broadening with multiple D isomers).

This complicated interpretation of the mass spectra,

as the m/z 149 (DE rings) fragment of Tm was

a�ected by contributions from the important m/z

149[151] fragment of C28baa sterane. While quanti-

®cation of the deuterium content was not possible,

a comparison of the mass spectra of non-deuterated

and deuterated Tm (not shown) revealed positive

evidence of extensive deuteration in the m/z 149

fragment, while the m/z 191 (AB rings) fragment

appeared to contain negligible quantities of deuter-

ium. This suggests that most deuterium is found in

the D or E rings of the molecule. In fact, despite

the problems of coelution, the Tm mass spectrum

suggests that up to 7 deuterium atoms (i.e.

Dmax=7) may have been incorporated into the DE

rings fragment, which contains eight possible lo-

cations for deuterium to enter into the E ring (i.e.

Fig. 6. Deuteration summary diagram of (a) the m/z 177 (DE ring) fragments and (b) the m/z 191 (ABring) fragments of the C29ba hopane generated in either H2O (control) or D2O from kerogens heated

under a variety of hydrous pyrolysis conditions (see text).


six positions entirely within ring E, and two sites onthe D/E ring junction).

The mass spectra (Fig. 5) and deuteration sum-

mary diagrams (Fig. 6) of C29ba hopane, and tabu-

lated data (Tables 2±5) for both the C29 hopanes,

show that deuteration has preferentially occurred in

the m/z 177 (DE rings) fragment of both molecules.

The values for Dmean, Dmode, Dmax and %D0

(Tables 2±5) are quite similar for the m/z 177 frag-

ment ions of both C29ab and ba hopanes for each

experiment performed, suggesting that stereochemi-

cal di�erences at C-17 and C-21 do not a�ect deu-

terium incorporation. For both the C29 hopanes,

Dmax reaches 10 in the pyrolysate with the most

extensive deuterium incorporation, this being the

number of possible locations on the side-chain plus

Fig. 7. Summed mass spectra of C27aaaR sterane generated during hydrous pyrolysis of Clavell's Hardkerogen at 3158C for 72 h in (a) H2O and (b) D2O. Major fragment ions are m/z 149 and 217. Note

that deuteration levels in (b) are similar in both fragments.


E ring, without any deuterium atoms reaching theD ring (including the D/E ring junction). This ob-servation suggests that propagation of deuterium in-

corporation beyond the E ring and into the D ringmay not occur during these experiments.

Preferred deuteration of the m/z 219 (DE rings

plus side-chain) fragment could also be observed inthe mass spectra of C32abR hopane. However, dueto the low abundance of C32 and higher hopane

homologues it is not practical to report any quanti-®ed amounts of deuterium incorporation.

Low levels of ``apparent deuteration'' were noted

in the m/z 191 (AB rings) fragments which are com-mon to all of the hopane compounds discussedhere. While low, these values are still slightly higher

than for the control m/z 191 fragments. Although

the important hopane fragment ions are predomi-nantly from the fragmentations shown in Fig. 4,minor alternative fragmentations can produce ions

of the same mass, but from other parts of the mol-ecule. While it is possible that the point taken forbackground subtraction may have biased results, it

is more likely that the observed apparent deutera-tion in the m/z 191 (AB rings) fragment probablyoriginates from alternative fragmentations, such as

those shown in Fig. 4 for the acyclic isoprenoids.The results presented here for hopane hydrocar-

bons con®rm and clarify the less speci®c con-

clusions of Hoering (1984). He interpreted the lowlevels of deuteration observed in the m/z 191 (ABrings) fragment of a C30 triterpane compared with

the high levels of deuteration in the molecular ion

Fig. 8. Deuteration summary diagram of (a) the m/z 149 (AB ring) fragments and (b) the m/z 217(ABC ring) fragments of the C27aaaR sterane generated in either H2O (control) or D2O from kerogens

heated under a variety of hydrous pyrolysis conditions (see text).


(M+412) to indicate that the bulk of the deuterium

incorporation was in the DE rings and side-chain

fragment. Deuteration was extensive, with a maxi-

mum of at least 12 deuterium atoms present in the

molecular ion (Hoering, 1984).

The mechanism by which hopane precursors are

bound into kerogen has been studied in greater

detail than for acyclic isoprenoid alkanes. One of

the major identi®ed precursors, bacteriohopanete-

trol, is considered to become incorporated into

kerogen by attachment via the functional groups on

the side-chain (either through ether or sulphur

links), although the possibility of hitherto unidenti-

®ed selectively preserved hopanoid-containing bio-

macromolecules (de Leeuw and Largeau, 1993)

might form an additional component of kerogen-

bound hopanoids. Our data, and those of Hoering

(1984), are consistent with the deuteration patterns

expected of hopanoid moieties bound through their

side chains being cleaved from the kerogen matrix

during hydrous pyrolysis. Side chain binding was

also suggested by Mycke and Michaelis (1986) and

Mycke et al. (1987), who chemically degraded

Messel Shale kerogen by catalytic hydrogenation

with rhodium-on-charcoal in the presence of D2O

and D2. This technique (normally with H2O and

H2) cleaves ether bonds in macromolecules. Mycke

and Michaelis (1986) found that, in hopanes cleaved

from kerogen by chemical degradation with deuter-

ium-labelled reagents, virtually no deuterium was

present in the molecular ion of the hopane mol-

ecule, while the DE rings and side-chain fragment

and molecular ion contained 1±3 deuterium atoms.

It was therefore proposed that the hopane precursor

was attached to kerogen by ether linkages in the

side-chain of the bacteriohopane(tetrol) (diplopterol

was not studied). This was subsequently con®rmed

by Mycke et al. (1987) in a similar series of exper-

iments in which bacteriohopanetetrol was recovered

from the alcohol fraction of a similarly degraded

Messel Shale kerogen. Chemical degradation using

Raney nickel to cleave C±S bonds has also shown

that the C32 bacteriohopanoid precursor may in

some cases be attached to kerogen by a series of up

to four sulphur links in the side chain, which may

have resulted from vulcanisation of the -OH groups

(or unsaturated dehydration products) during early

diagenesis in a sediment of high sulphur content

(Sinninghe DamsteÂ and de Leeuw, 1990).

Steranes. The two sterane hydrocarbons chosen

for study were C27aaaR and C27baa. Both steranes

have diagnostic mass spectral ions of m/z 149 (and

151) from the AB rings fragment, and m/z 217 (and

218) representing the ABC rings fragment (Fig. 7).

The m/z 151 and 218 ions are fragment ions which

result from rearrangement and hydrogen transfer

reactions during fragmentation. The presence of

these additional fragment ions in the sterane spectra

has the e�ect of making levels of deuteration

appear much higher than they actually are, thus

making quanti®cation di�cult, so that values

reported tend to be over-estimated. Hence, no cor-

rections were made for the relatively minor presence

of 13C for the steranes.

Despite the additional ions in the sterane mass

spectra, the steranes produced in the D2O pyroly-

sates clearly display signi®cant deuterium incorpor-

ation relative to the control mass spectra (Fig. 7).

However, for the steranes we do not have major

ions which are diagnostic for di�erent parts of the

molecule, as was the case when observing deutera-

tion in the regular acyclic isoprenoids and hopanes.

Instead, both major fragments contain the AB rings

of the molecule. Deuteration observed in the m/z

149 (AB rings) fragment and m/z 217 (ABC rings)

fragment appears similar (Fig. 8; Tables 2±5)

suggesting that there is little or no deuteration in

the C ring of the molecule. This is consistent with

the observation that the steroid precursors (e.g.

sterols) are functionalised at the C-3 position of the

A ring, and that this is the most likely site of ster-

oid attachment to kerogen. Steroid incorporation

into kerogen via sulphur links is an additional

mode of binding; many naturally-occurring sterenes

have the double bond in the A or B ring (d2, d4and d5), again consistent with our deuteration pat-

terns, although compounds unsaturated in the side

chain are known, o�ering additional potential sites

for binding into the kerogen matrix. As was

observed for the C29 hopanes, there were no ap-

preciable di�erences in the levels of deuteration in

the two sterane isomers (C27aaaR and baa), indicat-ing that the stereochemistry at C-5 may not in¯u-

ence deuteration.

Hoering (1984) also observed preferential deu-

teration in the AB rings of a C30 sterane, which he

presumed was due to the presence of an -OH func-

tional group on the A ring which could bond the

sterol precursor to kerogen during diagenesis, in a

manner similar to the functional groups present in

bacteriohopanetetrol. Similarly, in the chemical

degradation study of Messel Shale kerogen by

Mycke and Michaelis (1986), an isolated C304a-methylsterane was found to contain one deuterium

atom in the AB rings fragment. These latter authors

used a technique that selectively cleaves ether links.

Their results suggested that the C304a-methylsterane

must have been joined to the kerogen by the oxyge-

nated functional group in the A ring of the precur-

sor sterol. It would also appear that during

chemical degradation only one deuterium atom is

incorporated where the ether link was broken,

unlike the hydrous (D2O) pyrolysis method used in

this and Hoering's (1984) experiments, where poly-

deuteration was observed due to the fundamentally

di�erent mechanism of cleavage. In addition, D-lab-

elling chemical degradation techniques have ident-

i®ed sulphur linkages to the AB rings fragments as


a potential mode of binding steranes into a largermacromolecule (Sinninghe DamsteÂ and de Leeuw,

1990).

CONCLUSIONS

In this study of hopane, sterane and regular acyc-lic isoprenoid hydrocarbons produced during hy-drous pyrolysis using deuterium enriched water, we

have indeed con®rmed much of the work ofHoering (1984), showing that this method can be auseful tool in the investigation of bond sites in hy-

drocarbon compounds cleaved from macromolecu-lar matter such as kerogens. It has also beenobserved that deuterium labelling of these speciesoccurs in the absence of any catalytic materials

such as clay minerals (Hoering used powderedwhole Messel shale). However, deuterium labellingof compounds cleaved from kerogen which has

undergone hydrous pyrolysis in D2O is not nearlyso site speci®c for the majority of chemical degra-dation procedures. Comparisons between deuterium

labelling chemolysis and hydrous pyrolysis showthat (a) a larger number of deuterium atoms are in-corporated per cleaved bond in hydrous pyrolysis,

and (b) any bond type binding a biomarker mol-ecule into a macromolecular structure may be bro-ken during hydrous pyrolysis (C0O, C0S etc.)

The actual extent of the role of water H versus

kerogen and organic matter H in the saturation ofbiomarkers and hydrocarbon compounds releasedduring hydrous pyrolysis and natural generation

from kerogen cannot be determined from these ex-periments, but would appear to warrant furtherstudy. Although expensive incorporation of water-

derived hydrogen (deuterium in our case) into hy-drocarbon products generated during hydrous py-rolysis of kerogen has been clearly demonstrated,the mechanism(s) by which this incorporation

occurs has not been seriously addressed here. Aseries of pyrolysis experiments were conducted withboth H2O and D2O, to which reaction modi®ers

(e.g. radical inhibitors and initiators) had beenadded, but these results will be described elsewhere.

Associate Editor Ð R. L. Patience

AcknowledgementsÐThis research was encouraged andfunded by the Petroleum Science and TechnologyInstitute, UK. Technical assistance was provided byRobert Hunter, Paul Donohoe and Ian Harrison, NRG.We would also like to thank Walter Michaelis and TimEglinton for their constructive reviews of the manuscript.We would also like to thank Richard Patience for his com-ments and attention to detail!

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Biomarker binding into kerogens: evidence from hydrous pyrolysis using heavy water (D2O)