Structural investigations of sulphur-rich macromolecular oil fractions and a kerogen by sequential chemical degradation

Advances in Orgalic Geochemistry1991

0146-6380/92$5.00+ 0.00 Copyright© 1992PergamonPress Ltd

Org. Geochem. Vol. 19, Nos 4-6, pp. 351-370, 1992 Printed in Great Britain. All rights reserved

Structural investigations of sulphur-rich macromolecular oil fractions and a kerogen by sequential chemical degradation H. H. RICHNOW,A. JENmCtt and W. MICHAELIS Institute of Biogeochemistry and Marine Chemistry, University of Hamburg, BundesstraBe 55, D-2000 Hamburg 13, Germany Almtract--Structural studies of a sulphur-rich kerogen and macromolecular oil fractions from the Monterey Formation were performed by selective sequential chemical degradation. The method provides low-molecular weight compounds as former building blocks of the network which allow detailed analyses on a molecular level. The degradation sequence is based on three subsequently performed reactions--a selective cleavage of sulphur bonds in the first step carried out with Ni(0)cene/LiAID4, an ether and ester bond cleavage (BC13), and an oxidation of aromatic entities by ruthenium tetroxide as a final step. Each step of this sequence afforded a considerable amount of low-molecular weight material which was separated chromatographically and studied by GC and GC/MS, while the high-molecular weight or insoluble fractions were subjected to the next reaction step. The chemical degradation products--hydrocarbons and carboxylic acids--are discussed in terms of incorporation into the macromolecular structure, distribution of heteroatomic bridges and the genetic relationships between the different macromolecular crude oil fractions and kerogen. Labelling experiments with deuterium provided evidence for a simultaneous linkage by oxygen and sulphur functionalities or by aromatic units and sulphur bonds of cross-linking macromolecular network constituents. The determination of sulphur positions in the macromolecule suggests early diagenetic sulphur incorporation into the biological precursor compounds and subsequent formation of a cross-linked network. Key words--sequential chemical degradation, sulphur-rich macromolecules, resins, asphaltenes, kerogen, hiomarkers

INTRODUCTION Many attempts have been made to at least partly understand the structure of macromolecular sedimentary organic matter. A part of these structural investigations is based on non-destructive spectroscopic techniques such as i.r. and NMR. These methods reveal bulk data about the abundance of functional groups and of aromatic and aliphatic carbon atoms, but yield only limited information on the detailed macromolecular chemical structures (see review by Rullk6tter and Michaelis, 1990). Recently, interesting studies have been carried out to demonstrate the occurrence of sulphide moieties in sulphur-rich asphaltenes and coals with X-ray spectroscopic techniques (George and Gorbaty, 1989; Kelemen et al., 1990; Waldo et al., 1991). Progress in structural analysis of macromolecular material was achieved by the application of appropriate chemical degradation techniques (cf. Rullkttter and Michaelis, 1990). These techniques were developed to study representative building blocks of the parent macromolecular structures. With specific techniques for the chemical degradation of sulphur bonds, such as Raney Nickel, the presence of sulphur bonds in asphaltenes and resins ool9/4:6-o

could be demonstrated (Schmid, 1986; Sinninghe Damst6 et al., 1988). Evidence for polysulphide linkages in immature sulphur-rich macromolecular oil fractions has been obtained by MeLi/MeI and LiAIH4 chemolysis (Kohnen et al., 1991; Adam et al., 1991). Reagents, such as Li/EtND 2 and Ni(0)cene/ LiAID4, provide possibilities for the degradation of sulphur bonds in asphaltenes and kerogens. These reagents label the position of former sulphur linkages with deuterium during the desulphurization (Hoffmann et al., 1992; Richnow et al., 1992). Oxygen-linked structural units can be investigated by several ether- and ester-bond degrading reagents. Ester bonds are simply cleaved by acidic or alkaline hydrolysis. BCI3/LiAIH4 degradation has been used to prove the presence of ester and ether linkages in macromolecular organic matter (Michaelis et al., 1988; Jenisch et al., 1990). The bonding site of ether-linked aliphatic compounds could be traced by using LiAID4 in these degradation reactions (Chappe et al., 1982). Aromatic and aliphatic structural entities of fossil macromolecules have often been studied by a number of oxidation techniques (Vitorovit, 1980; Barakat and Yen, 1988). Further progress has been made by the application of ruthenium tetroxide, which 351

H.H. Racm~ow et al.

352

oxidizes aromatic structural units liberating aliphatic substituents as carboxylic acids (Trifilieff et al., 1992; Standen et al., 1991a, b). For this study, we examined the selectivity and reactivity of several chemical degradation methods and developed a degradation sequence by combining convenient techniques to differentiate between sulphur-, oxygen- and aromatic-bound aliphatic structural units. The sequence presented here is particularly designed for sulphur-rich macromolecules. Sulphur linkages are cleaved selectively, followed by the degradation of oxygen bonds. Subsequently, aromatic structural units are oxidized liberating alkyi substituents as aliphatic carboxylic acids. The low-molecular weight fractions obtained by this sequential chemical degradation of the macromolecular substrates are discussed in terms of binding sites, biomarker contents and condensation processes of sulphur-rich macromolecules. EXPERIMENTAL

Samples and geology The Miocene Monterey Formation is the most important petroleum source rock of California. Important amounts of crude oils have been produced from fractured Monterey kerogen (Isaacs, 1984; Curiale et al., 1985; Kruge, 1986). The oils have a high sulphur content and derive from clastic-poor source rocks deposited in highly reducing environments (cf. Curiale et al., 1985). Presumably, the Monterey petroleums are mixtures of early diagenetic and more mature migrated oils (Kruge, 1986). In this study a sulphur-rich Monterey oil (5.5 wt% S) and an outcrop sample from the carbonaceous marl member of the Monterey Formation were used.

Methods Extraction andfractionation. The sediment sample was extracted extensively with dichloromethane/ methanol (3:1; v/v). The kerogen was prepared following the procedure described by Orr (1986). According to Pelet et al. (1986) the asphaltenes were precipitated four times by a 50-fold excess of n-heptane from the sediment extract or the oil. Soluble compounds were separated by column chromatography to obtain hydrocarbons (n-hexane), aromatic and sulphur containing compounds (5% diethyl ether in n-hexane), and a polar fraction (dichloromethane). The resins were eluted by deactivation of the silica column with a mixture of dichloromethane/methanol/water (70:25:5; v/v/v). Nickel(O)cene degradation. Aliquots of the macromolecular substrates were desulphurized with nickel(0)cene [bis(1,5-cyclopentadiene)nickel(0); Fluka] which is soluble in organic solvents like tetrahydrofurane (THF). Details including deuteriation experiments in the desulphurization are given by Eisch et al. (1983). Briefly, the nickel(0)cene degra-

dation experiment was performed using molar ratios of approx. 1 Mol organically bonded sulphur/4 Mol nickel(0)cene/4 Mol LiA1H4. The sample and the reagent were dissolved in anhydrous THF. The reaction mixture was refluxed for 48 h, and quenched with H20. Aluminium hydroxides were destroyed by adding 6 n HCI. The reaction mixture was dissolved in an excess of CH2C12 and washed with H20. The organic phase was dried with anhydrous Na2SO4 before further separation. Labelling experiments were carried out with LiA1D4, and CH3COOD was used to scavenge the reaction mixture. The reaction products were separated on a silica column as described above. Compared to the Raney nickel desulphurization our procedure is simple to carry out and allows deuteriation of the reaction products with high specificity. In the case of liquid or solid substrates phase-transfer problems are minimized compared to the Raney Nickel method because the organic nickel complex is solvent soluble. Condensation of cyclopentadiene ligands is a side reaction (Diels-Alder reaction) which sometimes interferes with the yields of the degradation products, in particular in cases where the amounts of degradation products are low. During the work reported in this study the side reaction was found to occur only to a minor extent, because the studied macromolecular organic substrates resulted in high degradation yields. Boron trichloride degradation. The cleavage of oxygen linkages was performed by adding an excess of BC13at - 20°C to the macromolecular organic matter in dichloromethane. The mixture was allowed to react for at least 12 h at room temperature. After removal of BC13 the chlorides were converted to hydrocarbons with LiAIH4 in anhydrous tetrahydrofurane under reflux. The reaction mixture was separated by column chromatography as above. Ruthenium tetroxide degradation. Typically, a 20fold excess of sodium periodate, calculated on the organic carbon content of the sample (100 mg), was used as co-oxidant. The reactants were dissolved in dichloromethane/acetonitrile/water (5: 5 : 7; v/v/v). 25 mg of ruthenium trichloride hydrate (RuCI3 x 3 H20; Aldrich) were added and the mixture was stirred vigorously for 24 h at room temperature. The organic phase of the reaction mixture was extracted with dichloromethane and diethyl ether, dried with anhydrous Na2SO4, and filtered through Celite (Sigma). The carboxylic acids were esterified with boron trifluoride/methanol (BFa/MeOH) or diazomethane and purified by thin layer chromatography.

Analytical techniques Sulphur determinations were performed on a Carlo Erba Elemental Analyzer model 1500. Hydrocarbons and methyl esters were analyzed on a Carlo Erba 4160 gas chromatograph equipped with a fused silica capillary column (DB-5, 30m x 0.25 mm, J&W Scientific); the temperature

Sulphur-rich kerogen and maeromoleeular oil fraction structures

I RESIN,ASPHALTENE"' I

I

S-BONDS

Ni(0)eene/LiAID4

I

IHydrocarbon fraction

I

I IIRs'DOI I BCI3/LiAIH4 O.BONDS

]

GC/MS

I

Hydrocarbon fraction

I r°mt'°ll

[ fraction

CC

RESIDUE

described above. In the last step, this material was subjected to the ruthenium tetroxide degradation. Products of the RuO4-oxidation were extracted and esterified. The sequential chemical degradation procedure for the kerogen was modified as shown in the flow diagram (Fig. 2). In this case, products were exhaustively extracted from the solid reaction residue and the solvent soluble compounds were separated into a hydrocarbon, an aromatic and a polar fraction. The residual kerogen was then subjected to the next degradation step. RESULTS AND DISCUSSION Desuiphurization

I

I

GC/MS

353

RutheniumtitrT°LX~deO

I Ao=. I GC/MS Fig. I. Flow diagram of the sequential chemical degradation of soluble macromoleeular fractions (CC = column chromatography, TLC = thin layer chromatography).

program was: 80°C, 3min isothermal; 80-300°C, 3°C/min; 300°C, 20 min isothermal; injection mode: on-column; carrier gas: H2. GC/MS analyses were performed on a Carlo Erba 4160 gas chromatograph coupled to a Varian CH7A mass spectrometer. MS conditions: 70 eV ionization energy; source temperature 250°C; mass range m/z 50-800; resolution 1000. GC temperature program: 80°C, 5 min isothermal; 80-300°C, 3°C/min; 300°C, 20 min isothermal; injection mode: on-column; carrier gas: He. Sequential chemical degradation The sequential chemical degradation procedure is summarized in a flow diagram (Fig. 1). In the first step, the asphaltene or resin fraction was subjected to the nickel(0)cene desulphurization. The reaction products were separated by column chromatography. The column was eluted with n-hexane (to obtain hydrocarbons) and 5% diethyl ether in n-hexane (aromatic fraction). Deactivation of the column with a solvent mixture of dichloromethane/methanol/ water (70:25: 5; v/v/v) gave a polar fraction. This polar fraction, the "residue" of the desulphurization reaction, was subjected to the next chemical degradation step, the cleavage of ether and ester bonds by BC13. After BCI3/LiAIH4 the low-molecular weight reaction products (hydrocarbons, aromatic compounds) were separated by column chromatography from the resistant polar macromolecular "residue" as

The nickel(0)cene reagent desulphurizes sulphides and thiols (Chan et al., 1985). The selectivity, reactivity and the labelling efliciencies with deuterium were tested with standard compounds (Fig. 3). Aliphatic and aromatic thioethers are cleaved with moderate to good yields. Thiophene and thiolane structures are desulphurized between 44 and 73 wt%. Based on the degree of deuteriation the labelling with deuterium allows a distinction between the different sulphur containing structural units of the macromolecular fractions. Thioethers are basically labelled with one deuterium atom, thiolanes with 2 and thiophenes with mainly 4-6 deuterium atoms during the Ni(0)cene/LiAID4 desulphurization. The experiments with reference compounds show that oxygen ethers are not degraded by Ni(0)cene/LiAIH4 under the conditions used. Oxygen selective degradation Boron trichloride has been applied on a wide variety of geomacromolecules to cleave oxygenlinked compounds (Michaelis et al., 1988; Jenisch et al., 1990). The reagent cleaves ethers such as alkyl aryl ethers, mixed aliphatic ethers and certain types of esters giving good to excellent yields (Bhatt and Kulkarni, 1983). Results of reactivity tests of this reagent on dialkyl ethers are shown in Fig. 3. The cleavage of ethers with boron trichloride produces chlorides and alcohols as major products. The chlorides are reduced to hydrocarbons by LiALH4. Alcohols are not reduced under the conditions applied. Esters are hydrolyzed to corresponding acids and alcohols by this method (Jenisch et al., 1990). Experiments with standard compounds provide evidence that boron trichloride does not degrade carbonsulphur and sulphur-sulphur bonds. Ruthenium tetroxide degradation Ruthenium tetroxide has been previously applied in catalytic oxidation experiments of coals (Stock and Tse, 1983; Blanc and Albrecht, 1990), kerogens (Standen et al., 1991a) and asphaltenes (Trifilieff, 1987). It oxidizes certain types of aromatic structures releasing aliphatic alkyi substituents as carboxylic

354

H.H. RICtmowet ai.

I KEROGEN I

I I

Ni(0)cene/LiAID 4

I

S-BONDS

I IRESIDUE [

Ice

i

I fraction

I

Polar I fraction

fraction

GC/MS

I IEXTRACTI

RESIDUE I

I

CC

I

l

I Hydrocarbon fraction

O-BONDS

I

onl I--ti [

I BCI3/LiAIH 4

Ruthenium tetroxide

l

[ Aromatic fraction

Polar fraction

©

TLC

GC/MS

Acids

I

GC/MS Fig. 2. Flow diagram of the sequential chemical degradation of the kerogen (CC = column chromatography, TLC = thin layer chromatography). acids in high yields (Stock and Tse, 1983). These aliphatic carboxylic acids receive an additional carbon atom from the aromatic ring system (Fig. 4). In case of aliphatic moieties linking two aromatic structures the carbon chains of the degraded aliphatic dicarboxylic acids are extended by two further carbon atoms. Furthermore, this reagent oxidizes different types of ethers, but we assume that most of the ether bonds present in the macromolecular material have

been cleaved by the preceding BCI3 treatment. Other functional groups such as primary alcohols and olefins are oxidized to carboxylic acids (Carlsen et al., 1981; Choi et al., 1988). Yields

For this study, we performed our selective sequential chemical degradation technique on macromolecular oil fractions and a kerogen from the Monterey

BCIs RDS

S

R

Ni(0)cene LiA1H4/D4

R~S~R

No products

=

~ R

Alkanes 70-82 %

R~O

Alkanes 44-73 %

R

R

=

N o products

Fig. 3. Chemicaldegradation of standard compounds (R -- alkyl substituent).

Sulphur-rich kerogen and macromolecular oil fraction structures

355

,'/~,/(Ci2)n

MATRIX

RuO 4

HOOC ~

(CH2)n

(CH2) n MATRIX

MATRIX

RuO 4

(CH2)n ~

COOH

HOOC "

Fig. 4. Formation of carboxylic acids by ruthenium tetroxide oxidation of macromolecularly bound alkanoic entities. Formation. The yields obtained by the degradation sequence of a macromolecular sediment bitumen fraction (resin), of the corresponding kerogen, and of the oil resin and oil asphaltene fraction are listed in Table I. Significant amounts of GC-amenable compounds are derived from the high-molecular weight substrates by this degradation sequence, ranging from 19 to 39 wt% (Table l). The low-molecular weight aromatic and acidic fraction which is important in terms of degradation yields will not be discussed in this paper, and in case of the kerogen degradation we also obtained a soluble polar fraction which has not yet been analyzed. Generally, the yields obtained by desulphurization are higher than by oxygen bond cleavage indicating a high abundance of sulphur linkages in the studied samples. Hydrocarbons are found in higher concentrations after the degradation of heteroatomic bonds of the resin fractions, while higher amounts of carboxylic acids are released from the degradation of aromatic entities of the asphaltene and kerogen network. Therefore, heteroatomic bonds linking structural units are more abundant in the resin macromolecules, while alkyl substituted aromatic systems prevail as structural elements in the kerogens and asphaltenes.

Solvent soluble macromolecular fractions (resin, asphaltene) Relative concentrations of aliphatic hydrocarbons extracted from the sediment sample are shown in

comparison to the aliphatic components obtained by the stepwise degradation of the macromolecular bitumen fraction (resin) [Fig. 5(A-D)]. The sediment extract is characterized by high concentrations of 25, 28,30-trisnorhopane, 28,30-bisnorhopane and lower amounts of phytane and pristane, n-Alkanes with no distinct even-over-odd carbon number predominance and steranes are observed in low concentrations. Desulphurization of the resin yielded high relative concentrations of phytane and a series of n-alkanes

Table 1. Yields of chromatographic fractions released by sequential chemical degradation Monterery oil Monterey sediment resin asphaltene resin kerogen [wt%] Ni(O)cene HC ARe RES BCI3 HC ARe RES

7.8 18.2 74.0

1.7 9.3 85.8

1.9 8.2 80.3

0.7 8.2 13.6

2.5 7.0 45.2

I.I I.I 83.0

1.0 6.3 ND

0.1 1.0 2.1

13.9 3.3

31.8 8.7

14.2 1.5

30.5 8.7

38.8

21.9

18.9

18.7

RuO4 AC ME total LMW (GC-amenable)

ND ffi not determined; HC ffi hydrocarbons; ARe ffi aromatic compounds; RES ffi residue or in case of the kerogen polar fraction; AC ffi acids; ME ffi methyl esters; LMW ffi low-molecular weight material; wt% were determined by gravity.

356

H . H . RlCH~OW et al.

25,28,30-

A

trisnorhopane 28,30bisnorhopane

Ph

I

[

Pr 19

25

Ph

B

0

16

24

0

0

0

I olooo] I 0 O

O 0 O"

0

"

16

C O

0

24 o

O

0

s

Uo

0

E

0

0

16

12 0

D 0

o 0

O

I"Z

v

o o l o 24 Oo

> RT

Fig. 5. Gas chromatograms of the aliphatic hydrocarbons obtained from the sediment extract (A) and the respective resin degradation products after desuiphurization (B) and ether cleavage (C), and of the carboxylic acids (methyl esters) after RuO4 oxidation (D). (O) = n-alkanes (A-C) or n-monocarboxylic acids (D); numbers indicate the number of carbon atoms; S ffi steranes; Ph = phytane; Pr -- pristane; C4o = C+o biphytanes; v = isoprenoidal monocarboxylic acids.

Sulphur-rich kerogen and macromolecular oil fraction structures ranging from 16 to 34 carbon atoms with an evenover-odd carbon number predominance. Steranes and C40 isoprenoids (1,1'-biphytane skeleton) are found in considerable amounts. The aliphatic hydrocarbons released by the following degradation of oxygen bonds mainly comprise high concentrations of n-alkanes with a strong preference of compounds with even carbon numbers. The abundances of phytane, steranes and C40 isoprenoids relative to those of n-alkanes are lower than in the desulphurization products. 25,28,30-tris- and 28,30-bisnorhopane

357

could not be detected in the degradation products, indicating that these compounds are not structural constituents of the macromolecular network. In contrast, the C4o isoprenoid lipids are found exclusively chemically bound in the macromolecular fractions. The products of the subsequent oxidation of aromatic structures in the non-degraded macromolecular material yielded high amounts of linear carboxylic acids [Fig. 5(D)]. Acids with isoprenoidal carbon skeletons (v) and benzene carboxylic acids (not

A Ph

P~ o

o

I

I

iJ

o

o

Ooo O

30

O

O

O O

B o

Ph

o

,o

O

o 16

0

o

o

3O

° o o o

o

37 o 38 o

o oO

o o

o

o

°I

,

16

o

C

o

>p.i--i

o

Z ILl

I-

Z I--i

I

o

I

O

l

I

J O

O

O

,

,

i

I |

I

I I,I

__

° o o

o

3U

, I , 9oOooooooO

oo

o

> RT

Fig. 6. Gas chromatograms of the aliphatic hydrocarbons obtained from the Monterey oil (A), after the

desulphurization (B) and ether cleavage (C) of the respective oil resin fraction. (O) = n-alkanes, numbers indicate the number of carbon atoms; Ph = phytane; Pr = pristane.

358

H. H, I~CHNOWet ai.

indicated) were found in relatively low concentrations in this fraction. As an example of the sequential cleavage of heteroatomic bonds in macromolecular oil fractions, the aliphatic hydrocarbons of the oil resin degradation products are compared with those of the respective oil [Fig. 6(A-C)]. The oil contains n-alkanes in the range of C~3 to C3s. Phytane is the predominant isoprenoid. In the desulphurization products phytane is less abundant and n-alkanes with more than 30 carbon atoms occur in higher relative concentration than in the oil. Remarkably high concentrations of n-C37 and n-C3s occur in the desulphurization products. Sulphur-bound C37 and C3s n-alkanes have been

100

found earlier in sulphur-rich macromolecular fractions (Sinninghe Damst~ et aJ., 1988). The presence of these compounds suggests a significant input of lipids from biological sources like algae (Prymnesiophytae sp.) which are known to synthesize alkenones of those carbon chain lengths (Volkman et al., 1980; Marlowe et al., 1984). The oxygen selective degradation resulted in n-alkanes with a strong predominance of the C16 and Cls homologues [Fig. 6(C)]. In this fraction, C37 and C3s compounds did not appear in high abundance, which could well mean that they are not bonded to a great extent via oxygen linkages to the network of the oil resin. This might indicate, that if the polyunsaturated alkenones are the precursors,

57

A

71

197

B5

50 ,.J i

i , , , , i , , , • i wr,,

r,

, , , i 0 , , , I , ° ,-,

i

99 /

113

l~,~.~, |

/

....................

po|l

50

100.

......... i 350

225

I mllHllll

~. . . . . . . . .

spectra

253

I IHSlllll

338 'd2

I g~Hllll,

,., ._, .~...~,..~.....:.

50 7. Mass

~'~"÷"~'t~'"'"~'~l 200

B

I

Fig.

40

57

71

0

?

141

I IS i I l l

I I,,~HIb,

I Sill,Hi,

,.., . , , .. . . . . . . . . . . .

n-tctracosan¢

obtained

upon

.,.,

350

200 of

I. S H , I

the

Ni(0)cene/LiA]D

4 dcsu]phurization

subsequent BCI3/LiAIH4degradation (B) of the oil resin.

(A)

and

Sulphur-rich kerogen and macromolocularoil fraction structures they are preferentially linked by sulphur bonds to organic macromolecules. The different distributions of heteroatom-bound alkanes in the two resins [Fig. 5(B-C) and 6(B-C)] already demonstrate the wide range of structural variability between similar sulphur-rich macromolecular fractions. Linear carbon skeletons

Major products of the chemical degradation of the resins, asphaltenes and the kerogen are aliphatic compounds with a linear carbon skeleton. These substances are bonded via sulphur, oxygen or aromatic structural units to the macromolecular network. The binding sites of n-alkanes to the macromolecular materials are demonstrated by the mass spectra of n-tetracosane derived from the oil resin degradation experiments. Former linkages are labelled by deuterium incorporation during desulphurization. It is difficult to determine the precise location of deuterium atoms from the mass spectrum of n-tetracosane after desulphurization. However, displacement of fragment ions seems to indicate a deuteriation at one end of the molecule [Fig. 7(A)]. The incorporation of mainly 1 to 4 deuterium atoms can be interpreted by degraded sulphur functionalities located in terminal positions. In desulphurization experiments with thioethers, thiolanes and thiophenes as reference compounds we found that the resulting alkanes mainly incorporated 1, 2 and 4-6 deuterium atoms, respectively. The polydeuteriation of the degraded n-tetracosane may indicate a cross-linking of this compound via thioethers, but a contribution of sulphur-bound thiolanes and thiophenes especially for compounds with more than 3 deuterium atoms cannot be excluded. If a thiolane or thiophene is linked via a sulphur bond

359

to a macromolecule it can be expected that these compounds incorporate 3 or 5-7 deuterium atoms during desulphurization, respectively. According to the observed polydeuteriation pattern [Fig. 7(A)] the relative amount of compounds with more than 4 deuterium atoms is low. Thus, thiophenic structures additionally bonded by sulphur to the macromolecular network are not very abundant in the studied samples. The deuteriation signature of n-tetracosane subsequently released by the degradation of oxygen bonds indicates additional sulphur linkages [Fig. 7(B)]. The molecular ion of the n-C24 alkane is partly non-deuteriated. This compound must arise from the degradation of an oxygen linkage. Furthermore, some signals in the molecular ion region of the n-C24 alkane show an uptake of 1-2 deuterium atoms. The deuteriated n-tetracosane compounds could well have been attached to the macromolecular matrix by additional sulphur bonds. The relatively high concentration of n-tetracosane with 2 deuterium atoms incorporated can also be interpreted by a former alkanoic thiolane which was deuteriated in the desulphurization step but not released until the oxygen bond cleavage. Fragmentation patterns of the other n-alkanes show that a substantial amount of these compounds is linked simultaneously by oxygen and sulphur bonds to the macromolccular networks. Stepwise chemical degradation experiments of other macromolecular oil fractions--i.e, the Rozel Point oil-gave similar results (Richnow et al., 1992). It may be a common feature that n-alkanes are linked at the same time by oxygen and sulphur bonds to the macromolecular network in high sulphur macromolecules.

57

100

198 ./ ,-1 9 9

dl-2 I

71

183 t 50

85

253 t 25#

113

~991i27141 lr3198. ,199 254253283 284 50

150

250 m/z

Fig. 8. Mass spectrum and fragmentation pattern of the deuteriated 2,6,10,14-tetramethylhexadecane

(phytane) obtained from desulphurization of the oil resin.

360

H.H. RaCHNOWet al.

30

A

•~B 29 ~B 31

t

t 23 t ] 24

t

aB 35

t

r~

R

B

.

S

Ni(0)

~B

30

23

z3

zgl f--I

C

~B 32 (z8

>_

~B 35

BC] 3

aB

(zB Z

~"

-~ LJ

t

t

26 ~

28 ~

34 t , 1 30'

35'88

> RT Fig. 9. Ion chromatograms (m/z 191) depicting the distribution of triterpenoids in the hydrocarbon fraction of the Monterey oil (A) and of the following desulphurization (B) and subsequent ether cleavage products (C) of the resin fraction (t = tricyclic terpenoids; black peak = 28,30-bisnorhopane; Greek letters refer to the stereochemistry of hopanes; numbers indicate the number of carbon atoms of the compounds).

Sulphur-rich kerogen and macromolecular oil fraction structures

361

Acyclic isoprenoids

Triterpenoids

Phytane and other acyclic isoprenoids could be liberated by desulphurization and oygen bond cleavage. C40 isoprenoids (biphytane skeleton) seem to be linked via oxygen or sulphur in the macromolecular fractions of the sediment sample [Fig. 5(B, C)]. These isoprenoids are well known archaebacterial lipids and build the carbon backbone of glycerol ether membrane constituents (Langworthy, 1985). Alkyl ethers of this type are thought to be diagenetically stable. In various macromolecular organic substances biphytanes have only been found as oxygen-linked entities (Chappe et al., 1982; Pauly and van Vleet, 1986; Michaelis et al., 1990). Therefore, we were surprised to find these compounds in the low-molecular weight products after the desulphurization. They may have been incorporated into the macromolecular matrix via a reaction between sulphur and functionalized unsaturated derivatives of bacterial ether lipids. The mass spectrum of a deuteriated phytane obtained by desulphurization revealed the position of the former sulphur linkage. Deuterium incorporation between carbon atoms C-I 1 and C-16 or at C-20 (as indicated in Fig. 8) could give rise to the observed fragmentation pattern. Kohnen et al. (1991) found phytane to be linked by polysulphides in polar fractions of immature organic material at similar positions with a preference of a polysulphide linkage at the carbon atoms C-14 and C-15. The biological precursor of macromolecular-bound phytane might be the phytyl side chain of chlorophyll or probably unsaturated phytanyl ethers derived from archaebacteria. Nevertheless, the deuteriation experiments revealed that the site of linkage of phytane is in the same area of the molecules as the functional group in the possible biological precursors.

The distribution of tricyclic terpanes and bopanes in the oil and in the degradation products of the corresponding resin are displayed in Fig. 9. A series of tricyclic terpanes with 21-30 carbon atoms was tentatively identified. The relative abundance of individual compounds varies slightly between the oil and the products after desulphurization and ether cleavage. The binding site of sulphur-linked tricyclic terpenoids was investigated by labelling the positions of sulphur linkages with deuterium atoms during desulphurization. The mass spectrum of the Cz3 compound displays that fragments M + and M+-I5 are labelled with 1 or 2 deuterium atoms, while the fragments m/z 261, 191 and 123 are not shifted to higher masses (Fig. 10). Therefore, the side chain is deuteriated where the former sulphur bond was located. Oxygenlinked tricyclic terpenoids have similar binding sites in low-sulphur asphaltenes and resins (Richnow, 1991). The biological precursor of tricyclic triterpenoids is unknown but it is thought to be tricyclohexaprenol, which can be formed anaerobically by cyclization of hexaprenol, a universal cell constituent (Ourisson et al., 1982; Aqulno Neto et al., 1983). The proposed precursor has an unsaturated, functionalized side chain that might react with active sulphur species and become linked by sulphur bonds to macromolecules (Fig. 11). A series of 17¢¢(H),21//(H)-hopanes in the range of 29 to 35 carbon atoms with a dominance of the 22S over 22R isomers is present in the oil [Fig. 9(A)]. The distribution of hopanes in the degradation products is drastically different. Desulphurization products contain high concentrations of 17=(H),21//(H)pentakishomohopanes (22S/22R). The cleavage of

191

100.

95 I

I

123 9, J

5O

261

123

'rl''

l

''''I''

L

°''''''I'''''''''|'°'''''

100

I''''''

200

300 m/z

Fig. lO. Mass spectrum and fragmentation pattern of a deuteriated tricyclic terpenoid.

H.H. PdCRNOWet al.

362

oxygen bonds yields mainly 17~(H),21fl(H)diastereomers with a high contribution of the 17,¢(H),21fl(H)-bishomohopanes (22S/22R) and lower amounts of 17a(H),21/7(H)-, 17fl(H),21a(H)and 17fl(H),21fl(H)-pentakishomohopanes. The high relative abundance of the 22R-hopane isomers and of the 17fl(H),21~(H)- and 17fl(H),21fl(H)-pentakishomohopanes in the degradation products mirrors a lower degree of maturity than compared to the free hopanes of the oil. It has been observed in several studies that free, extractable alkanes display a more mature distribution than alkanes obtained from pyrolysis or degradation of macromolecular fractions of the same sample (Rubinstein et al., 1979; Mycke and Michaelis, 1986; Adam et al., 1992). There are no experimental results available to support the interpretation of these findings at present. A reasonable explanation is that molecules incorporated into a macromolecular matrix are less accessible to a reaction partner or catalyst. Therefore, the stereocbemical configuration of the biological precursor should be altered at a smaller rate. An alternative explanation might be that the thermodynamic equilibrium is different for a free and a bound hopanoid when the site of linkage between hopanoid and macromolecular matrix is close to the C-atom where stereochemical changes occur. 28,30-Bisnorhopane, abundant in the extractable hydrocarbons, has not been detected in the degradation products of resins, asphaltenes and kerogen.

This compound is probably not a structural unit of organic macromolecules. Saturated hydrocarbons of this type may be produced as such by microbial processes and are not incorporated into macromolecules during early diagenesis due to the lack of functional groups. According to Schoell et aL (1992) 28,30-bisnorhopane may derive from chemoautotrophic bacteria, possibly not yet identified H2S oxidizers. The distribution of hopanoic acids, released in the last step of the asphaltene degradation sequence by the oxidation of aromatic structural entities is displayed by the mass chromatogram of m/z 191 (Fig. 12). A series of 17~(H),21fl(H)-hopanoic acids with 28 to 36 carbon atoms and maximum concentration for the C32 homologue appears. Hopanoic acids with more than 30 carbon atoms are present as 22R- and 22S-diastereomers.

Comparison of kerogen and resin degradation products The distribution of linear carboxylic acids, released by the oxidation of aromatic structural units of the sediment resin fraction are compared with those of the corresponding kerogen (Fig. 13). The main compounds are linear monocarboxylic acids ranging between C9 and C2s with a predominance of the Ct6 carboxylic acid. There are some variations in the relative amounts of individual monocarboxylic acids between the different macromolecular structures as the sediment resin degradation products reveal a

~

OH

cyclisot~.i/~ S-BONDS

active sulphur species

Ni(O)cene/Li/AID4/,/"~emicol degradation DEUTERIATION

Fig. I 1. Proposed scheme for the cyclisation of hexaprenol, the sulphur incorporation and linkage of tricyclic compounds to a macromolecular matrix via sulphur bonds, and their subsequent cleavage by chemical degradation.

Sulphur-rich kerogen and macromolecular oil fraction structures higher relative concentration of Ct0, C12 and C~6 carboxylic acids. Major differences between macromolecular structures exist in the fraction of linear dicarboxylic acids, which occur with up to 22 carbon atoms. The dicarboxylic acids are abundant in the kerogen degradation products but not in the corresponding resin fraction. Dicarboxylic acids are thought to arise from alkyl bridges linking two aromatic entities. The high relative concentration of these compounds provides evidence for a higher degree of cross-linking within the macromolecular structure of kerogen. Our results from chemical degradation are in accordance with a highly crosslinked polymethylene chain network as suggested earlier by Barakat and Yen (1988). Steroids

The distribution of steroids released by chemical degradation of the kerogen is compared with the steranes present in the extract (Fig. 14). A predominance of 5a(H),14~(H),17~t(H)-20R-diastereomers with 26 to 30 carbon atoms and low relative concentrations of the corresponding 20S-isomers in the different fractions is obvious. Furthermore, low abundances of 5a (H), 14//(H), 17//(H)-steranes in the degradation products are observed. These results again suggest a low maturity of the macromolecular

363

organic matter. Differences in the stereochemical configurations between free and bound steroids seem to be caused by comparable mechanisms as discussed for the hopanoids. Significant concentrations of 5//(H)-steranes appear in the degradation products after sulphur and oxygen bond cleavage (Fig. 14). 5//(H)-Cholestan3//-ol can arise from cholesterol as an anaerobic bacterial transformation product (Taylor et al., 1981). If microbial activity resulted in 5//(H)-steroids the bacterial alteration must have taken place before the 5//(H)-steroids were incorporated into the kerogen. Furthermore, side chain reduced Cu steranes are detected in high concentrations only in the oxygenbound fraction. C24 steroids are not known to occur in significant concentrations in organisms. Bacterial degradation of cholesterol under anaerobic conditions results in side chain degraded steroids (Taylor et al., 1981). The occurrence of C24 steroids in the degradation products might be another indication for early diagenetic biodegradation of steroids prior to the linkage of these compounds to the macromolecular network. Compared to the extracts an unusually high amount of steroids with 30 carbon atoms is present in the degradation products. 4-Desmethyl steroids of

=8 32

m/z 191

", RT

Fig. 12. Ion chromatogram (m/z 191) showing the distribution of hopanoic acids (methyl esters) after the oxidation of aromatic structural units of the asphaitene fraction in the last degradation step (Greek letters indicate the stereochemistry; numbers refer to numbers of carbon atoms in the hopanoic acids).

364

H. H, Ricm~ow et al.

100

.

.

.

.

.

.

.

,

.

.

.

.

.

.

.

.

.

I

.

.

.

.

.

.

.

.

~

|

.

.

.

~

.

.

.

.

.

I

.

.

.

.

,

w

.

,

. I

.

.

.

.

.

.

.

.

.

.

j

16

KEROGEN

RuO 4

14

9

18

11

I It, 100

13

KEROGEN 10

15

21

0

.

.

.

.

.

.

.

i

.

.

.

.

.

.

.

.

.

i

.

.

.

.

.

.

.

.

.

,

.

.

.

.

.

.

.

.

,

.

.

.

.

.

.

.

i

.

.

.

.

.

.

.

.

i

16

RESIN

Z

z ^ m

I °

2 ~,JL

RuO 4

12

14

I

I

2S

m/z74 28

.........

1_

m/z 98

--.--> SCAN NUMBER Fig. 13. Ion chromatograms monitoring the distribution and abundance of monocarboxylic acids (m/z 74) and dicarboxylic acids (m/z 98) measured as methyl esters yielded by the oxidation of aromatic structural units of kerogen (with m/z 98 enlarged; above) and of sediment resin Co¢low). Numbers refer to carbon numbers of the alkanoic acids.

Sulphur-rich kerogen and macromoleeular oil fraction structures this structural type could account for marine biological sources (Moldowan et al., 1985). C26 sterols have a wide distribution in samples from marine environments (e.g. Delseth et al., 1979; Bergquist et al., 1986). The relatively high concentration of C26 and

A

28 R

27

m/z 217

29

I r,

B m/z 218

Ni(o)

m/z 217

28 R 29

27

C BCI 3

•26 i 30

28

D

R

365

C30 compounds might be characteristic indicators for the marine depositional environment of the Monterey samples. The position of sulphur bonds which link steroids to the macromolecular network can be investigated by labelling experiments with deuterium atoms during the nickel(0)cene degradation. The mass spectra of 5~(H),14~(H),lT~(H)-24-norcholestane and 5~(I-I), 14~(H),17~(H)-24-propylcholestane demonstrate a deuterium incorporation into the A,B-ring system. These molecules could have been attached to the network via the A,B-ring possibly at the C-2 and C-3 position (Fig. 15) as has been shown in particular for polysulphide-bonded steroidal compounds of macromolecular bitumen fractions (Adam et al., 1991; Kohnen et al., 1991) and for sulphur-bound steroids in kerogens (Hoffmann et al., 1992). Side chain deuteriated steroids are not present in significant amounts. Steroids with a sulphur linkage at the side chain as found by Kohnen et al. (1991) should be present in low concentrations in the investigated macromolecular material. Therefore, sulphur-bound steroids are linked to the macromolecular network similar to oxygen-linked steroids which are attached via the functional group of the biological precursor to the macromolecular material (Mycke and Michaelis, 1986). Steroids linked to aromatic structures are studied by oxidation of the remaining macromolecular network. A series of 3fl-carboxysteranes with 27-31 carbon atoms and a high abundance of the 20Rdiastereomers is obtained by RuO4 oxidation (Fig. 14). 3fl-Carboxysteranes were found previously in ruthenium tetroxide degradation products of macromolecular oil fractions (Trifilieff, 1987; Dany et al., 1990; Richnow et al., 1992). The carboxylic group of the steroid acids derives from the oxidation of an aromatic structural unit of the macromolecular network. The principal site of bonding between the steroid skeleton and the aromatic unit is the A-ring. In general, steroidal skeletons are attached by heteroatomic bonds mainly at the A/B-ring or are bonded to aromatic structural units via this part of the molecule which shows a clear relation to the functional group of the biological precursor. Network/cross -linking

m/z 275 S

In labelling experiments, we investigated in detail the type and site of macromolecularly linked

3O

RuO4

i

=

27

31

> RT

Fig. 14. Ion chromatograms showing the distribution of steroidal compounds in the aliphatic hydrocarbons of the sediment extract (m/z 217; A) and the corresponding kerogen degradation products after desulphurization (m/z 218, due to deuteriation of the A/B-ring; B) and ether cleavage (m/z 217; C) and the carboxylic acids (methyl esters) after the subsequent RuO4 oxidation (m/z 275; D). Black peaks = 14fl(H),17fl(H)-steranes; white peaks = 14=(H), 17=(H)-steranes, R/S=2OR/20S diastereomers; (O)= 5#(H)-steranes; numbers refer to number of carbon atoms of the compounds.

366

H . H . Ricxsow et al.

hopanoids by performing the desulphurization with Ni(0)cene/LiAID4. Similar results have been recently obtained by sequential degradation of macromolecular oil fractions from the Rozel Point oil (Richnow et al., 1992). In Fig. 16 the possible modes of linkages of hopanoids to the macromolecular organic matter are summarized. The biological precursors of hopanoids are pentakishomohopanepolyols (Ourisson et al.,

1987) which are known to be linked to macromolecules via oxygen bonds (Mycke et aL, 1987). The sequential chemical degradation of the sulphur-rich macromolecules reveals further types of linkages. The pentakishomohopanes obtained after desulphurization with Ni(0)cene/LiAID4 are labelled with 2-4 deuterium atoms, indicating that these compounds were linked to the macromolecular network via 2-4 sulphur functionalities at the side chain. In the

5~(H), 14~(H), 17~(H)-24-norcholestane-dI

100 218

o2,8.

150

0

,ll~ll!

50

i

150

250

350

5~(H),14~(H),17~(H)-24-propylcholestane-d 1

100. 218

D1

~

~

C~ .H7

218

150 415

100

201

300

400 m/z

Fig. 15. Mass spectra and fragmentation pattern of 5~(H),140c(H),lT~(H)-24-norcho|estane-dI (above) and 5~(H), 14~(H), ] 7~(H)-24-propylcholcstane-d~ (below) after dcsulphurization of the kerogcn displaying the deuterium incorporation.

Sulphur-rich kerogen and macromolecular oil fraction structures subsequent step, oxygen-bound compounds are released with BCI3 and reduced to hydrocarbons with LiAIH4. A portion of the resulting pentakishomohopanes is non-deuteriated. These compounds were linked exclusively by oxygen bonds. The bulk of the C35 hopanes shows an incorporation of several deuterium atoms which can only derive from the preceding desulphurization step. These labelled compounds must have been linked simultaneously by sulphur and oxygen bonds at the side chain to the macromolecular matrix. Hopanoic acids released during the following oxidation were linked to aromatic entities of the macromolecular network. However, we cannot exclude completely that a minor part of ether linkages in the macromolecular matrix resists the BC13 cleavage reaction. These ethers could give rise to carboxylic acids in our subsequent RuO4 degradation of the residual macromolecular fraction. Mass spectrometric investigation of the hopanoic acids reveal no evidence for deuterium incorporation

FORMATION

OF

suggesting a single alkyl aromatic bond at the side chain. Because we find high relative concentrations of C35 hopanes in the degradation products after desulphurization, the process leading to the formation of sulphur-bonded compounds seems to preserve the carbon skeleton of the biologically derived side chain. Incorporation of inorganic sulphur species into unsaturated lipids might explain the formation of sulphur bonds as suggested by Sinninghe Damst~ et aL (1989). However, the fact that hopanoids are simultaneously bonded by oxygen and sulphur functionalities points to an additional mechanism, a type of reaction which exchanges sulphur for oxygen functionalities but not at all positions. The hopanoids which can be degraded by the oxygen selective technique have been linked via oxygen bonds to macromolecular organic matter. The presence of 17p(H),21p(H)-pentakishomohopane in the oxygen degradation products (Fig. 9) indicates

GEOMACROMOLECULES

OH OH

S

S 0

"

R

"~~SS

I

" OH

-/-l,

_/z_/.. S

367

~

R~~S

o o "~l

"

0 l !

/

\.\"

I

j

Io Ni(O)cene/LiAID4

I. Ni(O)cene/LiAID4

s"

Ill. RuO,

II. BCI3/LiAIH4

D

D

D

H D

H

H

H

SEQUENTIAL CHEMICAL DEGRADATION Fig. 16. Summary of proposed bacteriohopanetetroi incorporation into macromolecular organic matter. Types of bonding and sites of linkage of pentakishomohopanes within the macrocolecular matrix and resulting deuteriation patterns after chemical degradation. 0(3 19/4:6--E

368

H.H. Rac~rNow et al.

that a portion of the bacterial membrane constituents is incorporated into the macromolecular networks without any significant alteration. In comparison to the desulphurization products the hopanoids which were linked by oxygen bonds reveal a trend to higher relative concentrations of hopanes with shorter side chains (Fig. 9). Prior to the attachment of these compounds via oxygen bonds to the macromolecules the side chain should have been degraded. A possible mechanism may be an early diagenetic oxidation of the side chain of C35 hopanepolyols which leads to hopanoic acids, ketones or alcohols. These compounds are observed frequently in extracts from recent sediments (Dastillung et al., 1980; Quirk et al., 1984). Thus, it is possible that side chain degraded compounds could be subsequently linked by oxygen bonds to the macromolecular organic matter. Another pathway of hopane diagenesis may proceed via the incorporation of hopanes into the macromolecular matrix by an attachment to aromatic structural units. The binding of hopanoids to macromolecular aromatic entities could be explained by Friedel~Craft-type reactions claimed by Trifilieff et al. (1992). During chemical oxidation of C35 hopanoids as aromatic substituents the resulting compounds get an additional carbon atom from the aromatic network resulting in a C36 hopanoic acid. The distribution of hopanoic acids from our RuO4 degradation experiments reveals relatively high concentrations of the C31~C33 homologues. Hopanoic ketones, aldehydes, alcohols and olefins of recent sediments already have a degraded side chain. Ketones, aldehydes, alcohols and olefins are well known substrates in FriedeI-Craft-type reactions forming alkyl aromatic substituents. An acid (clay) catalysis may enhance this alkylation reaction (Trifilieff et al., 1992). Therefore, we believe that the observed distribution can be the result of alkylation reactions of early diagenetic hopanoic derivatives. Still, it is difficult to understand how this alkylation could proceed in the presence of water which suppresses this reaction and is abundant even in mature sediments. n-Alkanoic structures also occur as cross-linking units. They can be linked by several sulphur bonds, simultaneously by sulphur and oxygen linkages or by two alkyl aromatic bonds to the macromolecular network. Thus, these compounds are not only present in terminal parts of macromolecules but, concerning their high relative abundance in the degradation products, build up a framework to which other compounds, e.g. steroids, can be attached. CONCLUSIONS The sequential chemical degradation technique described has been shown to be a versatile tool for the evaluation of structural elements in geological macromolecules and was found to be effective in terms of degradation yields.

This technique performed on resins, an asphaltene and a kerogen of the Monterey Formation allowed us to distinguish between the different types of linkages of structural building blocks in the macromolecular structures. Some biomarkers are linked exclusively via sulphur, oxygen or aromatic entities to the networks. We could prove that n-alkanes, hopanoids and isoprenoids are attached to the macromolecules by more than one linkage. Furthermore, some compounds such as hopanoids or n-alkanes are bound simultaneously by sulphur and oxygen bonds. Aliphatic compounds are attached to the macromolecular matrix at the position where the functional group in the biological precursors is found. Differences in the distributions of distinct compound classes are characteristic for each degradation step. Selective diagenetic reactions may account for the different types of bonds of the respective compounds in the process of geomacromolecule formation. The contents of "free" hydrocarbons in an oil and a sediment bitumen are not necessarily representative for the composition of macromolecular materials like oil resins, asphaltenes and kerogens. Distinct structural differences exist between the resin and asphaltene fraction and the kerogen. Thus, macromolecular soluble fractions can not be regarded as sub-units of the kerogen. Acknowledgements--We thank D. Vehlhaber, H. Lamp¢,

U. Kruse and M. Sternhagcn for technical assistance. J. W. de Leeuw (Delft) is acknowledged for providing a number of synthetic standard compounds. We gratefully acknowledge J. R. Maxwell and J. S. Sinninghc Damst6 for reviewing the manuscript. This project was supported by the Deutsche Forschungsgemeinschaft (Mi 157 7-1/2).

REFERENCES

Adam P., Schmid J. C., Connan J. and Albrecht P. (1991) 2,, and 3// steroid thiols from reductive cleavage of macromolecular petroleum fraction. Tetrahedron Lett. 32, 2955-2958. Adam P., Schmid J. C., Myckc B., Strazielle C., Connan J., Huc A., Riva A. and Albrecht P. (1992) Structural investigation of non-polar sulfur cross-linked macromolecules in petroleum. Geochim. Cosmochim. Acta. In press.

Aquino Neto F. R., Trendel J. M., Restle A., Connan J. and Albrecht P. 0983) Occurrence and formation of tricyclic and tetracyclic terpanes in sediments and petroleums. In Advances in Organic Geochemistry 1981 (Edited by M. Bjoroy et al.), pp. 659-667. Wiley, London. Barakat A. O. and Yen T. F. (1988) Preliminary analysis of Monterey kerogen by mild stepwis¢ oxidation with sodium dichromate in glacial acetic acid. Geochim. Cosmochim. Acta 52, 359-363. Bergqulst P. R., Lavis A. and Cambie R. C. (1986) Sterol composition and classification of the porifera. Biochem. Systematics Ecol. 14(1), 105-112. Bhatt M. V. and Kulkarni S. U. 0983) Cleavage of ethers. Synthesis 1983, 249-289. Blanc P. and Albrecht P. (1990) Molecular markers in bitumen and macromolecular matrix of coals. Their evaluation as rank parameters. In Advanced

Sulphur-rich kerogen and macromolecular oil fraction structures Methodologies in Coal Characterization (Edited by H. Charcosset), pp. 53-82. Elsevier, Amsterdam. Carlsen P. H. J., Katsukiti T. and Sharpless K. B. (1981) A greatly improved procedure for ruthenium tetroxide catalyzed oxidation of organic compounds. J. Org. Chem. 46, 3936-3938. Chan M.-C., Cheng K.-M., Li M. K. and Luh T.-Y. (1985) Nickelocene-lithium aluminum hydride. A new effective desulphurization reagent. J. Chem. Soc. Chem. Commun. 1985, 1610-1611. Chappe B., Albrecht P. and Michaelis W. (1982) Polar lipids of Archaebacteria in sediments and petroleum. Science 217, 65-66. Choi C.-Y., Wang S.-H. and Stock L. M, (1988) Ruthenium tetroxide catalyzed oxidation of maceral groups. Energy Fuels 2(1), 37-48. Curiale J. A., Cameron D. and Davis D. V. (1985) Biological marker distribution and significance in oils and rocks of the Monterey Formation, California. Geochim. Cosmochim. Acta 49, 271-288. Dany F., Riolo J., Trendel J. M. and Albrecht P. (1990) 3fl-Carboxysteranes, a novel family of fossil steroids. J. Chem. Soc. Chem. Commun. 1990, 1228-1230. Dastillung P. M., Albrecht P. and Ourisson G. (1980) Cetones aliphatiques et polycycliques dans les sediments: aldehydes et cetones en C27-C35 de la serie hopanique. J. Chem. Res. (M) 1980, 2319-2327. Delseth C., Tolela L., Seheuer P. J., Wells R. J. and Djerassi C. (1979) 14,Sct-24-Norcholestan-3fl-ol and (24 Z)-stigmasta-5,7,24(28)-trien-3fl-ol, two new marine sterols from the Pacific sponges Terpios zeteki and Dysidea herbacea. Helv. Chim. Acta 62(1), 101-109. Eisch J. J., Hallenbeck L. E. and Han K. I. (1983) Desulfurization of dibenzothiophenes by Nickel(0) complexes: evidence for electron transfer in oxidative additions. J. Org. Chem. 48, 2963-2968. George G. N. and Gorbaty M. L. (1989) Sulphur K-edge X-ray adsorption spectroscopy of petroleum asphaltenes and model compounds. J. Am. Chem. Soc. 111, 3182-3186. Hoffmann I. C., Hutchison J., Robson J. N., Chicarelli M. I. and Maxwell J. R. (1992) Evidence for sulphide links in a crude oil asphaltene and kerogens from reductive cleavage by lithium in ethylamine. In Advances in Organic Geochemistry 1991 (Edited by Eckardt C. B., Larter S. R., Manning D. A. and Maxwell J. R.), pp. 371-387. Isaacs C. M. (1984) Monterey--key to offshore California boom. Oil Gas J. 82, 2, 75-81. Jenisch A., Richnow H. H. and Michaelis W. (1990) Chemical structural units of macromolecular coal components. In Advances in Organic Geochemistry 1989 (Edited by Durand B. and Behar F.). Org. Geochem. 16, 917-929. Pergamon Press, Oxford. Kelemen S. R., George G. N. and Gorbaty M. L. (1990) Direct determinations and quantification of sulphur forms in heavy petroleum and coals. 1. The X-ray photoelectron spectroscopy (XPS) approach. Fuel 69, 939-944. Kohnen M. E. L., Sinninghe Damst6 J. S., Kock-van Dalen A. C. and de Lceuw J. W. (1991) Di- or polysulphidebound biomarkers in sulphur-rich geomacromolccules as revealed by selective chemolysis. Geochim. Cosmochim. Acta 55, 1375-1394. Kruge M. A. (1986) Biomarker geochemistry of the Miocene Monterey Formation, West San Joaquin Basin, California: implications for petroleum generation. In Advances in Organic Geochemistry 1985 (Edited by D. Leythaeuser and J. Rullk6tter). Org. Geoehem. 10, 517-530. Pergamon Press, Oxford. Langworthy T. A. (1985) Lipids of Archaebacteria. In The Bacteria VIII (Edited by R. P. Gunsalis), pp. 459--497. Academic Press, New York.

369

Marlowe I. T., Brassell S. C., Eglinton G. and Green J. C. (1984) Long-chain unsaturated ketones and esters in living algae and marine sediments. Org. Geoehem. 4, 135-141. Michaelis W., Jenisch A. and Richnow H. H. (1990) Hydrothermal petroleum generation in Red Sea sediments from the Kebrit and Shaban deeps. Appl. Geochem. 5, 103-114. Michaelis W., Jenisch A., Richnow H. H., Kruse U. and Mycke B. (1988) Organofazies des Olschiefers yon Messel. Cour. Forsch. Inst. Senckenberg 107, 89-103. Moldowan J. M., Seifert W. K. and Gallegos E. J. (1985) Relationship between petroleum composition and depositional environment of petroleum source rocks. Am. Assoc. Pet. Geol. Bull. 69, 1255-1268. Mycke B. and Michaelis W. (1986) Molecular fossils from chemical degradation of macromolecular organic matter. In Advances in Organic Geochemistry 1985 (Edited by Leythaeuser D. and Rullk6tter J.). Org. Geochem. 10, 847-858. Pergamon Press, Oxford. Mycke B., Narjes F. and Michaelis W. (1987) Bacteriohopanetetrol from chemical degradation of an oil shale kerogen. Nature 326, 179-181. Orr W. L. (1986) Kerogen/asphaltene/sulfur relationship in sulfur-rich Monterey oils. In Advances in Organic Geochemistry 1985 (Edited by Lcythaeuser D. and Rullk6tter J.). Org. Geochem. 10, 499-516. Pergamon Press, Oxford. Ourisson G., Albrecht P. and Rohmer M. (1987) The hopanoids. Pure Appl. Chem. 51, 709-729. Ourisson G., Albrecht P. and Rohmer M. (1982) Predictive microbial biochemistry--from molecular fossils to procaryotic membranes. Trends Biochem. Sci. 7, 236-239. Pauly G. G. and van Vleet E. S. (1986) Archaebacterial ether lipids: natural tracers of biog¢ochemical processes. In Advances in Organic Geochemistry 1985 (Edited by Lcythaeuser D. and Rullkttter J.), pp. 859-867. Pergamon Press, Oxford. Pelet R., Behar F. and Monin J. C. (1986) Resins and asphaltenes in the generation and migration of petroleum. In Advances in Organic Geochemistry 1985 (Edited by Leythaeuser D. and Rullk6tter J.), pp. 481-498. Pergamon Press, Oxford. Quirk M. M., Wardroper A. M. K., Whealey R. E. and Maxwell J. R. (1984) Extended hopanoids in peat environments. Chem. Geol. 42, 25-43. Richnow H. H. (1991) Die chemische Charakterisierung yon heteroatom-gebundenen Strukturelementen in makromolekularen Substanzen der Geosph/ire. Ph.D. dissertation, Univ. Hamburg. Richnow H. H., Jenisch A. and Michaelis W. (1992) The chemical structure of macromolecular oil fractions of the sulphur-rich Rozel Point oil. Geochim. Cosmochim. Acta. In press. Rubinstein I., Spyckerelle C. and Strausz O. P. (1979) Pyrolysis of asphaltenes: a source of geochemical information. Geochim. Cosmochim. Acta 43, 1~. Rullkttter J. and Michaelis W. (1990) The structure of kerogen and related materials--a review of recent progress and future work. In Advances in Organic Geochemistry 1989 (Edited by Durand B. and Behar F.), pp. 829-852. Pergamon Press, Oxford. Sehmid J. C. (1986) Marqueurs biologiques soufrts dans les pttroles. Ph.D. dissertation, Universit6 Louis Pasteur, Strasbourg. Sehoell M., McCaffrey M. A., Fago F. J. and Moldowan J. M. (1992) Carbon isotopic compositions of 28,30-bisnorhopanes and other biological markers in a Monterey crude oil. Geochim. Cosmochim. Acta 56, 1391-1399. Sinninghe Damst6 J. S., Rijpstra W. I. C., de Leeuw J. W. and Schenck P. A. (1988) Origin of organic sulphur compounds and sulphur-containing high molecular weight substances in sediments and immature crude oils. In Advances in Organic Geochemistry 1987 (Edited by

370

H.H. P-dCHNOWet al.

Mattavelli L. and Novelli L.), pp. 593-606. Pergamon Press, Oxford. Sinninghe Damst6 J. S., Rijpstra W. I. C., Kock-van Dalen A. C., de Leeuw J. W. and Schenck P. A. (1989) Quenching of labile functionalised lipids by inorganic sulphur species: evidence for the formation ofsedimentary organic sulphur compounds at the early stage of diagenesis. Geochim. Cosmochim. Acta 53, 1343-1355. Standen G., Boucher R. J., Rafalska-Bloch J. and Eglinton G. (1991a) Ruthenium tetroxide oxidation of natural organic macromolecules: Messel kerogen. Chem. Geol. 91, 297-313. Standen G., Patience R. L., Boucher R. J. and Eglinton G. (1991 b) Ruthenium tetroxide oxidation: the assessment of putative petroleum source rocks. In Organic Geochemistry. Advances and Applications in the Natural Environment (Edited by Manning D. A. C.), pp. 454-457. University Press, Manchester. Stock L M. and Tse K. (1983) Ruthenium tetroxide catalysed oxidation of Illinois No. 6 coal and some representative hydrocarbons. Fuel 62, 974-976. Taylor C. D., Smith S. O. and Gagosian R. B. (1981) Use of microbial enrichments for the study of the anaerobic degradation of cholesterol. Geochim. Cosmochim. Acta 45, 2161-2168.

Trifilieff S. (1987) Etude de la structure des fractions polaires de p6troles (r~sines, asphalt6nes) par d6gradations chimiques s~lectives. Ph.D. dissertation, Universit6 Louis Pasteur, Strasbourg. Trifllieff S., Sieskind O. and Albrecht P. (1992) Biological markers in petroleum asphaltenes: possible mode of incorporation. In Biological Markers in Sediments and Petroleum--A Tribute to Wolfgang K. Seifert (Edited by Moldowan J. M., Albrecht P. and Philp R. P.), pp. 350-369. Prentice Hall, Englewood Cliffs. Vitorovi~ D. (1980) Structural elucidation of kerogen by chemical methods. In Kerogen. Insoluble Organic Matter from Sedimentary Rocks (Edited by B. Durand), pp. 301-338. Editions Technip, Paris. Volkman J. K., Eglinton G., Corner E. D. S. and Sargen J. R. (1980) Novel unsaturated straight-chain C3~-C39 methyl and ethyl ketones in marine sediments and a coccolithophore Emiliania huxleyi. In Advances in Organic Geochemistry 1979 (Edited by Douglas A. G. and Maxwell J. R.), pp. 219-227. Pergamon Press, Oxford. Waldo (3. S., Carlson R. M. K., Moldowan J. M., Peters K. E. and Penner-Hahn J. E. (1991) Sulfur speciation in heavy petroleums: information from X-ray adsorption near-edge structure. Geochim. Cosmochim. Acta 55, 801-814.