Chapter 1 Introduction 1.

1 Biomarkers, bound biomarkers and geochemical fractions

1.1.1 Biomarkers and Bound biomarkers Biomarkers are organic compounds with a chemical structure that has an unambiguous link to a natural biological product. Most biomarkers are lipid components that have lost oxygen-containing functional groups that were present in the original biological precursor during burial and diagenesis and have been converted into hydrocarbons that are preserved in the fossil record. Biomarkers are often referred to as chemical fossils as they are the direct fossil remains of once living organisms. Biomarkers present in the hydrocarbon component of petroleum and sedimentary organic matter can be analysed by gas chromatography and gas chromatography mass spectrometry (Peters and Moldowan, 1993). Biomarkers are also present in the pyrolysis and chemical degradation products of the non-hydrocarbon fraction of petroleum and sedimentary organic matter (Gallegos, 1975; Seifert, 1978; Rubinstein et al., 1979; Mycke and Michaelis, 1986). The biomarkers present in the non-hydrocarbon fractions are not analysed by the methods most commonly used to analyse biomarkers; this is because they are covalently bound into larger molecules that can not be analysed by gas chromatography. As a large quantity of biomarkers can be present in kerogens and related materials (Eglinton and Douglas, 1988) the potential exists for a significant component of the biomarkers present in the fossil record to be routinely overlooked. The term bound biomarker is used to describe biomarkers present in the nonhydrocarbon component, the majority of which are linked by covalent bonds within a macromolecular network and are, thus, chemically bound into a host matrix. These macromolecules can be separated from petroleum and sedimentary organic matter into different geochemical fractions based on their chemical characteristics.

1.1.2 Geochemical fractions In organic geochemistry, the laboratory fractionation of petroleum and sedimentary organic matter is achieved by treating a sample with gradually less polar solvents; this has led to the adoption by most workers of a fractionation scheme similar to the one shown in Figure 1.1 (redrawn from Tissot and Welte, 1984). The kerogen, asphaltene and resin fractions are the non-hydrocarbon components that are the highest molecular weight constituents of sedimentary organic matter that are not normally analysed in the molecular characterisation of biomarkers.

Sedimentary organic matter Extraction extracted residue Kerogen EOM kerogen

Bitumen n-heptane insoluble Asphaltene asph & resins Resin Aromatic Hydrocarbons Aliphatic Hydrocarbons aro ali

Maltene Column Chromatography

Figure 1.1. Based on Tissot and Welte (1984). The composition of organic matter as defined by the solubility of its components in organic solvents (left), and the relative proportions of the different fractions present in sedimentary organic matter (right).

The different fractions in Figure 1.1 are operationally defined, but because different solvents and procedures can be used to separate similar fractions, the same name or terms are often applied to the products of varying fractionation procedures. This is most

often encountered for asphaltenes where both different solvents and procedures can be used as described in Chapter 2. Kerogen is the insoluble organic matter dispersed in sediments and insoluble in organic solvents (Durand, 1980), and the term is used to describe the largest most insoluble, polymeric material left after solvent extraction of sedimentary organic matter. The isolation of a clean kerogen fraction requires solvent extraction of the bitumen I fraction, prior to a stage of demineralisation and further solvent extraction to remove the bitumen II fraction. The term asphaltene is applied to the least soluble fraction of oils and extracts (kerogen comprises the bulk of the organic matter in Figure 1.1) that precipitates in petroleum ether, and the term maltene to the fraction that is soluble. Asphaltene fractionation procedures can vary considerably and these are described further in Chapter 2. The term resin was originally applied to bitumen constituents that were soluble in petroleum ether but that adsorbed onto fullers earth. More commonly the term is applied to the non-hydrocarbon component of the maltene fraction that elutes in the most polar solvents in separation by chromatographic methods. As shown on the right hand side of Figure 1.1, the kerogen fraction is quantitatively the largest fraction. The bitumen fraction, including the asphaltene and resin fractions, comprises a much lesser proportion of the total sedimentary organic matter (Tissot and Welte, 1984). Solvent extracted biomarkers usually only comprise a very small proportion of the bitumen fraction (Peters and Moldowan, 1993), and hence an even smaller proportion of the sedimentary organic matter.

1.2 Kerogen and related materials

Kerogen, asphaltenes and resins are often referred to as related materials (Rullktter and Michaelis, 1990) as they have overlapping elemental and chemical compositions and are interpreted to have structural similarities.

1.2.1 Overlapping elemental composition

Kerogen and related materials are chiefly composed of carbon and hydrogen with minor amounts of oxygen, sulphur and nitrogen, and the elemental composition reflects the level of thermal maturity as well as the chemical composition of organic matter and redox conditions in the environment of deposition. Its elemental composition and its tendency to generate oil or gas can be used to classify kerogen as one of three types: Type I kerogen contains the remains of algal organic matter and amorphous material that has been extensively reworked by bacteria. It has the highest H/C ratio and the lowest O/C ratio of the three groups and is most prone to generate oil and gas. Type II kerogen has inputs from autochthonous phytoplankton, zooplankton and bacterially derived organic matter that may have undergone extensive reworking. Allochthonous material can also be present in relatively small proportions and may comprise higher plant debris and plant membrane secretions, as well as highly oxidised inert material. Type II kerogens have H/C ratios slightly lower than Type I, and higher O/C ratios; additionally, some Type II kerogens contain large amounts of sulphur (atomic S/C ratio > 0.04; Type II S). Type III kerogens are formed predominantly from the remains of vascular plants. They contain the identifiable remains of vegetation debris in addition to microbially-degraded and oxidised material. Type III kerogens have lower H/C ratios and higher O/C ratios than other kerogen types of similar maturity and are most prone to generate gas. The thermal evolution of the three types of kerogen can be represented diagrammatically on a Van Krevelen diagram (Figure 1.2, adapted from Killops and Killops, 1993). This charts the change in the H/C and O/C ratio of the three kerogen types during thermal evolution. The H/C and O/C ratio of a number of oil asphaltenes and resins are plotted in Figure 1.2. It can be seen that the H/C ratios of oil asphaltenes and Type II kerogens are virtually the same at the onset of oil generation (Bandurski, 1982), although the asphaltene O/C ratios are much lower than those of the kerogen. This is interpreted to represent the creation of asphaltenes during kerogen cracking by 4

the loss of oxygen, which forms covalent cross links between different structural units in the original parent kerogen macromolecules (Pelet et al., 1985). A continuation of the same pattern is observed in the generally lower resin O/C values, suggesting that there is less cross-linking in resins than in asphaltenes. However, unlike the asphaltenes, the resins have much higher H/C ratios than kerogens, which have been interpreted to represent greater aromatisation in asphaltenes than in resins (Koots and Speight, 1975).

AsphUC ResUC type I AsphLC ResLC type II AsphC ResC AsphUD type III ResUD AsphLD 0 0.02 O/C 0.04 ResLD

1.5 H/C 1 0.5

Figure 1.2. Van Krevelen diagram showing the classification of kerogens by their H/C and O/C ratios, and how these change with thermal evolution. H/C and O/C values of resins and asphaltenes are also plotted (Koots and Speight, 1975). Asph = asphaltene; Res = resin. Reservoir age: UC = Upper Cretaceous, LC = lower Cretaceous, C = Carboniferous, UD = Upper Devonian, LD = lower Devonian.

Asphaltenes and resins can be separated into a number of different cuts (or subfractions), based on their relative solubility in different solvents. The H/C values (Pelet et al., 1985) of these different cuts of resin and asphaltene fractions, as well as the range of H/C ratios expected from a Type II kerogen are plotted in Figure 1.3. With the exception of the top 20th percentile (above 80% in the graph on the left hand side of Figure 1.4) of the asphaltene fraction that are the most soluble in the least polar solvents and have values closest to those of the resin fraction, the asphaltene and kerogen data are relatively homogeneous and have a fairly uniform distribution of H/C ratios. The resins appear to be far more heterogeneous and show considerably more variation.

1.5

H/C ratio

1.4

free resin res asph

H/C ratio

1.3 1.2

asphaltene
1.1 1 0 20 40 60 80 100

Avg kerogen

kerogen 100 10 Cumulative % of Organic Matter 1

Cumulative weight % of fraction

Figure 1.3. The graph on the left hand side shows the distribution of H/C values for the different fractions in sedimentary organic matter, the graph on the right hand side represents an idealised view of sedimentary organic matter in which the H/C values of the different fractions overlap. Resin and asphaltene data are from Pelet et al. (1986), and were obtained by separating resin and asphaltene fractions using different polarity solvents and mixtures of solvents to subdivide resin and asphaltene fractions. Kerogen data corresponds to the range of H/C values of a Type II kerogen during mid catagenesis (Killops and Killops, 1993).

Taking the idea of a continuum in elemental composition further, it can be envisaged that a range of macromolecular compounds exist with overlapping elemental compositions and sizes (right hand side of Figure 1.3). The compounds at the kerogen end are the largest molecules and have the lowest H/C values, while the compounds at the resin end have the highest H/C values and the smallest molecular size. One explanation given for this is that larger macromolecules are converted into smaller macromolecules by the thermal cracking and loss of hydrogen and heteroatoms during thermal evolution (Pelet et al., 1986); the overlap in H/C values represents the genetic link between kerogens, asphaltenes and resins. Another possible explanation for the existence of different-sized asphaltene and resin macromolecules with distinctive H/C and O/C ratios is that they are generated from different types of kerogen constituents (macerals). Three pieces of evidence support this: 1) The large step between the asphaltene and resin H/C values. Only the smallest/least polar asphaltenes overlap the resins in Figure 1.3. The two populations in Figure 1.3 are actually quite different, as can be seen on the graph in Figure 1.2, and do not really overlap as commonly reported (Koots and Speight, 1975).

2) Resins (even those from Type II and Type III organic matter) plot next to Type I kerogens in a Van Krevelen diagram (Pelet et al., 1986), whereas asphaltenes from different types of organic matter plot next to Type II kerogens (i.e. they all have similar H/C values). 3) Resins comprise the greatest proportion of an extract in Type I source rocks, and asphaltenes the greatest proportion in Type II source rocks (Pelet et al., 1996). These factors suggest a genetic link between resins and Type I kerogen, and asphaltenes and Type II kerogen. As most organic-rich mudstones contain a range of kerogen types (i.e. is composed of organic matter from different biological sources from different environments), they contain both resins and asphaltenes in their bitumens. 1.2.2 Structural similarities Study of the structure of kerogen and related materials has normally been closely linked to the study of petroleum generation seeking to match compounds observed in extracts and bitumens to equivalent moieties present in kerogen degradation products (Rullktter and Michaelis, 1990). In this context bound biomarkers represent very small structural components that are themselves only constituents of larger structural base units. Kerogen is a three-dimensional macromolecule consisting of a number of base units of aromatic nuclei with bridge-like alkyl chain cross links. Various carbon, oxygen and sulphur species serve to covalently cross-link structural components together. The nuclei consist of varying numbers of condensed rings which can possess a range of types of N, S, O, alkyl chain, cycloalkanes or other functional group substitution (Killops and Killops, 1993). The proposed structure of a kerogen nucleus (Figure 1.4) is very similar to that of an asphaltene molecule or aggregate (Yen, 1981).

aromatic sheet units

alkyl chains

n
Figure 1.4. Conceptual model of the base units that comprise kerogen, n > 1 for kerogens, n ~ 1 for asphaltenes, and n < 1 for resins (Yen 1981). Aromatic sheets are represented by rectangles, and alkyl chains by zigzags. N, S, O atoms can form covalent bonds that link compounds to the aromatic and alkyl structures, as well as cross links between different units to make larger structures.

From a geochemical point of view, kerogen is thought to comprise asphaltene-like subunits bound together by oxygen (Pelet et al., 1986) and sulphur (Sinninghe Damste et al., 1989). The breaking of these bonds releases asphaltenes, which possess structural similarities to kerogen. Resins have been suggested to arise from the breaking of asphaltene molecules, which gives rise to a smaller more heterogeneous range of compounds (Pelet et al., 1986). However, resins could also be generated directly from kerogen (see previous discussion), and small quantities of asphaltenes and resins might also form during kerogen formation or enter into the fossil record prior to oil generation or catagenesis as macromolecules that are the same molecular weight and have the same range of solubilities in solvents as the resins and asphaltenes that are formed during early catagenesis. It is important to realise that kerogen, asphaltene and resin molecules not only have different structural compositions, but that their structures also undergo different structural evolution with rising thermal maturity. Across the oil window and during catagenesis kerogen undergoes a structural evolution that involves four main changes: 1) aromatisation; 2) the removal of heteroatoms and rise in proportion of carbon-carbon cross links; 3) the loss of aliphatic units and other functional groups, and rise in proportion of methyl groups; 4) a shortening in the average aliphatic chain length (Table 1.1). This represents a reordering of the basic kerogen structure to a more ordered and less bulky one, and is accompanied by a rise in % Ro and changes in other optical properties (e.g. spore colouration index) as the structure becomes darker, more dense and graphitic (Killops and Killops, 1993). 8

Table 1.1. Changes in structure with thermal evolution

Aromatic carbon Aliphatic carbon Heteroatom content Aliphatic chain-length Based on Michels et al. (2000) Kerogen Increase Decrease Decrease Decrease Asphaltene Increase Decrease Increase Decrease Resin Increase Decrease Little Change Increase

A recent artificial maturation study of kerogen, asphaltenes and resins across the oil window suggests that some of these changes occur in the resin and asphaltene fractions but not others (Michels et al., 2000). Structural features that evolve in a similar way in kerogen, asphaltenes and resins include the general loss of aliphatic units and rise in aromaticity. Structural features that do not evolve in a similar way in the four fractions include average aliphatic chain length and oxygen content. Average aliphatic chain length increases in the resin fraction and decreases in the kerogen and asphaltene fractions. Oxygen content is low in the resin fraction and remains so, whilst the proportion of oxygen functional groups may actually increase in the asphaltene fraction and decreases in the kerogen fraction. The hydrogen index decreases fairly constantly in the asphaltene and kerogen fractions, whilst changing much less in the resin fraction. These observations suggest that kerogens, asphaltenes and resins share structural similarities, but that subtle differences exist and thermal evolution will bring about different structural changes in each fraction. In summary, kerogen and related materials have similar elemental compositions and a common structure, although small differences exist in both the elemental composition, and the effects of thermal evolution on the macromolecule structure and elemental composition.

Free HC kerogen asphaltenes resins

lighter HC heavier HC

High H/C kerogen Low H/C

Figure 1.5. Two models of asphaltene and resin formation during thermal maturation. The one in the left is from Pelet et al. (1986), and shows kerogen generating asphaltenes, that can crack to make resins, which in turn crack to make hydrocarbons. The other figure on the right hand side is based on a review of published data and suggests that resins and asphaltenes are generated from different kerogen types, the implication being that the fractions might reflect different biological inputs.

Resin, asphaltene and kerogen bulk data can be interpreted in one of two ways (Figure 1.5). One, consistent with structural models, has asphaltenes as intermediates between kerogen and resins and hydrocarbons. The other model, proposed after a review of elemental data undertaken in this current study, considers asphaltenes and resins to be generated from different kerogen types. However, both models contain, and need to allow for elements found in the other, e.g. direct generation of resins in the first model and generation of resins from asphaltenes in the second, suggesting that neither can provide an all-encompassing description of the origins of the different fractions present in sedimentary organic matter. The implications of the second model are that the different polar fractions (resin and asphaltenes) of organic matter could be derived from different pools of sedimentary organic matter with different geological and biological associations; this could imply that they might contain different bound biomarker pools which could also reflect these varying associations. Additionally, as the different fractions respond to thermal maturation in different ways, perhaps their biomarker pools will also respond in different ways.

asphaltenes
10

resins

1.3 Origin of bound biomarkers

Biomarkers are present in geomacromolecules because they were present in the original biomacromolecule, or because they have become incorporated into the macromolecular component of sedimentary organic matter during subsequent phases of diagenesis and kerogen formation. There are three kerogen forming mechanisms during which biomarkers can become incorporated into the macromolecular component of sedimentary organic matter, along with an additional method by which biomarkers can be present in the polar macromolecules (resins and asphaltenes) (Killops and Killops, 1993); these are shown in Figure 1.6 and include selective preservation, condensation, vulcanisation and incorporation.

Bound biomolecules
rese rvat ion

Free biomolecules Selective preservation

Vu lc a nis ati on

Sele c

Co nd en

Incorporation Free biomarkers Soluble geomacromolecules

Insoluble geomacromolecules (kerogen) Biomarker generation

Figure 1.6. Routes for biomarker incorporation from biomolecules and biomacromolecules at the top, to geomacromolecules at the bottom.

1.3.1 Selective preservation Selective preservation proceeds by the removal of the labile components of organic matter such as proteins, carbohydrates and nucleic acids, and the accumulation of more recalcitrant biomacromolecules such as algaenans and lignins (Tegelaar et al., 1989). This is supported by the presence of algaenans in Tetrahedron algal cell wall material, of the Messel oil shale kerogen (Goth et al., 1988). Biomarkers are preserved in the form of the constituents of macromolecules, but resistant molecules such as hopanes

Diagenesis

tive p

sa tio n

&

11

and tricyclic terpanes can also be preserved as hydrocarbons (Peters and Moldowan, 1993). 1.3.2 Classical condensation mechanism In aquatic environments microbial degradation breaks down biomacromolecules in sedimentary organic matter into smaller units, which subsequently undergo an initial phase of condensation to make humic substances. Further polycondensation reactions act on humic compounds removing functional groups (alcohols and acids), and with increasing burial depth will produce larger and increasingly insoluble geopolymers (Figure 1.7). At the end of diagenesis kerogen-sized compounds have been produced that contain lipid components, including biomarkers that are attached and bound into the larger molecules at the sites of former acid and alcohol functional groups (Tissot and Welte, 1984).

Figure 1.7. Two important kerogen polymerisation reactions. Condensation involves the expulsion of water and involves reaction between two oxygen containing functional groups. Vulcanisation proceeds via sulphur incorporation into lipids at the locations of unsaturation, leading to the creation of sulphur bonds between molecules to create high molecular weight compounds.

1.3.3 Vulcanisation Sulphur incorporation into sedimentary organic matter occurs in environments where sulphide is not exclusively taken up by Fe2+, and free hydrogen sulphide and polysulphides are produced. Reactions between hydrogen sulphide and unsaturated C=C bonds produce thiol groups. High molecular weight compounds, and eventually

12

sulphur rich macromolecules, are produced by intermolecular addition which proceeds by the reaction of thiol groups with unsaturation sites on other molecules (Sinninghe Damste et al., 1989). Sulphur incorporation is also an important mechanism for the preservation of extended C35 hopanes and C40 isorenieratene derivatives in macromolecular sedimentary organic matter (Peters and Moldowan, 1991; Kohnen et al., 1989). 1.3.4 Non-kerogen bound biomarkers The preservation and incorporation of biomarkers into soluble organic matter (either as free hydrocarbons or in macromolecules) is not a quantitatively important process in most marine mudstones. However, a number of studies have shown significant quantities of bound biomarkers to be present in the non-hydrocarbon fractions of soluble organic matter. These include the Green River Shale (Eglinton and Douglas, 1989), where the bitumen phase was found to contain a large proportion of biomarkers and a Messinian Marl from the Gesso-Solfifera formation (Sinninghe Damste et al., 1989; Kohnen et al., 1991; Schaeffer et al., 1998) which is also unusual because of the large amount of extractable polar material that it contains. In general, at the onset of oil generation, most of the bitumen phase (especially polar material) is thought to have been predominantly derived from the kerogen fraction (Pelet et al., 1986). 1.3.5 Bound biomarkers and macromolecule structure Bound biomarkers are often represented as shown in Figure 1.8, with biomarkers shown as discrete entities, bound either onto or into the host macromolecular network by one or more binding sites (Rullktter and Michaelis, 1990). The type of link is important as it will relate to the process responsible for the incorporation of biomarkers into macromolecules, and to the nature and position of the functional groups that were originally present in the precursor biomarker. Cross-linking oxygen groups develop as a consequence of condensation reactions via the classical kerogen formation process but may also be present in the original biological materials, while sulphur bonds develop during intra-molecular sulphur incorporation. Carbon-carbon bonds and direct bonds to aromatic nuclei, although present in sedimentary organic matter in low proportions prior to burial (e.g. in lignins), become gradually more important at latter stages of burial and diagenesis due to the elimination of other bond types and the formation of new aromatic units.

13

Biomarkers may be bound at terminal positions if they only contain one binding position as is the case with most steranes, or serve as bridges between different areas of the macromolecular network when they contain more than one binding position (Hoffman et al., 1991; Kohnen et al., 1991). It is also important to note that different bond types may bind the same biomarker into a macromolecule, and that in many instances more than one bond type will bind the same biomarker (Richnow et al., 1991; Koopmans et al., 1996c).
R = biomarker X= X R X X R S O C O C O

Figure 1.8. Schematic representation of biomarker binding mechanisms. Oxygen bonds are important for the incorporation of biomarkers into macromolecules via the classical and selective preservation processes, and sulphur bonds are important for the incorporation of biomarkers during vulcanization. Kerogen bonds and carbon links to aromatic units, although present in some starting materials such as lignins, increase in importance at latter stages of burial and diagenesis.

1.4 Biomarkers
The following section reviews the biomarker types for which results are presented in this thesis. For each biomarker type background information is presented for reference purposes as well as important and frequently overlooked work on bound biomarkers. 1.4.1 Sterane
1.4.1 Sterane precursors

Sterols, the precursors of steranes, are tetracyclic saturated terpanes with a double bond located in the B ring (stenols) and hydroxyl functionality located at C-3 on the A ring. Two sterols, cholest-5-en-3-ol present in animals and plants and stigmasterol present in higher plants are shown in Figure 1.9. Various sterols can have side chains of different lengths, and the usual homologous series of steranes ranges from C27 to C30. A 14

double bond is commonly situated between the C-5 and C-6 position in the B ring, but can also be present between the C-7 and C-8 position, or at both positions in the case of ergosterol (Killops and Killops, 1993; Peters and Moldowan, 1993). Other steroid structural variations include double bonds present in the side chains of a number of sterols (Figure 1.9), an additional methyl group situated at the C-4 position (4methylsterol) and at the C-23 position (dinosterol). Sterols are thought to be biosynthesised by a range of eukaryotes as membrane rigidifiers; their limited occurrence in other organisms is thought to represent uptake rather than biosynthesis, or limited production in some methanotrophic bacteria (Schouten et al., 2001).

OH

29 28

OH
21 20 18 12 11 19 1 2 3 4 10 5 6 9 8 7 14 13 17

22 23

24 25 27

26

16 15

Figure 1.9. Structures of Cholesterol and Stigmasterol, present in algae and higher plants, that are two of the precursors for C27 and C29 steranes. The numbering system used for steranes is also shown

15

1.4.1.2 Bound steranes

A review of previous literature would suggest that steranes in macromolecules are predominately bound by a single bond located in their A-ring (Figure 1.10) via an ether or sulphur linkage located at the position of the alcohol group in the cholesterol precursor (Mycke and Michaelis 1986; Kohnen et al., 1991; Hofmann et al., 1991; Richnow 1991; Peng et al., 1997; Peng et al., 1999; Strausz et al., 1999). However, a number of studies suggest that limited binding can also occur at side chain positions (Hoffman et al., 1991; Kohnen et al., 1991; Peng et al., 1997; Strausz et al., 1999), and perhaps even B-ring positions (Kohnen et al., 1991), resulting from sulphur incorporation at the positions of unsaturation in the sidechain, and C-5 and C-6 positions respectively.

D and CO2CH3

D
Figure 1.10. Sites of deuterium incorporation (Hofmann, et al., 1991; Richnow et al., 1991). By far the most common binding site reported for steranes is in the A ring (assumed to be the C-3 OH group site). Side chain positions have been reported for sulphur binding in the larger C29 steranes (note double bond position in Figure 1.9 and discussions on sulphur incorporation in Figure 1.5).

The dominance of the A-ring binding position has led to the general conclusion that most steranes will be located at terminal positions of their macromolecular networks (Mycke and Michaelis 1986; Kohnen et al., 1991), and therefore they do not take part significantly in cross linking between different macromolecular sub-units. Bound steranes and methyl steranes have been reported (Gallegos, 1975; Mycke and Michaelis, 1986), whereas diasteranes are generally thought not to be bound into the sedimentary macromolecular organic matter present in source rocks (Seifert, 1978). Diasteranes form via diasterenes, in which the unsaturation (C=C bond) located between the C-X and C-Y positions is in a hindered position within the ring structure,

16

this probably hinders and or retards reactions that lead to the binding of steranes into macromolecules (De Leeuw et al., 1989).
1.4.1.3 Fossil steranes

Three major reaction pathways are thought to act on biologically derived steroids during diagenesis. One involves the loss of the A-ring alcohol group and double bond saturation and leads to the regular series of C27 to C30 fossil steranes. Other pathways involve clay-catalysed backbone rearrangement of a series of sterenes to create diasterenes the precursors of diasteranes (De Leeuw et al., 1989; van Kaam- Peters et al., 1998), and the aromatisation of the sterane C-ring, and then A- and B-rings to create triaromatic steroids. Pregnanes, and other steranes with carbon numbers lower than most known biological cholesterol precursors, are present in many oils and source rocks. These have been proposed to be related to the effects of thermal maturation and cracking of higher carbon number homologues (Mackenzie et al., 1982), although more recent work suggests that these compounds and their aromatic analogues could be source-specific indicators (Li and Jiang, 2001). Other work has shown that the proportion of these compounds in hydrous pyrolysates, relative to higher sterane homologues, can be linked to the severity of pyrolysis conditions (Fowler and Brooks, 1987). However, this does not mean that they are entirely derived from the cracking of higher homologues, and bound pregnanes have been reported in chemical degradation studies of both immature and mature oil asphaltenes (Peng et al., 1997; Strausz et al., 1999).
1.4.1.4 Sterane isomerisation

Four chiral centres are important in C27 to C30 fossil regular steranes, at C-5, C-14 and C-17 positions in the ring system positions and at C-20 in the side chain, as shown in Figure 1.11. A C-5 chiral centre is not present in the cholesterol biologically derived precursors, but is present in fossil steranes after the loss of the double bond. Thermal evolution post diagenesis brings about a rise in the proportion of the (20S) isomer relative to the biologically synthesised (20R) isomer to a composition of 55 % (20S)/ (20S)+(20R), and a rise in the proportion of the 5(H), 14(H), 17(H) isomer relative to the 5(H),14(H),17(H) isomer to approximately 80 %. The 5() isomer is dominant in most fossil steranes, although the 5(H) isomer is found in relatively 17

immature samples as a transformation product (Taylor et al., 1991) and, rather importantly as far as this study is concerned, in the degradation products of macromolecules (Richnow et al., 1991). The isomer composition of bound steranes is generally less thermally mature than their free counterparts (Rubinstein et al., 1979), and sterane isomerisation appears to proceed more slowly in kerogen-bound steranes compared with free steranes across the oil window (Murray et al., 1998).

20

17

14

5 Figure 1.11. Main isomerisation centres in steranes. Major cyclic centres occur at the C-5, C-14, C17 (H) positions. Isomer shown is 5a(H), 14a(H), 17a(H) with an unspecified C-20 configuration. 1.4.1.5 Steranes as source indicators

The relative proportion of C27-C29 compounds plotted on a ternary diagram can be used for source input discrimination (Grantham and Wakefield, 1988), whilst other steranes have more specific associations. C29 steranes (24-ethylcholestanes) are often associated with C29 sterols derived from higher plants, and the C27/C30 ratio is often used as an indicator for marine conditions as C30 steranes are derived from 24-n-propylcholestanes present in Chrysophyte algae, which are exclusively marine organisms (Peters and Moldowan, 1993). Dinosteranes have a clear association with dinosterol which is found in dinoflagellates (largely marine and exclusively aquatic organisms). Pregnane distributions have been used for source discrimination, even though they have been proposed to be diagenetic products sensitive to thermal maturation (Li and Jiang, 2001). 1.4.2 Hopanes
1.4.2.1 Hopane precursors

18

Fossil hopanes are derived from bacteriohopanepolyols, diploptene and diplopterol precursors, although it is the C35 bacteriohopanepolyols such as bacteriohopanetetrol that constitute the major triterpenoids in eubacteria (Rohmer et al., 1984). Figure 1.12 shows the structures of the biologically produced C30 and C35 hopanoids, which can have a wide variety of functional groups on their side chains as well as methyl groups at the C-2 or C-3 position and double bonds at the C-6 and/or C-11 position (Rohmer, 1993). Hopanoids are solely produced by certain types of aerobic eubacteria, and the presence of hopanoids in some higher plants and mosses is generally attributable to hopanoid-producing bacteria living on leaves (Rohmer, 1993). Hopanoids appear to fulfil a similar role to steranes as cell membrane rigidifiers.

Figure 1.12. C30 diploptene, C30 diplopterol and C35 bacteriohopanepolyol fossil hopane precursors, with different side chain lengths, and numbers of functional groups that can take part in macromolecule binding. 1.4.2.2 Bound hopanes

Based on the results of deuterium incorporation in chemical degradation studies (Hofmann et al., 1991; Richnow et al., 1991), and the recovery of intact bacteriohopaneterol (Mycke et al., 1987), hopanoids appear to be bound into organic macromolecules by one or more cross links located at functional group positions on their side chains (Mycke and Michaelis, 1986; Mycke et al., 1987; Hoffman et al., 1991; Richnow et al., 1991; Peng et al., 1997; Peng et al., 1999; Strausz et al., 1999). In general, the extent of deuterium incorporation during chemical degradation and 19

pyrolysis studies is related to the length of hopane side chain and carbon number, and all hopanes greater than C30 can possess more than one binding site (Mycke and Michaelis, 1986; Peng et al., 1997). This means that unlike steranes, hopanes can take part in cross linking and can be bound into the centre of macromolecules.

D to D4

Figure 1.13. Deuterium incorporation during chemical degradation (based on Hofmann et al., 1991; Richnow et al., 1991) and the position of hopanoid binding sites situated on the side chain.

A number of studies have noted that hopanes bound by different heteroatoms, and into different fractions can have distinctive carbon number distributions. In Monterey Formation-sourced oils the dominant hopane carbon number of the resin degradation products varies depending on the type of bond cleavage used (Richnow et al., 1991); C32 for ether-bound hopanes and C35 for sulphur-bound hopanes, free hopanes are dominated by the C30 component. Kerogen hydropyrolysates have been noted to be dominated by lower carbon number homologues than their free fraction bitumen counterparts (Bishop et al., 1998), an effect seen in steranes released by hydrous pyrolysis (Fowler and Brooks, 1987), although different kerogens generally have a degree of variety in their hopane carbon number distributions showing that they are sensitive to source input and/or diagenesis.

1.4.2.3 Fossil hopanes

Fossil hopanes are ubiquitous in oils, extracts and pyrolysates and have a range of carbon numbers (C27 to C35) not seen in biological hopane precursors. These different carbon numbers correspond to different lengths of side chain; C31 to C35 hopanes are derived solely from C35 precursors whilst C27 to C30 hopanes could be derived from both

20

C30 and C35 precursors. A variety of other hopane structures are found in oils and source rocks including C27 and C29 Ts (neohopanes), hopanes in which the C-28 methyl group is located in the C-17 position, and the norhopanes, 25,28,30-trisnorhopane, 28,30bisnorhopane, and 25-norhopanes, that lack the specified methyl groups. Hopenes with double bonds located at the C-13(18) and C-17(21) positions, are also found. A-ring C2 or C-3 methylhopanes are fossil products of methylated precursors associated with methylotrophic bacteria and cyanobacteria (Summons et al., 1994).
19 12 11 25 1 2 3 4 23 24 10 5 6 9 26 8 7 27 13 14 28 15 18 17 16 20 21 22 30 29 31 32 33 34 35

Figure 1.14. Carbon numbering in C35 hopane

Unlike diasteranes, which have been shown in a number of instances to be absent from the bound fractions of source rocks, the state of knowledge on the presence of rearranged hopanes, and other hopanes originating during diagenesis in macromolecules is less clear. Bisnorhopanes (Richnow et al., 1991) as well as C27 and C29 Ts hopanes (Fowler and Brooks, 1987) have been reported to be absent in bound fractions when present in extracts, although many other studies have not reported this to be the case (Seifert, 1978; Philp and Gilbert, 1985; Murray et al., 1998). One study has noted that the neohopanes (Ts and C29 Ts hopanes) had more affinity for extracts, and that in general significant concentrations were only present in bitumen phases of source rocks (Bishop et al., 1998). Thus, it is not possible to definitively say, based on current geochemical literature, whether rearranged hopanes and other hopanes derived from diagenesis and biodegradation are consistently absent in degradation and pyrolysis products of macromolecules and are only present due to adsorption or whether they are actually representative of bound biomarkers.

1.4.2.4 Hopane isomerisation

21

There are three chiral centres that are important for hopanes, two of these are at the C17 and C-21 cyclic ring positions and are present in C29 to C35 hopanes, whilst the C-22 chiral centre is only present for the C31 to C35 hopanes. The biological configuration of bacteriohopanetetrol and other (C35 biohopanoids) is predominantly 17(H),21(H) (22 R), although hopanes with this configuration are not found in the extracts of oil window maturity samples where and especially (22S) and (22R) hopanes predominate. The S/S+R and /+ parameters can be used as maturity indicators and these have limits of 55-60% and 80% respectively (Killops and Killops, 1991; Peters and Moldowan, 1993). For some time bound hopanes have been known to have lower values for these maturity parameters than their free counterparts (Seifert, 1978), e.g. higher proportions of moretanes (hopanes with the 17(H),21() configuration). Recent hydropyrolysis work has also shown that 17 (H),21(H) hopanes can also be present in the kerogen when they are absent in the free fractions (Love et al., 1995), and that moretane/hopane ratios and the % 22S/S+R parameter change far more slowly in the kerogen fraction than in the free fraction (Murray et al., 1998).

21 17 22

Figure 1.15. The significant hopane isomerisation positions at C-17, C-21 and C-22, shown is the 17(H), 21(H) isomer with an unspecified C-22 configuration.

1.4.2.5 Hopanes as source indicators

Hopanes are used as source indicators, many of which involve comparison with other biomarker types. For example hopane/sterane ratios can be used as a paleoenvironmental tool to assess the relative contributions from bacteria and eukaryotes (Peters and Moldowan, 1993), and hopane/sterane ratios have been applied

22

to biomarker sequence stratigraphy in organic matter rich mudstones and source rocks (Robinson and Engel, 1993). Enrichment in moretanes ( hopanes) has been associated with terrestrial input (Hoffman et al., 1984), as have increased proportions of C29 hopane. High relative proportions of C35 hopanes can be related to anoxic sulphide rich depositional conditions in the depositional environment (Peters and Moldowan, 1991; Peters and Moldowan, 1993), with increased sulphur-binding resulting in the preservation of C35 hopane.

OH

12 11 1 2 3 4 16 15 17 10 5 6 9 18 8 7 13 14

19

21 20 22 23

24 25

26 27 28

29 30

Figure 1.16. C30 Hexaprenol, a potential precursor for C30 and lower tricyclic terpanes. Numbering shown for C30 tricyclic terpane.

1.4.3 Tricyclic terpanes

1.4.3.1 Tricyclic terpane precursors

Despite structural similarities, and a similarly widespread occurrence as steranes and hopanes, tricyclic terpanes have no clear analogue in recent sediments or living organisms. They are thought to derive from the cyclisation of polyprenol precursors (Figure 1.16), a common membrane constituent (Aquino Neto et al., 1983). High tricyclic terpane concentrations in sediments have been associated with bloom conditions of algae, and particularly Tasmanites algae, although tricyclic terpanes are found across a very wide time range and variety of geological deposits (Peters and Moldowan, 1993) which suggests that many other biological sources could exist.
1.4.3.2 Bound tricyclic terpanes

As with the sterane and hopane biomarkers, double bonds and functional groups in the biological precursors correspond to the macromolecule binding sites observed for 23

tricyclic terpanes (McCaffrey et al., 1993). Sulphur binding in the C ring, while a theoretical possibility, is not observed in practice and tricyclic terpanes are bound into macromolecules by the isoprenoid chain (Richnow et al., 1991; McCaffery et al., 1993; Peng et al., 1997; Peng et al., 1999; Strausz et al., 1999). The isoprenol side chain has two possible types of attachment site, double bond locations suitable for sulphur incorporation, and the alcohol functionality; deuterium labelling studies indicate that both sites are involved. It is also noteworthy that the extent of deuterium incorporation and the number of binding sites increases with the isoprenoid side chain length of precursors (compare spectra for C23 and C39 tricyclic terpanes in Richnow et al., 1991; McCaffrey et al., 1993), the effect of which is that the extended tricyclic terpanes have a higher number of potential attachment sites. The tricyclic terpanes, often appear to be enriched in the asphaltene and resin fractions relative to hopanes (m/z 191 traces in Jenisch et al., 1989; Richnow et al., 1991; Chapter 3).

D to D3

Figure 1.17. Binding sites in the cyclised tricyclic terpane precursor, denoted by deuterium incorporation in the side chain, although limited sulphur incorporation can occur at the unsaturation (C-13(14)) in the C ring. 1.4.3.3 Fossil tricyclic terpanes

A wide range of tricyclic terpane homologues with a carbon number range from C19 to C54 can be present in oils and source rocks (de Grande et al., 1993). The C19 to C29 range is most common, and the C30+ extended tricyclic terpanes are generally more difficult to identify in gas chromatograms of aliphatic fractions due to co-elution with hopanes (Moldowan et al., 1983). Other structural variations include B- and C-ring aromatised (Azevedo et al., 1992), and C-ring C-13(14) unsaturated tricyclic terpanes (McCaffery et al., 1993).
1.4.3.4 Tricyclic terpane isomerisation

24

No original biological configuration can be assigned to tricyclic terpanes as they are not present in living organisms, although a range of isomers are found in oils and extracts. Two chiral centres at C-13 and C-14 (Figure 1.18) allow for 4 possible configurations; the 13(H),14(H) isomer dominates in oils and oil window maturity source rocks and the 13(H),14(H) isomer is only found in pre-oil window source rocks (Chicarelli et al., 1988; Farrimond et al., 1999), whilst the and isomers are only found in C20 and C21 tricyclic terpanes (Chicarelli et al., 1988; Jincai et al., 1999). Chiral carbons are also present in the alkyl chains of C25+ homologues, although the (22S) and (22R) isomers are only clearly resolved by gas chromatography from the C26 homologues and above. Many of the other isomers possible in the extended tricyclic terpanes C27, 32,
37

etc. are not seen until C35 or C36 and even then are often obscured by co-elution with C31+ hopanes (Moldowan et al., 1983). Recent work would suggest that the (22S) isomer elutes before the (22R) isomer (Peters, 1999), although this has not been authenticated by co-injection of standards.

Figure 1.18. Ring and chain isomerisation in tricyclic terpanes.

No information is available on the extent of isomerisation of bound tricyclic terpanes. The relative rate of generation of tricyclic terpanes from kerogen is thought to be less than that of hopanes, and they are released later in the oil window (Peters et al., 1990). This is a possible cause behind the increase of the tricyclics/ 17a(H) hopane maturity parameter (Peters and Moldowan, 1993).
1.4.3.5 Tricyclic terpanes as source indicators

Based on their structures C19 to C20 tricyclic terpanes could be derived from higher plant resins; however, the majority of tricyclic terpanes are probably derived from polyprenol precursors (Peters and Moldowan, 1993). There are also associations between high tricyclic terpane concentrations and Tasmanites algae (Simoneit et al., 1993), and possibly algal bloom conditions (Kruge et al., 1990). Tricyclic terpanes in general also

25

appear to be very resistant to biodegradation (Palacas et al., 1986), and can be used for fingerprinting oils when other biomarkers have been degraded. The tricyclic/hopane ratios can be used for correlation purposes, although this parameter is also sensitive to source inputs and compares a biomarker that may be derived from algal and bacterial sources to a biomarker of exclusively bacterial origin (Peters and Moldowan, 1993). 1.4.4 Isorenieratane
1.4.4.1 Isorenieratane precursors

Isorenieratene (Figure 1.19) is the main precursor of isorenieratane (Figure 1.20) (Summons and Powell, 1986) and also an important precursor for 1-alkyl-2,3,6trimethylbenzenes (ATMB); see Figure 1.19 (Summons and Powell, 1987; Requejo et al., 1992). Isorenieratene is found in chlorobiaceae, photosynthetic green sulphur bacteria, where it serves as an accessory pigment to chlorophyll during photosynthesis.

16 17 3 4 5 2 1 6 18 7 8

19 9 10 11 12

20 13 14 15

Figure 1.19. Isorenieratene, the precursor of Isorenieratane and aryl isoprenoids. Also shown is the ring numbering system used by Summons and Powell (1987) in the identification of 1-alkyl-2,3,6trimethylbenzenes (ATMB).

As a group, aryl isoprenoid structural isomers can have a range of possible precursors beside isorenieratene, although most of these have different benzene substitution patterns to ATMB (Summons and Powell, 1987). However -isorenieratane, a derivative of -carotene can also give rise to ATMB, and the ATMB of a North Sea oil were shown to have 13C (o/oo) values closer to those of -carotene derivatives than isorenieratene (Koopmans et al., 1996b). Thus, without isotopic evidence, or the presence of isorenieratane itself, ATMB are not robust evidence for the green sulphur bacteria Chlorobiaceae and associated euxinic conditions.

26

Figure 1.20. Isorenieratane and 1-alkyl-2,3,6-trimethylbenzenes (ATMB) 1.4.4.2 Bound isorenieratane

ATMB and isorenieratane (as well as other associated derivatives) have been found in the desulphurisation products of a range of source rock and sediment polar fractions (Kenig et al., 1995; Koopmans et al., 1996a; Van Kaam-Peters 1997). However, very little has been reported about the nature, positions and number of binding sites. Expected sulphur binding positions for isorenieratane would be the double bond locations situated on the long isoprenoid side chain, via the sulphur incorporation routes described previously (Sinninghe Damste et al., 1989).
1.4.4.3 Fossil isorenieratane

Despite the possibility of isorenieratene-derived ATMB, many reported instances of C13-C31 ATMB show clear isotopic associations to, or possible derivation from, isorenieratane precursors (Summons and Powell, 1986; Requejo et al., 1992; Kenig et al., 1995), whereas -carotane or -isorenieratane derived ATMB has been less frequently reported (Koopmans et al., 1996b). As ATMB can be present in higher maturity source rocks when isorenieratane is not (Summons and Powell, 1987), its use as a potential indicator of euxinic conditions should not be completely overlooked, especially when supported by isotopic composition data. Other diagenetic isorenieratene products include diaryl isoprenoids, compounds with mid chain benzene and thiophene units, as well as more extensively cyclised C40 compounds (Koopmans et al., 1996a).
1.4.4.4 Isorenieratane isomerisation

Isorenieratane has been reported to have four isomerised chiral carbons (Koopmans et al., 1996a), although in practice the various diastereomers are not resolved and they elute as a broad peak at the end of a typical gas chromatogram. The two aryl isoprenoid

27

isomers that have been most commonly reported are the ATMB (2-alkyl-1,3,4trimethylbenzene) series which can constitute major peaks in a m/z 133 mass chromatogram, and another unidentified series of smaller peaks possessing similar spectra but with a greater m/z 134 intensity that elute just before (Summons and Powell, 1986; Requejo et al., 1991).
1.4.4.5 Isorenieratane as a source indicator

Without isotope data ATMB are considered a much less rigorous marker for euxinic conditions (Koopmans et al., 1996b) than isorenieratane, which is used to infer an input from Chlorobiaceae, a photosynthetic green-sulphur bacteria (Summons and Powell, 1986). Chlorobiaceae occupy a unique ecological niche, requiring high light intensity and anoxic aquatic environments with high concentrations of hydrogen sulphide (e.g. euxinic conditions). However, both compound types can be used in oil source correlations (Requejo et al., 1991). 1.4.5 Summary In summary it would seem that most biomarkers have macromolecularly bound equivalents, and that these are bound into host macromolecules at the sites of unsaturation or functional groups in the original biological precursor. Biomarkers that do not have bound equivalents often have carbon skeletons that originate from biodegradation or diagenetic processes in which original functionality is lost. One conclusion that can be drawn from the observations of biomarker binding mechanisms is that a proportion of biomarkers very likely enter the sedimentary organic matter fossil record as bound biomarkers. This could cause the different fractions of sedimentary organic matter to possess different biomarker structures and histories, particularly in early catagenesis and before significant oil generation and biomarker release has occurred, and during subsequent episodes of thermal maturation and biodegradation.

28

1.5 Methods for analysing bound biomarkers

In order to analyse bound biomarkers degradation methods are needed to break apart the covalent bonds that bind them into their host macromolecules. Many of the methods, whilst good tools within their context (e.g. maturity assessment, kerogen structure investigation etc.), are not always ideal for bound biomarker analysis. For each bound biomarker analysis technique there are three main issues and areas of concern: 1) The degree of hydrogen disproportionation between released products and the residue. The breaking of covalent bonds creates free valencies, that unless capped (e.g. by hydrogen), can lead to secondary reactions or the creation of unsaturated compounds not originally present in oils and extracts. The effects of pyrolytic cracking in air (free oxygen containing environment) are shown in Figure 1.21, as are other possible structural rearrangements driven by hydrogen scavenging (Figure 1.22), that are more likely to occur in an environment in which both oxygen and hydrogen were restricted.

H H C H

H C H

H C H

H C H

H C H

H C H H

Pyrolysis in presence of oxygen e.g. in air Air (inc O2)

Pyrolysis in absence of oxygen e.g. closed system(He) Hydrogen in macromolecule H H H C H H C H H C H H

H H C H

H C H

H C H

H C H

H C H

H C H H H

H C H

H C H

H C H

H H C H

H C H H CO2 H

H C H

H C H

H C H H Cyclised and aromatised residue

Figure 1.21. Hydrogen disproportionation; breaking of covalent bonds leads to a hydrogen deficit that will be balanced by scavenging. The major flaw, common in many pyrolysis studies, is that this

29

deficit is meet by scavenging from products or residue. This could lead to cracking and the creation of sites of unsaturation in products, and the cyclisation and aromatisation of the residue.

H2

H2

Figure 1.22. Two structural rearrangements, cyclisation and aromatisation, that can occur in pyrolysis products to provide hydrogen to cap binding sites.

2) The overall conversion rate of the kerogen, asphaltene or resin macromolecular feedstock, and the yield of analysable (hydrocarbon) products. Two measures of the degree to which degradation and pyrolysis reactions have proceeded are used. One is the conversion rate of the macromolecular feedstock; this is important as it allows a consideration of the extent to which all the organic matter has been treated and the thoroughness with which all biomarkers have been accounted for. Methods with low conversion rates do not accurately represent the quantities of biomarkers present, and their products will be most prone to contamination by occluded free compounds. Second is the product yield, which is either presented as the weight % of DCM soluble products, or as mg/g (ppt) weight TOC. The product yield does not take account of the degree to which degradation or pyrolysis has proceeded, and therefore the thoroughness with which all biomarkers have been accounted for. In addition, non-GC amenable compounds and heavy polar compounds (similar to resins and asphaltenes) may contain biomarkers and ought not to be considered in the product yield. Unless yield data is expressed as a proportion of the total sediment TOC or whole oil, no assessment can be made of how representative the data is of the sediment or sample as a whole.

weight product . = conversion rate weight feedstock

(1)

30

weight products. x 100 = % yield weight feedstock mg weight products . = ppt TOC g TOC in sample

(2)

(3)

3) Secondary reactions such as cracking and isomerisation. Two common secondary reactions are a decrease in the carbon number of products (e.g. steranes and hopanes), and the alteration of thermally sensitive isomers to those normally associated with higher geological temperatures. These are caused by the exposure of pyrolysis or degradation products to high temperatures or aggressive chemical environments that are used in many methods. A closed vessel system greatly exacerbates the problem, whereas an on-line system that ensures the removal of products and a slow heating program will help to minimise the effects of higher temperatures. An additional consideration in pyrolysis and chemical degradation studies is analytical precision, which is particularly difficult to evaluate for a number of reasons. Even if a degradation or pyrolysis method is relatively precise, the separation of its products and their analysis by GC-FID or GC-MS might not be. Furthermore, many geological materials are very heterogeneous, and this could be another cause of apparently poor precision. In practice the selection of a pyrolysis or degradation method will also hinge on other issues pertaining to cost, time constraints and the availability of the expertise needed for the different methods. The main pyrolysis and degradation methods used for biomarker analysis are described in the following sections, divided into pyrolysis, chemical degradation, hydrous pyrolysis, thermolysis TMAH and hydropyrolysis. 1.5.1 Pyrolysis Pyrolysis involves the pyrolytic breaking of covalent cross links in macromolecules at high temperatures (up to 770 0C), and has seen use as a tool to simulate and asses petroleum maturation (Tissot et al., 1974), in the molecular typing of kerogens (Larter and Senftle, 1985) and to release and analyse bound biomarkers (Gallegos, 1975).

31

A range of pyrolysis techniques have been used in bound biomarker analysis: sealed vessel pyrolysis (Seifert, 1978), open system and online pyrolysis (Behar and Pelet, 1985) and Curie point and pyroprobe pyrolysis (Philp and Gilbert, 1985). Open system and online flash pyrolysis methods are used to minimise the exposure of temperaturesensitive bound biomarkers to high pyrolysis temperatures, and so minimise the secondary reactions that are generally extensive in closed systems. However, online pyrolysis methods are not normally used for biomarker quantification, and when a method like Py-GC-MS is used the very low product yields produced can mean that data are not representative of the total kerogen or related material. This is because a large proportion of both of product and residue are a non-GC amenable heavy tar. In practice, aside from general questions of reproducibility, a number of artefacts are typically encountered in pyrolysis data (these are derived from both hydrogen disproportionation and thermally driven secondary reactions). Commonly encountered pyrolysis artefacts include the alteration of n-alkane envelope (with a tendency to reduce any pre-existing odd over even carbon preference, and loss of higher number nalkanes), the loss of both thermally-sensitive biomarker configurations (e.g. hopanes) and higher carbon number biomarker homologues (e.g. C35 hopanes), cracking of larger compounds leading to greatly elevated amounts of compounds such as 1,2,3,4 tetramethylbenzene and increased proportions of unsaturated compounds e.g. n-alkenes, hopenes etc. Bound biomarker analysis using pyrolysis methods requires careful selection of temperatures and heating rates for the biomarkers under consideration, and will inevitably require the careful balancing of overall biomarker yield and reproducibility against secondary reactions that lead to the cracking and isomerisation of products. However pyrolysis methods are relatively fast and inexpensive, thus they might be considered appropriate for purely qualitative bound biomarker work, or in situations where it is not important to accurately represent original biomarker structures. Probably the major failing of pyrolysis methods to release bound biomarkers, even more than their low product yields, is their inability to limit secondary reactions caused by hydrogen disproportionation. This is caused by the unavailability of hydrogen to cap former binding sites and can lead to a range of structural alterations. This fact even

32

compromises their ability to represent qualitatively the bound biomarker content of kerogen and related materials, and cannot be entirely overcome by adjusting temperature and heating rate. 1.5.2 Chemical degradation Early chemical degradation techniques suffered from many of the problems associated with pyrolysis methods: low yields and structural alteration of products. However, in the past fifteen years a number of chemical degradation procedures have risen to prominence in biomarker geochemistry: these are rhodium on charcoal or other similar catalyst (Rh-C/H2) hydrogenolysis, boron trichloride (BCl3) O-bond cleavage, Raney nickel Ni/H2 or Ni(0)cene desulphurisation and ruthenium ion catalysed oxidation (RICO). Hydrogenolysis involves the mild heating (max 200 oC to 300 oC) of feedstock in the presence of a catalyst (such as rhodium on charcoal) in a closed hydrogen atmosphere (moderate H2 pressures of 5-7 MPa). It was initially used to recover phenolic compounds from lignin-derived material present in coals and humic substances, but has been successfully applied to the recovery of biomarkers from kerogens and other materials (Mycke and Michaelis, 1986). Much of the early bound biomarker labelling work detailing biomarker binding sites, was undertaken using hydrogenolysis with deuterium as the reaction medium (Mycke and Michaelis, 1985; Michaelis et al., 1989; Michaelis et al., 1990). The major advantages of hydrogenolysis over pyrolysis are that conversion rates can be high (up to 70 %) with yields of as much as 35 % by weight of soluble products, and that some temperature sensitive biomarkers such as C35 hopane can be recovered intact. However, high hydrogenolysis yields depend on both the type of macromolecular material, as well as the proportion of non-ether bonds present. High feedstock conversion rates of 70 % and soluble product yields of 35 % are unusual, conversion rates vary between 6 and 70 % depending on material, and kerogen and coal chemical degradation yields of 10 % or more are considered high (Mycke and Michaelis, 1985; Rullktter and Michaelis, 1990). Furthermore, when compared to other methods in sequential kerogen degradation studies, hydrogenolysis has been shown to yield only

33

low proportions of the total aliphatic material present as well low proportions of biomarkers (Love et al., 1995; Love et al., 1998). As a bound biomarker analysis tool hydrogenolysis only accurately represents the composition and quantities of biomarkers present in certain materials dominated by ether bonds (such as low sulphur coals, and lignin-rich humic materials). As described in the section detailing the structure of kerogen and related materials (section 1.2), a variety of heteroatomic bonds form the covalent-cross links between biomarkers and their host macromolecules. The key tenet of selective chemical degradation studies is that these bonds can be targeted sequentially to deduce structural information and achieve higher yields of released biomarkers than if only a single technique was applied. The four bond types considered in selective chemical degradation are sulphur, ester, ether, and benzene ring alkyl attachments. Sulphur bonds are reduced by Raney nickel (Ni/H2) or Ni(0)cene/ LiAlD4, ester bonds are targeted by hydrolysis and ether bonds by boron trichloride (BCl3) cleavage (e.g. Peng et al., 1997). In practice desulphurisation is typically followed by only a single treatment to cleave oxygen cross-links, often hydrogenolysis (Koopmans et al., 1996) or BCl3 treatment (Richnow et al., 1991). HI/LiAlH4 treatment can also be used to break both oxygen and sulphur bonds (Koopmans et al., 1996c). While the low temperatures involved, and the relatively mild nature of the reagents help to produce structurally unaltered biomarkers, the most important component of selective chemical degradation studies is the capping ability of most of the reactions. This addresses the problem of hydrogen disproportionation, the major failing of pyrolysis methods, and with the use of LiAlD4 can label binding positions. Additional bond types not addressed by the above chemical treatments, such as carboncarbon cross links, have been treated in various ways. Ruthenium ion catalysed oxidation is often used to recover a proportion of the carbon-bound material that contains an alkyl chain attached to an aromatic ring (Richnow et al., 1990; Strausz et al., 1999). Target aromatic rings are oxidised in a such a way as to create a carboxylic acid at the former site of attachment, and thus provide structural information, in 34

addition to capping the former site of attachment. Pyrolysis methods are also used to account for non-oxygen and non-sulphur bound biomarkers (Hold et al., 1999). Chemical degradation methods achieve highest biomarker yields from smaller macromolecular compounds. Jenisch et al. (1989) reported boron trichloride cleavage results where oil window maturity coal resin yields where ~50 %, asphaltene yields ~25 % and kerogen yields ~5 %. For sulphur-rich sediments and kerogens, desulphurisation techniques generate yields of similar orders of magnitude, a remarkably high 150 mg/g TOC has been reported for the Serpiano S-rich kerogens (Hoffman et al., 1992) and 1510 % for resins and 5-1 % for asphaltenes (Kohnen et al., 1991; Hold et al., 1999). The one exception to this general trend is ruthenium ion catalysed oxidation which produces higher yields from asphaltenes and kerogens than from resins (Richnow et al., 1991) on account of the greater amount of aromatic carbon in the former two fractions. These generally low yields, especially from kerogens, arise because reagents are not able to access the functional groups in sterically hindered positions at the centre of macromolecules (Rullktter and Michaelis, 1990), or because of bond types that do not react with the reagents used. This prevents reactions from going to completion. The biomarkers released by different chemical degradation methods (e.g. biomarkers bound by different bond types) often have different compositions (Michaelis et al., 1989; Richnow et al., 1991). Despite this, and although almost all of the chemical degradation techniques listed above are incapable of producing even moderate yields when applied on their own, they are still frequently applied singularly with pyrolysis being used to account for any remaining material. This is probably as a consequence of the time-intensive nature of chemical degradation studies, but will inevitably mean that both the composition and quantities of bound biomarkers produced are nonrepresentative. On one of the few instances that a sequential chemical degradation scheme capable of attacking all bond types has been applied (Richnow et al., 1991), yields whilst good for resins (38 %) were very low for oil and source rock resins and kerogens (< 20 %). To overcome these limitations chemical degradation studies normally concentrate on materials in which they will produce the highest yields, such as resins and asphaltenes 35

or immature sulphur-rich sediments with large amounts of extractable polar compounds (e.g. the Gibellina Messinian marl). This has the obvious implication that chemical degradation studies are not applied to the analysis of bound biomarkers present in kerogen, the quantitatively greatest pool of organic carbon on earth. 1.5.3 Hydrous Pyrolysis Hydrous pyrolysis refers to pyrolysis performed in a closed vessel under strictly controlled chemical conditions: rock chips are heated in the presence of liquid water at subcritical temperatures (<374 oC) in the absence of an oxygen atmosphere (Lewan et al., 1979). Despite the much greater heating rates and temperatures involved hydrous pyrolysis tends to simulate the chemical conditions present in petroleum generating sedimentary basins, and produces a free flowing oil that forms an oil phase above the water phase when the system has cooled to room temperature. Hydrous pyrolysis has been used to study the effects of thermal maturation on biomarkers (Lewan et al., 1986; Peters et al., 1990), and the effects of dia- and catagenesis on bound biomarkers (Koopmans et al., 1996c, 1997). Hydrous pyrolysis has also been successfully applied to the analysis of bound biomarkers (Eglinton and Douglas, 1987; Fowler and Brooks, 1987; Jones et al., 1988). The quantitative study of kerogen-bound biomarkers by Eglinton and Douglas (1987) was the first direct demonstration of the significant amounts of biomarkers that are present in (Kimmeridge, New Albany and Monterey) kerogens. Fowler and Brooks (1987) used hydrous pyrolysis to release asphaltene-bound biomarkers, whose sterane compositions were shown to be significantly different from that of their corresponding free hydrocarbon fractions. Their results also showed that the proportions of pregnanes and regular steranes, and C29 and C30 hopanes varied with the severity of pyrolysis conditions. Hydrous pyrolysis can be successfully applied to biomarker analysis, with careful control of temperature and heating time (Eglinton and Douglas, 1987). This is due to the relatively mild temperatures used and the ability of water to act as a mild acid and donate hydrogen to cap liberated products. However, hydrous pyrolysis produces an oillike phase which contains asphaltenes and resins (Koopmans et al., 1996c) that also contain bound-biomarkers; these can only be analysed by the use of a two stage method 36

or more extreme heating times and rates. The latter will increase the likelihood of secondary reactions occurring, whilst a two stage process increases the work required per sample. Additionally, the closed vessel nature of hydrous pyrolysis means that thermal alteration of products can occur, particularly if longer heating times are used to maximise conversion rates (Fowler and Brooks, 1987). These factors mean that hydrous pyrolysis is limited in its ability to accurately represent the quantities and compositions of bound biomarkers present in kerogen and related materials. 1.5.4 Thermolysis TMAH One method to help overcome the shortcomings of pyrolysis techniques is to add catalysts or reactants that can aid bond cleavage and/or cap former binding sites of biomarkers as they are released. Tetramethylammonium hydroxide (TMAH) has been used to increase the recovery of ether-linked lignins in coals and similar materials (Hatcher and Clifford, 1997), as well as to study asphaltene and kerogen-bound hopanes and hopanoic acids (Del Rio et al., 1996). Instant methyl-derivatisation helps to preserve structures and prevent hydrogen disproportionation, but overall yields of biomarkers and conversion rates obtained from other macromolecular materials can still be quite low. Most published bound biomarker studies that have used TMAH have tended to be non-quantitative. 1.5.5 Hydropyrolysis (note : Specific details of the hydropyrolysis method are given in Chapter 2) In catalytic hydropyrolysis high hydrogen pressures of ~15 MPa and relatively low maximum pyrolysis temperatures of 500 oC (note also slow heating rate of 4 oC min-1) are utilised in a fast-sweeping chamber to pyrolyse organic compounds in the presence of a dispersed sulphided molybdenum catalyst. High conversion rates (> 85 %) are achieved, and yields are also very high depending upon kerogen type (Love et al., 1995). Bound biomarkers with their original biological configuration can be recovered, and n-alkanes and biomarkers have carbon numbers similar to those present in oils and extracts e.g. suggesting that artificial thermal cracking has not occurred (Love et al., 1995; Love et al., 1998). Secondary reactions are significantly reduced in hydropyrolysis due to the extremely high reactor sweep rates, and the prevention of hydrogen disproportionation by the dense hydrogen atmosphere. These factors mean that hydropyrolysis products are both structurally and quantitatively representative of 37

the original bound biomarkers. This makes hydropyrolysis an ideal tool for bound biomarker analysis.

1.6 The state of knowledge on bound biomarkers

This section of the chapter discusses the advantages and reasons for using bound biomarkers, as well as differences between free and bound biomarker compositions and abundance, and the difficulties posed in applying bound biomarker data to oil correlation and maturity assessment. 1.6.1 The advantages and reasons for using bound biomarker data There a number of circumstances in which bound biomarker data will represent the only source of unaltered reliable biomarker information, e.g. when the free fraction hydrocarbon biomarkers have been degraded or contaminated. In other situations too, bound biomarkers might represent a more preferable source of biomarker information. Examples of this might include situations where biomarker information that can be guaranteed to be in situ is desirable, or when bound biomarkers are a more precise representation of true stratigraphy or biogenecity.
1.6.1.1 Resistance to biodegradation

For some time biomarkers have been known to be present in the pyrolysis and degradation products of asphaltenes separated from biodegraded oils, even when biomarkers are absent or heavily degraded in the free hydrocarbon fraction (Rubinstein et al., 1979; Jones et al., 1988; Fowler and Brooks, 1989; Peng et al., 1997). A number of authors have demonstrated pyrolysis and hydrous pyrolysis methods for using asphaltene-bound biomarkers for correlation purposes, where free biomarkers have been removed due to the effects of biodegradation (Rubinstein et al., 1979; Cassani and Eglinton, 1986; Jones et al., 1988).
1.6.1.2 Resistance to contamination

Kerogen is an immobile phase that effectively forms part of the rock matrix. Solvent extraction removes the mobile DCM-soluble bitumen phase and cleans the kerogen of any other DCM-soluble material, including contamination. Contamination may result from filtrate invasion during drilling by oil- and polymer- based drilling muds, or from oil migration. Hydropyrolysis products of solvent-extracted/cleaned kerogen fractions 38

from a drill core contaminated with an oil-based mud have been shown to be unaffected by contamination, and clean of contaminants present in the free fraction (Murray et al., 1998).
1.6.1.3 Better representation of biogenicity and stratigraphy

The pyrolysis and chemical degradation products of macromolecular fractions have been reported to better represent stratigraphy or biogenicity in a number of instances (Kenig et al., 1995; Schaeffer et al., 1995; Love et al., 1998). This has been attributed to quantitative factors, i.e. that the bound products in question constitute the greatest proportion of organic matter and are therefore more precise. Additional factors such as selective incorporation of biomarkers to a given fraction, and the better protection of key indicator compounds in some fractions and not others might also be important. 1.6.2 Causes of variation in the biomarker data obtained from different fractions of sedimentary organic matter Compositional variability in bound biomarker data is far more complicated to interpret than similar differences in free biomarker data, due to a number of reasons. One reason, touched upon in the discussion of the different pyrolysis and degradation techniques, is that the products of the different techniques are not comparable because of the different yields and conversion rates, secondary reactions and extent to which hydrogen disproportionation occurs. There are other more fundamental reasons for the differences in biomarker compositions which reflect the effects of thermal maturity, biomarker binding position and number and type of cross-linking covalent bonds. As described in the following section all these factors can cause differences in bound biomarker composition.
1.6.2.1 Differences in quantities of biomarkers in different fractions across the oil window

There have been relatively few studies of bound biomarkers in samples that cover the oil window. Hydrous pyrolysis studies have shown that very significant quantities of biomarkers are present in the kerogen fraction, and that the maximum release of bound steranes occurs at lower temperatures before that of hopanes (Eglinton and Douglas, 1988).

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Hydropyrolysis results from a Kimmeridge Clay maturity suite show that biomarkers are present in significant quantities in the kerogen fraction, both throughout and fairly late into the oil window (Murray et al., 1998). The study not only showed that different fractions contain different quantities of biomarkers at different thermal maturities, but also the importance of the number of binding sites present in different biomarkers. Quantitatively, kerogen-bound biomarkers are most significant at the beginning and end of the oil window (Figure 1.23), and biomarkers with more than one binding site (e.g. C35 hopane) are released later into the oil window than those with predominantly only a single site (e.g. steranes), although this will also depend on bond type.

ratio free/bound 0 0.35 0.4 0.45 Ro% 0.5 0.55 0.6 0.65 Figure 1.23. Data from Murray et al. (1998) that show the ratio of free to bound biomarkers. Note that bound biomarkers are most significant early and late into the oil window (e.g. when value <1). Also note the effect of number of binding sites; C 35 hopanes are retained by the kerogen fraction later into the oil window than biomarkers bound by only a single bond. 1.6.2.2 Impact of cross-linking bond types on bound biomarker distribution C29 R sterane C35 S+R hopane C30 hopane 2 4 6 8 10 12

Biomarkers released by the cleavage of different types of bonds, either during chemolysis or artificial maturation, often possess different distributions. Examples of this include the carbon number distribution of hopane and sterane homologues (Richnow et al., 1991), as well as the proportions of different types of biomarkers (Rullktter and Michaelis, 1989). Artificial maturation studies have shown that different bond types, e.g. sulphur, oxygen, and carbon bonds, are broken in distinct oil generation phases leading to different peak concentration and biomarker abundance maxima (Koopmans et al., 1996c). This suggests that both the type and strength of a biomarker bond, as well as the number biomarker binding sites, could be important factors in determining the proportions of various biomarker types that are present in the different geochemical fractions of organic matter at any given maturity. 40

1.6.2.3 Differences in the composition, proportions and quantities of bound biomarkers present in different fractions

The first study to demonstrate that the biomarkers present in different geochemical fractions have different compositions was that of Eglinton and Douglas (1988), which found kerogen and whole rock pyrolysate GC-MS traces to generally have more in common than the kerogen pyrolysate and unheated bitumen GC-MS traces. Other work has shown that the biomarker contents of the free hydrocarbon fraction are not representative of the macromolecular fractions, and that soluble macromolecular fractions (resins and asphaltenes) are not compositional analogues of kerogen (Fowler and Brooks, 1989; Jenisch et al., 1990; Kohnen et al., 1991; Richnow et al., 1991; Bishop et al., 1998; Murray et al., 1998; Hold et al., 1999). Commonly reported differences in biomarker composition among the three main biomarker types used in this study include the higher proportions of tricyclic terpanes relative to hopanes in the resin and asphaltene fractions, greater proportions of steranes relative to hopanes in the free fraction, and different sterane and hopane carbon number distributions. The proportions and types of isorenieratane derivatives also vary between different fractions (Koopmans et al., 1996a; 1996c).
1.6.2.4 Steric protection of biomarkers in macromolecular fractions

Bound biomarkers released by pyrolysis (Seifert, 1978; Rubinstein et al., 1979) chemical degradation (Mycke and Michaelis, 1986; Mycke et al., 1987) and hydropyrolysis (Love et al., 1995) have been shown to contain biomarkers with structural configurations that are thermally less mature than their free counterparts. The degree to which these sterically-protected bound biomarkers appear to be less mature than their free counterparts can depend on the pyrolysis or degradation methods used, but is mainly dependant on the maturity of the sample. A systematic study of how bound biomarker maturity parameters evolve and develop across the oil window suggests that they progress at a slower rate than their free counterparts, before eventually converging with them at the parameters endpoint (Figure 1.24; Murray et al., 1998). Overall therefore, the apparent degree of steric protection declines with maturity as the free and bound biomarker maturity parameters converge. What has yet to be reported are the values of biomarker maturity parameters measured in all the various geochemical fractions (i.e. the free, resin, asphaltene and kerogen fractions).

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(20S) / (20S) + (20R)

0.7 0.6 0.5 0.4 0.3 0.2 0.4 0.5 0.6 0.45 0.55 0.65 / +

0.6 0.5 0.4 0.3 0.2 0.1 0 0.4 0.5 0.35 0.45 0.55 0.6 0.65

%Ro

free

kerogen

%Ro

Figure 1.24. (20S)/(20S)+(20R) = C29 sterane 5(H), 14(H), 17(H) (20S)/(20S)+(20R); /+ = C30 hopane 14(H), 17(H) / 14 (H),17(H)+ 14 (H), 17(H). Both parameters converge in the kerogen and free fractions with increasing maturity (data redrawn from Murray et al., 1998).

1.6.3 Problems in using bound biomarker data A number of potential problems arise when using bound biomarker data. One of these is the way in which the absence of compounds commonly present in the free fraction is interpreted. Other issues relate to the impact on correlation studies of the compositional difference in the biomarker contents of the kerogen, asphaltene, resin and free fractions, and the observed differences in maturity parameters measured in bound and free fractions with identical thermal histories (e.g. fractions of the same sample).
1.6.3.1 Compounds not present in bound fractions

A number of biomarker compounds that are generally present in the free hydrocarbon fraction are either absent or present in very low abundance in the pyrolysis and degradation products of kerogens, asphaltenes and resins. Biomarkers for which this is the case (as previously noted in the discussion of the four main biomarker types), include diasteranes, trisnorneohopane and bisnorhopane which are thought to be the product of diagenetic reactions and other processes that occur exclusively in the free fraction such as biodegradation, or due to steric hindrance of reactive sites that prevents incorporation into macromolecule networks. However, it is difficult to conclusively prove, even with deuterium labelling techniques (due to possible hydrogen exchange), that a macromolecular fraction has been completely cleaned of free biomarkers and that the presence of a given biomarker in a pyrolysate is solely attributable to the breaking of covalent bonds and liberation from a macromolecule. Examples of this include the diasteranes which, although nearly every single bound biomarker study would support

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their absence from bound fractions, have in a few instances been reported in chemical degradation products (e.g. Peng et al., 1997). Similarly Ts and C29 Ts hopanes, although commonly reported to be absent from pyrolysates (e.g. Fowler and Brooks, 1989), have been found in the kerogen pyrolysis products of some samples but not others (Bishop et al., 1998). Gammacerane too has been reported to be absent in whole rock pyrolysates but present in polar fraction pyrolysis and chemical degradation products (Philp and Gilbert, 1985). However, other work has since shown gammacerane to be present in kerogen fraction hydropyrolysis products (Murray et al., 1998). Ensuring that fractionation procedures are as rigorous as possible will mean that the presence or absence of a compound in a pyrolysate of macromolecular organic matter is not attributable to preparation procedures or occluded free material. Using a method that produces high yields and kerogen conversion rates increases the proportions of originally bound material in the pyrolysis or degradation products and will swamp any occluded free material. An additional method for screening bound biomarkers liberated by hydropyrolysis for free fraction contamination is discussed in section 3.3.6 in Chapter 3.
1.6.3.2 Using bound biomarkers for correlation studies

Correlation studies comparing asphaltene to kerogen pyrolysis products rest on the theoretical grounds that asphaltenes are smaller structural analogues of kerogens (Bandurski, 1982; Behar et al., 1984; Behar and Pelet, 1985; Pelet et al., 1986). Later Py-GC studies that have correlated oil asphaltenes to source rock kerogens considered only very basic pyrolysis products that represent simple structural units, e.g. alkanes and benzene compounds (Hartgers et al., 1992; Garg and Philp, 1994; Mukhopadhyay et al., 1995). In biomarker analysis, structural alterations that are inconsequential for other types of quantitatively more abundant compounds, can radically alter geological interpretation in trace concentration (ppm) biomarkers (e.g. cracking of steranes). Although GC-MS fingerprints of biomarkers represent a detailed pool of precise information, they are sensitive to thermal and other alteration processes. Therefore any technique that does not accurately represent the structures, quantities or proportions of bound biomarkers present, could lead to misleading bound biomarker correlations.

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Even when reliable data are obtained, bound biomarker correlations can be more difficult to make than using biomarkers in the free hydrocarbon fraction. This is because the various different fractions can contain different proportions of biomarkers (Fowler and Brooks, 1989; Richnow et al., 1991), and the proportion and composition of these biomarkers, because of the varying binding types involved, are extremely sensitive to thermal maturity. Thus although bound biomarkers are sterically protected form alteration processes to some degree, and in heavily biodegraded oils will be the only source of biomarker data, their application to correlation studies is more complicated than that of their free counterparts.
1.6.3.3 Using bound biomarkers for maturity assessment

Although a number of authors have reported that biomarker maturity parameters measured in bound fractions can be used to distinguish samples of different thermal maturities (Philp and Gilbert, 1985; Bishop et al., 1998; Murray et al., 1998), this has only been achieved by the comparison of identical fractions. A hydropyrolysis study by Murray et al. (1998) noted that the differences in the values of maturity parameters between the free and kerogen-bound biomarkers appears to reduce (at least for moretane/hopane and sterane (20S)/(20S) + (20R) parameters) across the oil window. No such data is available for the resin and asphaltene fractions, which makes it difficult to integrate bound biomarker data measured in different fractions with the more commonly acquired free biomarker data. One way to overcome which would be to have a model of the thermal evolution of maturity ratios in each fraction, this would allow the conversion of a maturity parameter measured in one fraction to that in another.

1.6.3.4 Role of hydropyrolysis in addressing problems associated with applying bound biomarkers

Kerogen is the most suitable material with which to investigate the binding of compounds into macromolecular sedimentary organic matter, because: 1) kerogen is most easily cleaned of soluble free organic matter by solvent extraction, and additional more aggressive treatment phases if needed e.g. bitumen II extraction (Bishop et al., 1998) or pyridine extraction (Love et al., 1995); and 2) quantitatively kerogen pyrolysis or degradation products ought to swamp any occluded free material as kerogen

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comprises the bulk of organic matter. In comparison to hydropyrolysis most other degradation or pyrolysis methods achieve poor conversion rates of kerogen. The main problem in applying bound biomarkers to oil correlation studies is that they have different carbon number distributions to their free counterparts. These differences in the carbon number distribution of products can be further increased by the use of pyrolysis methods and associated secondary reactions, or the selective breaking of only a certain proportion and type of bonds in chemical degradation methods (e.g. sulphur bonds) which will yield biomarkers with distributions not representative of the entirety of the biomarkers present (Richnow et al., 1991). Hydropyrolysis does not significantly alter the carbon number distribution of biomarkers, and yet it also achieves high conversion rates and biomarker yields as it can break all bond types that bind the biomarkers into their host macromolecules. This means that hydropyrolysis is capable of providing a reliable data set, that encompasses all the biomarkers present in every fraction, that can be used to investigate and understand the differences between the biomarker distribution of each fraction. Such data could then be used to integrate biomarker data measured in different fractions of sedimentary organic matter. A similar problem to that encountered when applying bound biomarkers to oil correlation studies is also encountered when applying bound biomarkers to maturity assessment; biomarker maturity parameters measured in different fractions of equal maturity in sedimentary organic matter appear different (generally the isomeric composition of macromolecular fractions appear less mature). This would make elucidating source kitchen areas, using parameters measured in different fractions difficult. Measuring maturity parameters in pyrolysis products could be further complicated by secondary reactions and the thermal alteration of biomarkers by extreme temperature pyrolysis temperatures.

1.7 Aims and objectives of this study

The aim of this study is to investigate hydropyrolysis as a tool for molecularly characterising sedimentary organic matter and petroleum. To achieve this aim the sedimentary organic matter or petroleum of a number of sample suites (covering a range of geological ages and different thermal maturities) will first have to be split into

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different geochemical fractions (free solvent extracted hydrocarbon resin, asphaltene and kerogen fractions), and a methodology for doing this as precisely and as efficiently as possible established. The resin, asphaltene and kerogen fractions that contain bound biomarkers will then be subjected to hydropyrolysis, this is necessary to convert them into hydrocarbons and release biomarkers that are covalently bound into macromolecular networks for analysis by gas-chromatography and gas-chromatography mass spectrometry, in the same manner that is used to analyse the biomarkers present in the free hydrocarbon fractions of the solvent extracts of sediments, rocks and petroleum. The principal objective of this study is to molecularly characterise all of the sedimentary organic matter (e.g. all of its fractions), in terms of both its composition and the absolute quantities of biomarkers that are present (e.g. by comparison to a biomarker standard). Other objectives are to investigate and contribute to the following ongoing areas of bound biomarker research: 1) The effects of thermal maturation on biomarkers bound into the resin, asphaltene and kerogen fractions. 2) Assessing what differences there are in the biomarker parameters measured in the resin asphaltene and kerogen fractions. 3) Assessing how differences in the bound biomarker content of the different fractions might impact using biomarkers to interpret past environments and conduct oil correlation studies.

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