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Molecular analysis of petroleum in fluid inclusions: a practical methodology
Organic Geochemistry 31 (2000) 1163±1173
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Molecular analysis of petroleum in ¯uid inclusions: a practical methodology D.M. Jones *, G. Macleod 1 Fossil Fuels and Environmental Geochemistry (Postgraduate Institute): NRG, University of Newcastle upon Tyne, NE1 7RU, UK Received 21 February 2000; accepted 2 August 2000 (returned to author for revision 12 April 2000)
Abstract A method is described for extracting the petroleum from petroleum-bearing ¯uid inclusions hosted in the diagenetic cements of sedimentary rocks, whilst minimising contamination from petroleum in the pore space. A clean-up technique, involving the addition of extraction standards to the rock prior to analysis, has been developed that allows increased con®dence that the hydrocarbons extracted are only from included petroleum and are not mixtures of included petroleum and petroleum (or other organic material) adhering to the surface of the host minerals within the sample. Replicate quantitative analyses indicate that the technique produces highly reproducible results. The problems of analysing included petroleum and the clean-up method development are discussed and examples of analyses of included petroleums from carbonate reservoir and carrier units are given. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Petroleum; Fluid inclusions; Hydrocarbons; Biomarkers
1. Introduction Fluid inclusions are tiny vacuoles in minerals, containing ¯uids that were present when the mineral cement formed. They thus can be regarded as time capsules of geo¯uids and as such are invaluable for understanding the evolution and migration of petroleum in sedimentary basins (cf. Roedder, 1984; Karlsen et al., 1993). An example of their use in the elucidation of the ®lling history of a petroleum reservoir is given by Karlsen et al. (1993). Petroleum-bearing ¯uid inclusions can be studied using microthermometric methods to obtain the minimum trapping temperature of single petroleum inclusions (Goldstein and Reynolds, 1994) and the gross * Corresponding author. Tel.: +44-191-222-8628; fax: +44191-222-5431. E-mail address: [email protected] (D.M. Jones). 1 Present address: Shell Exploration and Production Technology Co., Bellaire Technology Center, PO Box 481, Houston, TX 77001-0481, USA.
composition of included petroleum can be derived by confocal microscopy and pressure-volume-temperature (PVT) simulation (Macleod et al., 1996; Aplin et al., 1997, 1999). Assorted beam techniques can also be used to determine information on the general composition of the included petroleum, such as bond types present in the included petroleum, and some bulk compositional data from ultra violet (UV) spectroscopic techniques (e.g. Kihle, 1995). There have been a number of studies of the molecular compositions of petroleums in ¯uid inclusions by various techniques including thermal decrepitation or crushing (e.g. Murray, 1957; Hors®eld and McLimans, 1984; Etminan and Homan, 1989; Jesenius and Burruss, 1990; Karlsen et al., 1993; Macleod et al., 1994; Bigge et al., 1995; Jones et al., 1996; George et al., 1995, 1997, 1998a,b,c). To date, no beam technique has been reported that allows the analysis of biomarker compounds directly in the inclusions, nor does any technique exist to extract a single petroleum inclusion and manipulate the minute quantity of extracted petroleum into gas chromatographic±mass spectrometric (GC±MS) or GC±MS±MS systems. There are two
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00115-7
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probable reasons for this. Firstly, although extracting a single inclusion and removing and transferring the small quantities of petroleum onto GC±MS systems is not impossible, there are many potential technical diculties. For example, such a small quantity of petroleum would adhere to syringe walls, or extraction lines as it was being transported to the inlet valve of the analytical unit, reducing the minute quantity of petroleum available even further. Such problems are not insurmountable, but would require a major investment of time and scienti®c ingenuity. Secondly, it may be possible to analyse single unusually large petroleum bearing inclusions in the laboratory, but petroleum inclusions hosted in typical diagenetic cements from sedimentary basins are often sub micron to 10 mm in length. If we assume that a single petroleum inclusion hosted in diagenetic cement, is spheroidal with a 10 mm diameter, the volume of the inclusion will be 524 mm3 ; if we assume that the vapour phase is 6% by volume, the volume of the liquid phase will be just under 500 mm3; if an average density of the petroleum is taken to be 0.8 g/cm3, then the mass of petroleum present in the inclusion will be approximately 410ÿ10g. With an average biomarker concentration in the petroleum of, say, 100 ppm, it is unlikely that many of the useful biomarker compounds could be detected to a satisfactory level to allow interpretations based on their distributions. The analysis of gasoline (and up to C21) range compounds from the combined products of the decrepitation of between 10 and 100 petroleum inclusions has been shown using laser micropyrolysis GC±MS (Greenwood et al., 1998). However, to our knowledge, it has not been possible to analyse the isomer distributions of C21+ biomarker compounds in petroleum-bearing ¯uid inclusions, other than by crushing the host mineral cements below solvent and extracting small quantities of petroleum, which will have been yielded from hundreds or thousands of inclusions (e.g. Karlsen et al., 1993; Macleod et al., 1994; Aplin et al., 1997; George et al., 1997, 1998a,b,c.). Such data are useful only if a careful petrographic and microthermometric study has been conducted beforehand. For example, it is possible that many different generations of ¯uid inclusions are being analysed at the molecular level and thus any molecular data derived from such an analyses will not be representative of a single generation of petroleum ¯uid inclusions (i.e. a single period in geologic time). Even if a careful petrographic study of the samples has been conducted before the included petroleum is extracted, there are still several problems associated with the extraction process. Most of these problems pertain to a simple question: is the petroleum being analysed truly included petroleum, or is it petroleum strongly adhered to the surface of the mineral cements or stuck in micro-®ssures in the cements, or more likely petroleum adhered to the surface of, for
example, quartz grains ? Organic material such as kerogen may also be present in the sample and could have adsorbed petroleum on its surfaces, or indeed, may degrade during work up to produce ``contaminant'' organic compounds. Hydrocarbons trapped/occluded in polar material strongly adsorbed onto mineral surfaces in reservoir sandstones have been detected using sequential extractions with increasing polar solvent mixtures by Wilhelms et al. (1996). Previously published studies of the extraction and analysis of included petroleum have used assorted methodologies for the cleaning of the host mineral cements, before they are crushed below solvent and worked-up in the usual manner for biomarker analysis and GC analysis. One clean-up process used to clean the mineral samples is solvent extraction, often with mixtures of dichloromethane and methanol using a Soxhlet apparatus (Karlsen et al., 1993; Macleod et al., 1994). Other, more severe clean-up methods are also used such as washing samples in hydrogen peroxide (Aplin et al., 1997; George et al., 1997, 1998b,c) or chromic acid (Karlsen et al., 1993; Macleod et al., 1994; George et al., 1998b,c). Indications of the success of the clean-up procedures have been obtained by observing the cleaned-up minerals under dark-®eld UV light microscopy for non-inclusion related ¯uorescence or by further Soxhlet extraction of the cleaned-up minerals and analysis of the extract by GC (Karlsen et al., 1993; George et al., 1997,1998a,b). Some procedures involve the disintegration of reservoir sandstones into individual grains which are then separated into mineral groups using sink-¯oat or magnetic separation techniques, before further clean-up (Karlsen et al., 1993; George et al., 1998b,c). With such small volumes of petroleum being analysed it only requires a small amount of contaminant petroleum to swamp that included and this has the potential to cause erroneous interpretations to be made. Therefore, in this work, a methodology and clean-up process has been developed that allows increased con®dence that truly included petroleum is being analysed and not mixtures of included petroleum and surface contamination. The method involves the addition of a suite of standard compounds to the surface of the rock or mineral grains that host the petroleum ¯uid inclusions of interest. The standards in the spike adhere to the surface of the samples and any particulate organic matter present, e.g. disseminated kerogen and organic-rich stylolytes. The samples are then put through a thorough cleaning process and only when the spiked standards have been completely removed, or are below the levels of detection that could interfere with the analyte hydrocarbons, are the samples crushed below solvent to release the included petroleum. The extracts from the ®nal clean-up stages are monitored for any presence of the standard compounds, to ensure that only truly included petroleum is analysed.
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2. Methods The samples required to yield the necessary information for the study can be selected after a petrographic and microthermometry study of the samples has been completed and a knowledge of the assorted petroleum ¯uid inclusion assemblages has been obtained (Goldstein and Reynolds, 1994). In this work 10±20 g samples of host rock or minerals are routinely used, though useable quantities of included petroleum have been extracted from ca. 2±3 g of host material. The amount of material required to liberate the necessary quantity of petroleum for a reproducible biomarker analysis is directly proportional to the abundance of petroleum inclusions present in the sample; hence the importance of the initial petrographic and microthermometry observations for selecting appropriate samples. In view of the very low amounts of hydrocarbons present in inclusions, strict precautions to avoid contamination are necessary; all solvents used are Distol grade (Fisher UK Ltd.) or redistilled on a 30 plate Oldershaw column. Alumina (Brockmann Grade 1 for column chromatography; BDH UK Ltd.) and silica gel (Merck Kieselgel, 60; BDH UK Ltd.) adsorbants and cotton wool plugs were pre-extracted before use and the adsorbants activated to 120 C overnight prior to use. All solvents and adsorbants were tested before use to ensure purity. All glassware used was cleaned using fresh chromic acid and rinsed in distilled water before use. Procedural blanks were run with each batch to monitor any contamination problems. 2.1. Sample preparation and crushing The sample clean-up methodology is shown schematically in Fig. 1. Firstly, the amount of petroleum adhering to the surface of the samples was determined, which also provided an aliquot of the so called ``free hydrocarbons'' for comparison with the included petroleum. For this, a sub-sample of the rock was extracted using dichloromethane (DCM) as follows. An aliquot (3 g) of the rock chips supplied was extracted (ultrasonic bath, 10 min) into DCM (5 ml). The total extract was cleaned up by elution through an activated alumina (1 cm bed) and silica gel (2 cm bed) Pasteur pipette mini-column with hexane (10 ml) and DCM (10 ml) in order to remove heavy polars, particulates etc. The eluate was then concentrated by rotary evaporation and analysed by gas chromatography (GC) in order to assess the amount of free hydrocarbons present in the sample and hence the amount of recovery (surrogate) standards to add to achieve comparable concentrations. The surrogate standard used was a 2 mg/ml solution of squalane (BDH Chemicals Ltd), 1,10 -binaphthyl (Kodak Ltd.) and n-phenylcarbazole (Aldrich UK Ltd.) in DCM.
Fig. 1. Analytical scheme.
The rock sample for analysis of included petroleum was ®rst disaggregated as gently as possible using a pestle and mortar so as not to cause excessive losses of ¯uid inclusions in mineral grains. The surrogate standard (diluted if necessary to achieve a volume of 1 ml) was added to an aliquot of the disaggregated rock sample (10±20 g) in a 30 ml vial, which was then allowed to dry at room temperature for at least 2 h. The sample was then ultrasonically extracted ®ve times with successive 20 ml amounts of DCM. The extracts were combined and saved. The sample was then Soxhlet extracted for 24 h using 250 ml of an azeotropic DCM/methanol
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(93:7) mixture. After this time the extract ¯ask was removed, the solvent rotary evaporated and the extract saved and combined with the ultrasonic extracts. Another ¯ask containing fresh solvent was then used to Soxhlet extract the sample for a further 24 h. After thorough drying (40 C, 48 h), the thoroughly extracted rock sample was transferred to a 200 ml beaker to which about 50 ml of hydrogen peroxide (100 volumes, i.e. about 30%; BDH Ltd. UK) was added. Some samples generated many small bubbles on addition of the hydrogen peroxide. After 24 h the hydrogen peroxide was decanted and a fresh aliquot of hydrogen peroxide added. If initial bubbling was minor then the samples were placed in an ultrasonic bath for 5 min. This procedure was repeated twice more giving a total of 4 hydrogen peroxide treatments over 5 days. Some samples partially decompose to a ®ne clayey sludge with sandy grains during the hydrogen peroxide treatment. This sludge was removed after the ®nal hydrogen peroxide treatment when the samples were washed (6) with distilled water. The remaining rock particles were then air dried at 40 C for 48 h. The solvent extracted and hydrogen peroxide treated dry rock particles were then further Soxhlet extracted twice. The ®nal Soxhlet extract was cleaned-up on a mini-column as described above, concentrated to 100 ml and analysed by GC and GC±MS. If the chromatograms still showed the presence of surrogate standards, then the hydrogen peroxide and subsequent Soxhlet extraction procedure was repeated. If the chromatogram showed no or negligible amounts of surrogate standards and other hydrocarbons, the rock particles were then crushed under 30 ml of DCM:methanol (9: 1) using a chromic acid cleaned pestle and mortar to release the hydrocarbons from any petroleum ¯uid inclusions in the rock particles. This was termed the crush-leach extraction. The resulting slurry was transferred to a glass centrifuge tube and centrifuged at 3000 rpm for 5 min. The supernatant was transferred to a round bottomed ¯ask and combined with the supernatants from two further DCM washes of the sludge in the centrifuge tube. Since the supernatants often still contained suspended clayey material they were carefully rotary evaporated just to dryness so that the suspended material remained stuck to the walls of the ¯ask. The ¯ask was then carefully rinsed (3) with 2 ml aliquots of DCM. This DCM extract was then reduced in volume to 0.5 ml and cleaned-up on a minicolumn as described above, concentrated to 100 ml and analysed by GC and GC±MS. For quantitative analysis, internal standards of n-heptadecylcyclohexane (ICN Biochemicals Ltd), p-terphenyl (Fluka Ltd), n-dodecylperhydroanthracene (a gift from BP) and D4 cholestane (synthesised in this laboratory) for quanti®cation of the saturated hydrocarbons, aromatic hydrocarbons, triterpanes and steranes, respectively, were added to the
vial containing the included hydrocarbons, just prior to GC and GC±MS analysis. These internal standards were chosen on the same basis as the surrogate standards i.e. that they do not generally coelute with other abundant oil components and they are unlikely to occur naturally in petroleums in signi®cant quantities (though caution is required since, for example, some oils can contain squalane). 2.2. GC and GC±MS Gas chromatographic analyses on the free hydrocarbon fractions were carried out on a Hewlett Packard 5890A-II instrument ®tted with a Hewlett Packard 7673 autosampler. A Hewlett Packard HP-1 polymethylsilicone coated (0.25 mm ®lm thickness) fused silica capillary column (25 m0.25 mm i.d.) was employed, using hydrogen as carrier gas and a ¯ame ionisation detector. Splitless injection was used and the oven was programmed from 50 C (2 min) to 300 C at 4 C minÿ1. The gas chromatographic analyses of the included oils were carried out using cold on-column injection onto a Carlo Erba Mega series 5360 gas chromatograph, ®tted with a Hewlett Packard HP-5 phenylmethylsilicone-coated (0.25 mm ®lm thickness) fused silica capillary column (25 m0.25 mm i.d.). Hydrogen was used as carrier gas and ¯ame ionisation detection was used. The GC oven program was as follows: 50 C for 2 min; 50±300 C at 6 C minÿ1; 300 C for 20 min. Data were acquired and processed using a VG Multichrom chromatography data system. Gas chromatography±mass spectrometry (GC±MS) analyses were carried out on a Hewlett Packard 5890-5972 MSD quadrupole instrument ®tted with a HP-1 coated (0.25 mm ®lm thickness) fused silica column (25 m0.25 mm i.d.). Splitless (1 min) injection was used and the GC oven temperature programmed from 40 C (held for 5 min) to 300 C at 4 C minÿ1 where it was held for 20 min. Data were mainly acquired in the selected ion monitoring (SIM) mode, though some full scan data were also acquired. Some analyses were also carried out, under similar analytical conditions, on a VG/Fisons Trio 1000 GC±MS system. 3. Results and discussion Some examples of the results of this included hydrocarbon analysis procedure are given below. The samples analysed were commercial cuttings from carbonate rocks from four wells in a South American basin on which background data is not available, but they are merely used as examples to demonstrate the data that can be produced using the method described. Microscopical screening analysis showed that the samples did not contain unusually abundant numbers of petroleum
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¯uid inclusions. These carbonate rocks samples contained stylolytes with abundant insoluble organic matter which was dicult to remove. However, since these samples were carbonates, the hydrogen peroxide oxidation procedure was necessary for the sample clean-up since an aggressive acidic oxidising agent (such as chromic acid) could not be used, though the latter would clearly be a very eective agent for the clean-up of quartz grains (e.g. Karlsen et al., 1993; George et al., 1998b). Furthermore, in samples where rock disaggregation and separation into single grains of individual minerals is possible (e.g. Karlsen et al., 1993; George et al., 1998b), then in addition to increasing the selectivity of the inclusions analysed this can also reduce the number of clean-up steps required and improve the clean-up eciency, since it is possible that standards added to composite grains may not easily penetrate microfractures or voids in mineral cements.
Fig. 2. Gas chromatograms of GA1 (a) and GA2 (b) S1 free total hydrocarbons. n-Alkanes are numbered, Pr is pristane, Ph is phytane, SPCZ, SBN and SSQ are the surrogate standard spikes n-phenylcarbazole, 1,10 -binaphthyl and squalane, respectively.
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3.1. Saturated hydrocarbons Examples of the gas chromatograms of the free hydrocarbons from the ®rst solvent extraction (S1) of the samples GA1 and GA2 are shown in Fig. 2, while Fig. 3 shows those of the included hydrocarbons released by the crush-leach procedure after exhaustive clean-up of these samples. Comparison of Figs. 2 and 3 clearly shows the major dierences in the saturated hydrocarbon distributions between the free (extractable) and the included fractions released by the crush-leach procedure. While the free saturated hydrocarbons are dominated by narrow ranges of n-alkanes (centered around C19 and C15 in samples GA1 and GA2, respectively) and contain signi®cant unresolved complex mixtures (UCMs) in their chromatograms, similar to those often seen in oil-based-mud contaminated samples, the distributions seen in the crush-leach fractions are much more like those found in crude oils. Although the chromatograms from the crush-leach hydrocarbon fractions also tend to show a ``humpy'' baseline, this is exaggerated
Fig. 3. Gas chromatograms of GA1 (a) and GA2 (b) crushleach (included petroleum) total hydrocarbon fractions. nAlkanes are numbered, Pr is pristane, Ph is phytane.
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by the very low signal intensities and the increased visibility of the baseline hump due to column bleed that occurs after about 55 min in the chromatograms. It is also clear from the chromatograms of the crush-leach hydrocarbons that the saturated distribution from the GA2 sample was signi®cantly dierent from the others that were analysed from this region, being much more waxy and containing much lower relative abundances of pristane and phytane. Quanti®cation of the individual hydrocarbons released by the crush-leach procedure (see Table 1) showed that they were generally in extremely low abundance in these samples, with many being found in the low ng/g (ppb) levels. In these particular samples, the individual included n-alkane concentrations ranged from below the detection limit of around 1 ng/g in some samples to over 100 ng/g for certain n-alkanes in the GA2 sample. In this latter sample, the ¯uid inclusion C15±C35 hydrocarbons were about an order of magnitude more abundant than some of the other samples analysed from this region. Assuming that the n-alkanes comprise about 10% of a typical petroleum, then from the summed nC15-to n-C35 concentrations in Table 1, the amounts of petroleum released by the crush-leach procedure was
calculated to be approximately 3000 and 14,000 ng/g rock for the GA1 and GA2 samples, respectively. Since approximately 10 g of each rock sample was crushed and we previously estimated that a typical petroleum ¯uid inclusion may contain 0.4 ng of petroleum, then it would appear that the number of inclusions analysed from the GA1 and GA2 samples were approximately 75,000 and 350,000, respectively. Clearly, with these numbers of inclusions being crushed, the presence of more than a single generation of inclusions would result in mixtures of hydrocarbons being analysed. 3.2. Aromatic hydrocarbons Alkylated naphthalenes, phenanthrenes and dibenzothiophenes were detected by GC±MS in the included hydrocarbon crush-leach fractions and ratios (see Table 2) commonly used for maturity assessment, such as those based on methylphenanthrenes and methyldibenzothiophenes were measured (e.g. Radke, 1987). Such aromatic hydrocarbon parameters have previously been reported from petroleum ¯uid inclusions (e.g. George et al., 1997, 1998a). Interestingly, anthracene and methylanthracenes were observed in many, but not all, of the included
Table 1 Included petroleum individual alkane concentrations (ng/g rock) from crush-leach total hydrocarbon fractions Analyte
GA1(ng/g)
GA2Ab(ng/g)
GA2Bb(ng/g)
GA3(ng/g)
nC15 nC16 nC17 nC18 nC19 nC20 nC21 nC22 nC23 nC24 nC25 nC26 nC27 nC28 nC29 nC30 nC31 nC32 nC33 nC34 nC35
10.8 14.9 21.8 26.7 25.4 28.0 25.5 21.7 18.4 17.4 15.0 13.6 11.4 10.1 10.1 9.0 8.0 7.4 5.4 5.4 5.8
110.2 106.2 103.0 91.3 82.5 87.0 82.9 78.4 69.9 69.2 65.8 67.3 58.5 57.0 48.0 55.9 46.6 47.8 31.0 25.4 25.6
120.2 108.9 102.6 88.2 79.5 81.9 78.1 73.1 69.5 64.4 61.0 63.3 54.2 53.4 44.1 52.0 43.8 40.9 28.2 23.6 19.3
11.6 8.1 14.3 21.5 25.3 29.1 27.3 26.8 22.2 19.9 17.3 16.4 15.3 11.7 15.3 12.0 10.1 8.1 6.5 5.6 5.0
Pristane Phytane
8.5 9.9
9.5 20.2
10.8 20.6
5.7 10.9
C29ab hopane C30ab hopane
0.97 1.29
24.59 11.96
25.75 11.61
a b
1.18 2.03
nm : Not measurable. GA2A and GA2B are two aliquots of the same sample analysed separately.
GA4(ng/g)
GA5(ng/g)
GA6(ng/g)
2.4 1.7 2.1 3.7 4.8 7.8 7.8 7.0 6.0 5.3 5.1 4.1 4.2 3.2 3.5 2.2 2.5 1.7 1.3 nma nm
13.5 21.4 25.0 26.9 23.9 24.1 22.8 23.2 20.1 21.1 20.0 21.4 18.5 19.0 14.5 18.3 12.9 15.0 9.7 8.3 7.7
12.1 14.4 15.5 15.6 16.1 16.1 14.8 13.6 11.7 10.7 9.8 9.1 10.8 11.1 12.4 10.4 9.1 6.6 4.7 3.2 3.0
0.8 1.4
2.7 6.0
9.3 5.8
11.94 10.52
0.16 0.21
nm nm
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Table 2 Geochemical parameters from included petroleums Sample
Pr/n-C17 Pr/Phc Ts/(Tm+Ts)d C29H/C30Hd C31 22S/(S+R) homohopane
C29 Sterane MPI-1f MPI-3f Rc(%)f 4-/1-MDBTg (bb/bb+aa)e
GA1 GA2A*b GA2B*b GA3 GA4 GA5 GA6
0.39 0.09 0.11 0.39 0.37 0.11 0.60
nm 0.56 0.59 nm nm nm nm
a b c d e f g
0.85 0.47 0.52 0.52 0.55 0.46 1.60
0.40 0.28 0.27 0.36 nma 0.13 0.55
0.75 2.06 2.22 0.58 nm 1.13 0.75
0.56 0.51 0.51 0.50 nm 0.54 nm
0.51 0.60 0.62 0.64 0.84 0.68 0.92
0.78 0.65 0.66 0.75 0.90 1.01 1.18
0.71 0.76 0.77 0.78 0.90 0.81 0.95
2.89 1.83 1.96 2.44 2.24 1.05 3.00
m=Not measurable. GA2A* and GA2B* are two aliquots of the same sample analysed separately. Pr and Ph are pristane and phytane, respectively. H denotes hopane, Ts and Tm are de®ned in Table 3. bb and aa refer to the isomeric positions at C-14 and C-17 of the C29 sterane. MPI-1, MPI-3 and Rc (%) are methylphenanthrene based maturity parameters de®ned in Radke (1987). 4- and 1-MDBT are methyldibenzothiophenes.
petroleum crush-leach extracts that were analysed in this and other ¯uid inclusion studies in this laboratory (e.g. see Fig. 4). They have also been reported in petroleum ¯uid inclusion studies by others (e.g. George et al., 1995, 1997, 1998a). Anthracenes are rarely associated with conventional crude oils but they have recently been noted to occur in unusual crude oils from the Canadian Williston Basin, where they were thought to have been generated by short term, high temperature pyrolysis reactions (Li et al., 1998). Anthracene and methylanthracenes have previously been detected in coaly sediments, especially those with vitrinite re¯ectance values below about 1.0% (Radke et al., 1982; Garrigues et al., 1988) and methylanthracenes were apparent (D. Karlsen, 2000, personal communication) in the aromatic hydrocarbon fraction of some of the more terrestriallyin¯uenced crude oils from the Haltenbanken region of the Norwegian continental shelf shown by Karlsen et al., (1993). The presence of anthracenes requires caution when using methylphenanthrene maturity parameters on included petroleum released by crush-leach techniques because of the coelution of 1-methylanthracene with the 9- or 1-methylphenanthrene, depending on the gas chromatographic stationary phase. Although present in a minority of included oils, typically less than 20% (S. George, 2000, personal communication), the relatively common occurrence of anthracenes in included petroleums compared to crude oils also raises a question of whether they could be analytical artefacts. For example, are they formed by some kind of very localised heating which occurs when the mineral grains are crushed to release the ¯uids from the inclusions, even though this was done under solvent and, in our case, by relatively gentle hand crushing using a pestle and mortar? It is possible, for example, that local temperatures near
Fig. 4. m/z 178 and m/z 192 mass chromatograms of GA1 crush-leach (included petroleum) total hydrocarbon fraction. P, A and 2-MA are phenanthrene, anthracene and 2-methylanthracene, respectively. The methylphenanthrenes in the m/z 192 mass chromatogram are numbered according to their methylation position.
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shearing grains may be very high. An alternative explanation may be that these aromatic hydrocarbons could have been generated from terrigenous organic matter, and were adsorbed onto mineral surfaces then preserved in inclusions. 3.3. Biomarkers Examples of the m/z 191 and 218 mass chromatograms of the included hydrocarbons released by the crush-leach procedure from samples GA1 and GA2 are shown in Figs. 5 and 6. The concentrations of the hopanes in many of these samples were very low (see Table 1), and were close to the detection limits of the GC±MS instrument used, resulting in poor signal to noise ratios in the mass chromatograms from some samples (e.g. GA1). Sterane concentrations in the crushleach fractions were even lower, with individual C29 sterane isomers being between 4 and about 70 times lower in abundance than the C3017a(H),21b(H)-hopane peak, and generally were too low to accurately quantify. They were thus of limited use for correlation purposes, though a higher sensitivity mass spectrometer may widen the useful range of concentrations of these compounds. However, even with the instrumentation used, the m/z 191 mass chromatogram showing the hopane distribution of the included petroleum from the GA2 sample
displayed a clear and distinctive distribution which was useful for correlation purposes. The C29 17a (H),21b(H)norhopane to C3017a(H),21b(H)-hopane ratio in this sample was also signi®cantly dierent from those of the other samples in this region and was consistent with the clear dierences seen in the gas chromatograms of these crush-leach extracts. 3.4. Free hydrocarbon input to crush-leach extracts An assessment of potential contamination by surface hydrocarbons was made by using mass chromatograms
Fig. 6. m/z 191 and m/z 218 mass chromatograms of GA2A crush-leach (included petroleum) total hydrocarbon fraction showing the presence of hopane and sterane distributions. Peak assignments are as for Fig. 5.
Table 3 Hopane peak assignments in m/z 191 mass chromatograms
Fig. 5. m/z 191 and m/z 218 mass chromatograms of GA1 crush-leach (included petroleum) total hydrocarbon fraction showing the presence of hopanes and steranes in abundances close to the detection limits of the GC±MS system used. Hopane peak assignments are given in Table 3, the retention time positions of the C27, C28 and C29 14b(H),17b(H) steranes in the m/z 218 mass chromatograms are marked.
Peak
Assignment
H1 H2 H3 H4 H5 H6
C27 18a(H)-trisnorneohopane (Ts) C27 17a(H)-trisnorhopane (Tm) C29 17a(H),21b(H)-norhopane C30 17a(H),21b(H)-hopane C31 17a(H),21b(H)-homohopane [22S] C31 17a(H),21b(H)-homohopane [22R]
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to monitor for the presence of surrogate standards which were used to spike the rock before analysis. The abundant peaks due to the n-phenylcarbazole (100 mg), 1,10 -binaphthyl (200 mg) and the squalane (500 mg) which were added to a 10 g aliquots of the sample GA2 prior to clean-up are clearly seen in the respective m/z 243, 253 and 57 mass chromatograms of the ®rst solvent extract of the free hydrocarbons of this sample which are shown in Fig. 7a. These peaks are no longer seen in the corresponding crush-leach hydrocarbon fraction mass chromatograms (Fig. 7b). A peak eluting around 55 min in the m/z 253 mass chromatogram of the crushleach hydrocarbon fraction has a similar retention time as that of 1,10 -binaphthyl, but is probably an unknown coelutant. A quantitative assessment of the likely amount of cross contamination of the included hydrocarbons from the crush-leach extraction by remaining free hydrocarbons in the samples was made by analysing the hydrocarbons in the ®nal Soxhlet extraction after the ®nal hydrogen peroxide treatment. The results of these are given in Table 4. They show that although none of the samples in this set were rich in petroleum ¯uid inclusions, and some were very lean, the concentrations of individual alkanes in the crush leach extracts in these samples were between about 100 (in the richest) and 3 (in the leanest) times greater than those in their corresponding ®nal Soxhlet cleanup extract. This
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Table 4 Individual alkane concentrations (ng/g rock) of free total hydrocarbons in the ®nal soxhlet extract after peroxide treatment Analyte GA1 GA2A*b GA2B*b GA3 GA4 GA5 GA6 (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) nC15 nC16 nC17 nC18 nC19 nC20 nC21 nC22 nC23 nC24 nC25 nC26 nC27 nC28 nC29 nC30 nC31 nC32 nC33 nC34 nC35 Pristane Phytane
2.4 2.2 4.6 3.1 1.4 2.1 2.3 2.6 2.3 2.3 1.6 2.3 1.3 1.4 1.5 1.1 nm nm nm nm nm 3.0 1.9
2.5 2.2 0.7 1.0 1.0 1.0 1.0 1.2 1.2 1.3 1.3 1.6 1.1 1.0 0.9 1.0 nm nm nm nm nm 0.5 0.4
0.9 1.8 1.4 2.0 1.2 1.0 1.4 1.1 1.0 1.2 1.4 1.5 2.5 3.5 4.2 4.3 4.0 3.2 2.5 1.7 1.1 1.4 1.0
0.8 2.7 2.2 2.2 1.4 nma 0.8 1.4 1.2 1.4 1.3 1.1 1.2 1.5 1.1 0.0 nm nm nm nm nm 2.2 1.2
0.0 0.2 0.2 0.8 2.2 1.4 1.5 1.4 1.3 1.1 0.8 1.1 1.5 1.0 2.7 0.9 nm nm nm nm nm nm nm
0.0 0.5 0.4 0.3 0.5 0.4 0.3 0.3 0.2 0.0 0.3 0.2 0.2 0.2 0.2 0.2 nm nm nm nm nm nm nm
0.6 0.7 1.1 0.5 0.6 0.4 0.4 0.4 0.4 0.4 0.5 0.2 0.3 0.2 0.3 0.2 nm nm nm nm nm nm nm
a
nm: Not measurable. *GA2A and *GA2B are two aliquots of the same sample analysed separately. b
Fig. 7. Comparison of mass chromatograms used to show the presence of the surrogate standard spike compounds in the GA2A free (a) and corresponding (b) crush-leach (included petroleum) total hydrocarbon fractions. Peak assignments are as given in Fig. 2.
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shows that the reliability of the included petroleum analyses is vastly improved in samples that contain more abundant ¯uid inclusions. 3.5. Reproducibility After initial disaggregation, sample GA2 was divided into two aliquots A and B, which were then cleaned-up and analysed separately as though they were two dierent samples. The quantitative analysis results given in Tables 1 and 2 show excellent reproducibility for this duplicate analysis both in terms of the quantities of hydrocarbons released and also the composition of them as shown by the various geochemical parameters and ratios measured. 4. Conclusions A method, based on spiking samples with a mixture of standard hydrocarbons, has been developed allowing the analysis of biomarker and other hydrocarbons in petroleum ¯uid inclusions in reservoir rocks and carrier systems to be made with more con®dence that the hydrocarbons extracted are free from extraneous hydrocarbons derived from the surface of the mineral. The method, which ultimately involves crushing the cleanedup mineral grains under solvent in order to extract the hydrocarbons released from the ¯uid inclusions (termed crush-leach), is very time-consuming but necessary to ensure a satisfactory clean-up. However, meaningful results from this method are also critically dependent on there being a sucient abundance of petroleum ¯uid inclusions in the sample and also that these inclusions are representative of a single period of geological time (i.e. a single generation of inclusions). This requires that suitable samples can only be selected after a careful petrographic and microthermometric study has been conducted. In some circumstances, the method therefore generates data which can be combined with microthermometry and paleo-PVT data in order to give a detailed picture of the source, maturity and physical properties of palaeo-petroleum. Acknowledgements We are grateful to M. Chen for her technical assistance in the method development and quantitation work. We also thank Steve Larter and Andy Aplin for their useful discussions and P. Donohoe, K. Noke, R. Hunter and I. Harrison for their technical support. We acknowledge the reviewers D. Karlsen and T. Ruble and associate editor S. George for their constructive criticism and helpful comments which considerably improved this paper. Associate EditorÐS.C. George
References Aplin, A.C., Macleod, G., Larter, S.R., Jones, M., Chen, M., Bigge, A. et al., 1997. Petroleum ¯uid inclusions: improved analytical methods enhance their use as a tool in petroleum geoscience. In: Hendry, J. Carey, P., Parnell, J., Ruell, A., Worden, R. (Eds.), Geo¯uids II '97 Extended Abstracts, Belfast, pp. 260±263. Aplin, A.C., Macleod, G., Larter, S.R., Pedersen, K.S., Sorensen, H., Booth, T., 1999. Combined use of confocal laser scanning microscopy and PVT simulation for estimating the composition and physical properties of petroleum in ¯uid inclusions. Marine & Petroleum Geology 16, 97±110. Bigge, M.A., Petch, G.S., Macleod, G., Larter, S.R., Aplin, A.C., 1995. Quantitative geochemical analysis of petroleum ¯uid inclusions. Problems and developments. In: Grimalt, J.O., Dorronsoro, C. (Eds.), Organic Geochemistry: Developments and Applications to Energy, Climate, Environment and Human History. A.I.G.O.A, Donostia, San Sebastian, pp. 757±759. Etminan, H., Homann, C.F., 1989. Biomarkers in ¯uid inclusions: a new tool for constraining source regimes and its implications for the genesis of Mississippi Valley-type deposits. Geology 17, 19±22. Garrigues, P., DeSury, R., Angelin, M.L., Bellocq, J., Oudin, J.L., Ewald, M., 1988. Relation of the methylated aromatic hydrocarbon distribution pattern to the maturity of organic matter in ancient sediments from the Mahakam Delta. Geochimica et Cosmochimica Acta 52, 375±384. George, S.C., Eadington, P.J., Hamilton, P.J., 1995. Organic geochemistry of petroleum-bearing ¯uid inclusions in quartz grains from a Cretaceous sandstone. In: Grimalt, J.O., Dorronsoro, C. (Eds.), Organic Geochemistry: Developments and Applications to Energy, Climate, Environment and Human History. A.I.G.O.A, Donostia, San Sebastian, pp. 282±283. George, S.C., Krieger, F.W., Eadington, P.J., Quezada, R.A., Greenwood, P.F., Eisenberg, L.I. et al., 1997. Geochemical comparison of oil-bearing ¯uid inclusions and produced oil from the Toro sandstone, Papua New Guinea. Organic Geochemistry 26, 155±173. George, S.C., Lisk, M., Summons, R.E., Quezada, R.A., 1988a. Constraining the oil charge history of the South Pepper oil®eld from the analysis of oil-bearing ¯uid inclusions. Organic Geochemistry 29, 631±648. George, S.C., Lisk, M., Eadington, P.J. Quezada, R.A. 1998b. Geochemistry of a palaeo-oil column, Octavius 2, Vulcan sub-basin. In: Purcell, P.G., Purcell, R.R. (Eds.), The Sedimentary Basins of Western Australia 2, Proceedings of the Petroleum Exploration Society of Australia Symposium, Perth, 1998, pp. 195±210. George, S.C., Eadington, P.J., Lisk, M., Quezada, R.A., 1998c. Geochemical comparison of oil trapped in ¯uid inclusions and reservoired oil in Blackback oil®eld, Gippsland Basin, Australia. Petroleum Exploration Society of Australia Journal 26, 64±82. Greenwood, P.F., George, S.C., Hall, K., 1998. Applications of laser micropyrolysis gas chromatography mass spectrometry. Organic Geochemistry 29, 1075±1089. Goldstein R.H. Reynolds T.J., 1994. Systematics of ¯uid inclusions in diagenetic minerals. Society for Sedimentary Geology, Short Course 31. 199 pp.
D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173 Hors®eld, B., McLimans, R.K., 1984. Geothermometry and geochemistry of aqueous and oil-bearing ¯uid inclusions from Fateh Field, Dubai. Organic Geochemistry 6, 733± 740. Jesenius, J., Burruss, R.C., 1990. Hydrocarbon Ð water interactions during brine migration: evidence from hydrocarbon inclusions in calcite cements from Danish North sea oils. Geochimica et Cosmochimica Acta 54, 705±740. Jones, D.M., Macleod, G., Larter, S.R., Hall, D.L., Aplin, A.C., Chen, M., 1996. Characterisation of the molecular composition of included petroleum. In: Brown, P.E., Hagemann, S.G. (Eds.), Proceedings of the Sixth Biennial PanAmerican Conference on Research on Fluid Inclusions, (PACROFI VI). Madison, Wisconsin, USA, pp. 64±65. Karlsen, D.A., Nedkvitne, T., Larter, S.R., Bjùrlykke, K., 1993. Hydrocarbon composition of authigenic inclusions: Application to elucidation of petroleum reservoir ®lling history. Geochimica et Cosmochimica Acta 57, 3641±3659. Kihle, J., 1995. Adaptation of ¯uorescence excitation-emission micro-spectroscopy for characterization of single hydrocarbon ¯uid inclusions. Organic Geochemistry 23, 1029± 1042. Li, M.W., Osadetz, K.G., Yao, H.X., Obermajer, M., Fowler, M.G., Snowdon, L.R. et al., 1998. Unusual crude oils in the
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Canadian Williston Basin, southeastern Saskatchewan. Organic Geochemistry 28, 477±488. Macleod, G., Petch, S., Larter, S.R., Aplin, A.C., 1994. Improved analysis of petroleum ¯uid inclusions: applications to reservoir studies. Abstracts of the American Chemical Society 207, 132. Macleod, G., Larter, S.R., Aplin, A.C., Pedersen, K.S., Booth, T.A., 1996. Determination of the eective composition of single petroleum inclusions using confocal scanning laser microscopy and PVT simulation. In: Brown, P.E., Hagemann, S.G. (Eds.), Proceedings of the Sixth Biennial PanAmerican Conference on Research on Fluid Inclusions, (PACROFI VI). Madison, Wisconsin, USA, pp. 81±82. Murray, R.C., 1957. Hydrocarbon ¯uid inclusions in quartz. Bulletin American Association of Petroleum Geologists 41, 950±956. Radke, M., 1987. Organic geochemistry of aromatic hydrocarbons. In: Brooks, J., Welte, D. (Eds.), Advances in Petroleum Geochemistry, Vol. 2. Academic Press, London, pp. 141±207. Roedder, E., 1984. Fluid inclusions. Reviews in Mineralogy 12. Wilhelms, A., Horstad, I., Karlsen, D., 1996. Sequential extraction Ð a useful tool for reservoir geochemistry? Organic Geochemistry 24, 1157±1172.
www.elsevier.nl/locate/orggeochem
Molecular analysis of petroleum in ¯uid inclusions: a practical methodology D.M. Jones *, G. Macleod 1 Fossil Fuels and Environmental Geochemistry (Postgraduate Institute): NRG, University of Newcastle upon Tyne, NE1 7RU, UK Received 21 February 2000; accepted 2 August 2000 (returned to author for revision 12 April 2000)
Abstract A method is described for extracting the petroleum from petroleum-bearing ¯uid inclusions hosted in the diagenetic cements of sedimentary rocks, whilst minimising contamination from petroleum in the pore space. A clean-up technique, involving the addition of extraction standards to the rock prior to analysis, has been developed that allows increased con®dence that the hydrocarbons extracted are only from included petroleum and are not mixtures of included petroleum and petroleum (or other organic material) adhering to the surface of the host minerals within the sample. Replicate quantitative analyses indicate that the technique produces highly reproducible results. The problems of analysing included petroleum and the clean-up method development are discussed and examples of analyses of included petroleums from carbonate reservoir and carrier units are given. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Petroleum; Fluid inclusions; Hydrocarbons; Biomarkers
1. Introduction Fluid inclusions are tiny vacuoles in minerals, containing ¯uids that were present when the mineral cement formed. They thus can be regarded as time capsules of geo¯uids and as such are invaluable for understanding the evolution and migration of petroleum in sedimentary basins (cf. Roedder, 1984; Karlsen et al., 1993). An example of their use in the elucidation of the ®lling history of a petroleum reservoir is given by Karlsen et al. (1993). Petroleum-bearing ¯uid inclusions can be studied using microthermometric methods to obtain the minimum trapping temperature of single petroleum inclusions (Goldstein and Reynolds, 1994) and the gross * Corresponding author. Tel.: +44-191-222-8628; fax: +44191-222-5431. E-mail address: [email protected] (D.M. Jones). 1 Present address: Shell Exploration and Production Technology Co., Bellaire Technology Center, PO Box 481, Houston, TX 77001-0481, USA.
composition of included petroleum can be derived by confocal microscopy and pressure-volume-temperature (PVT) simulation (Macleod et al., 1996; Aplin et al., 1997, 1999). Assorted beam techniques can also be used to determine information on the general composition of the included petroleum, such as bond types present in the included petroleum, and some bulk compositional data from ultra violet (UV) spectroscopic techniques (e.g. Kihle, 1995). There have been a number of studies of the molecular compositions of petroleums in ¯uid inclusions by various techniques including thermal decrepitation or crushing (e.g. Murray, 1957; Hors®eld and McLimans, 1984; Etminan and Homan, 1989; Jesenius and Burruss, 1990; Karlsen et al., 1993; Macleod et al., 1994; Bigge et al., 1995; Jones et al., 1996; George et al., 1995, 1997, 1998a,b,c). To date, no beam technique has been reported that allows the analysis of biomarker compounds directly in the inclusions, nor does any technique exist to extract a single petroleum inclusion and manipulate the minute quantity of extracted petroleum into gas chromatographic±mass spectrometric (GC±MS) or GC±MS±MS systems. There are two
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00115-7
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probable reasons for this. Firstly, although extracting a single inclusion and removing and transferring the small quantities of petroleum onto GC±MS systems is not impossible, there are many potential technical diculties. For example, such a small quantity of petroleum would adhere to syringe walls, or extraction lines as it was being transported to the inlet valve of the analytical unit, reducing the minute quantity of petroleum available even further. Such problems are not insurmountable, but would require a major investment of time and scienti®c ingenuity. Secondly, it may be possible to analyse single unusually large petroleum bearing inclusions in the laboratory, but petroleum inclusions hosted in typical diagenetic cements from sedimentary basins are often sub micron to 10 mm in length. If we assume that a single petroleum inclusion hosted in diagenetic cement, is spheroidal with a 10 mm diameter, the volume of the inclusion will be 524 mm3 ; if we assume that the vapour phase is 6% by volume, the volume of the liquid phase will be just under 500 mm3; if an average density of the petroleum is taken to be 0.8 g/cm3, then the mass of petroleum present in the inclusion will be approximately 410ÿ10g. With an average biomarker concentration in the petroleum of, say, 100 ppm, it is unlikely that many of the useful biomarker compounds could be detected to a satisfactory level to allow interpretations based on their distributions. The analysis of gasoline (and up to C21) range compounds from the combined products of the decrepitation of between 10 and 100 petroleum inclusions has been shown using laser micropyrolysis GC±MS (Greenwood et al., 1998). However, to our knowledge, it has not been possible to analyse the isomer distributions of C21+ biomarker compounds in petroleum-bearing ¯uid inclusions, other than by crushing the host mineral cements below solvent and extracting small quantities of petroleum, which will have been yielded from hundreds or thousands of inclusions (e.g. Karlsen et al., 1993; Macleod et al., 1994; Aplin et al., 1997; George et al., 1997, 1998a,b,c.). Such data are useful only if a careful petrographic and microthermometric study has been conducted beforehand. For example, it is possible that many different generations of ¯uid inclusions are being analysed at the molecular level and thus any molecular data derived from such an analyses will not be representative of a single generation of petroleum ¯uid inclusions (i.e. a single period in geologic time). Even if a careful petrographic study of the samples has been conducted before the included petroleum is extracted, there are still several problems associated with the extraction process. Most of these problems pertain to a simple question: is the petroleum being analysed truly included petroleum, or is it petroleum strongly adhered to the surface of the mineral cements or stuck in micro-®ssures in the cements, or more likely petroleum adhered to the surface of, for
example, quartz grains ? Organic material such as kerogen may also be present in the sample and could have adsorbed petroleum on its surfaces, or indeed, may degrade during work up to produce ``contaminant'' organic compounds. Hydrocarbons trapped/occluded in polar material strongly adsorbed onto mineral surfaces in reservoir sandstones have been detected using sequential extractions with increasing polar solvent mixtures by Wilhelms et al. (1996). Previously published studies of the extraction and analysis of included petroleum have used assorted methodologies for the cleaning of the host mineral cements, before they are crushed below solvent and worked-up in the usual manner for biomarker analysis and GC analysis. One clean-up process used to clean the mineral samples is solvent extraction, often with mixtures of dichloromethane and methanol using a Soxhlet apparatus (Karlsen et al., 1993; Macleod et al., 1994). Other, more severe clean-up methods are also used such as washing samples in hydrogen peroxide (Aplin et al., 1997; George et al., 1997, 1998b,c) or chromic acid (Karlsen et al., 1993; Macleod et al., 1994; George et al., 1998b,c). Indications of the success of the clean-up procedures have been obtained by observing the cleaned-up minerals under dark-®eld UV light microscopy for non-inclusion related ¯uorescence or by further Soxhlet extraction of the cleaned-up minerals and analysis of the extract by GC (Karlsen et al., 1993; George et al., 1997,1998a,b). Some procedures involve the disintegration of reservoir sandstones into individual grains which are then separated into mineral groups using sink-¯oat or magnetic separation techniques, before further clean-up (Karlsen et al., 1993; George et al., 1998b,c). With such small volumes of petroleum being analysed it only requires a small amount of contaminant petroleum to swamp that included and this has the potential to cause erroneous interpretations to be made. Therefore, in this work, a methodology and clean-up process has been developed that allows increased con®dence that truly included petroleum is being analysed and not mixtures of included petroleum and surface contamination. The method involves the addition of a suite of standard compounds to the surface of the rock or mineral grains that host the petroleum ¯uid inclusions of interest. The standards in the spike adhere to the surface of the samples and any particulate organic matter present, e.g. disseminated kerogen and organic-rich stylolytes. The samples are then put through a thorough cleaning process and only when the spiked standards have been completely removed, or are below the levels of detection that could interfere with the analyte hydrocarbons, are the samples crushed below solvent to release the included petroleum. The extracts from the ®nal clean-up stages are monitored for any presence of the standard compounds, to ensure that only truly included petroleum is analysed.
D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
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2. Methods The samples required to yield the necessary information for the study can be selected after a petrographic and microthermometry study of the samples has been completed and a knowledge of the assorted petroleum ¯uid inclusion assemblages has been obtained (Goldstein and Reynolds, 1994). In this work 10±20 g samples of host rock or minerals are routinely used, though useable quantities of included petroleum have been extracted from ca. 2±3 g of host material. The amount of material required to liberate the necessary quantity of petroleum for a reproducible biomarker analysis is directly proportional to the abundance of petroleum inclusions present in the sample; hence the importance of the initial petrographic and microthermometry observations for selecting appropriate samples. In view of the very low amounts of hydrocarbons present in inclusions, strict precautions to avoid contamination are necessary; all solvents used are Distol grade (Fisher UK Ltd.) or redistilled on a 30 plate Oldershaw column. Alumina (Brockmann Grade 1 for column chromatography; BDH UK Ltd.) and silica gel (Merck Kieselgel, 60; BDH UK Ltd.) adsorbants and cotton wool plugs were pre-extracted before use and the adsorbants activated to 120 C overnight prior to use. All solvents and adsorbants were tested before use to ensure purity. All glassware used was cleaned using fresh chromic acid and rinsed in distilled water before use. Procedural blanks were run with each batch to monitor any contamination problems. 2.1. Sample preparation and crushing The sample clean-up methodology is shown schematically in Fig. 1. Firstly, the amount of petroleum adhering to the surface of the samples was determined, which also provided an aliquot of the so called ``free hydrocarbons'' for comparison with the included petroleum. For this, a sub-sample of the rock was extracted using dichloromethane (DCM) as follows. An aliquot (3 g) of the rock chips supplied was extracted (ultrasonic bath, 10 min) into DCM (5 ml). The total extract was cleaned up by elution through an activated alumina (1 cm bed) and silica gel (2 cm bed) Pasteur pipette mini-column with hexane (10 ml) and DCM (10 ml) in order to remove heavy polars, particulates etc. The eluate was then concentrated by rotary evaporation and analysed by gas chromatography (GC) in order to assess the amount of free hydrocarbons present in the sample and hence the amount of recovery (surrogate) standards to add to achieve comparable concentrations. The surrogate standard used was a 2 mg/ml solution of squalane (BDH Chemicals Ltd), 1,10 -binaphthyl (Kodak Ltd.) and n-phenylcarbazole (Aldrich UK Ltd.) in DCM.
Fig. 1. Analytical scheme.
The rock sample for analysis of included petroleum was ®rst disaggregated as gently as possible using a pestle and mortar so as not to cause excessive losses of ¯uid inclusions in mineral grains. The surrogate standard (diluted if necessary to achieve a volume of 1 ml) was added to an aliquot of the disaggregated rock sample (10±20 g) in a 30 ml vial, which was then allowed to dry at room temperature for at least 2 h. The sample was then ultrasonically extracted ®ve times with successive 20 ml amounts of DCM. The extracts were combined and saved. The sample was then Soxhlet extracted for 24 h using 250 ml of an azeotropic DCM/methanol
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(93:7) mixture. After this time the extract ¯ask was removed, the solvent rotary evaporated and the extract saved and combined with the ultrasonic extracts. Another ¯ask containing fresh solvent was then used to Soxhlet extract the sample for a further 24 h. After thorough drying (40 C, 48 h), the thoroughly extracted rock sample was transferred to a 200 ml beaker to which about 50 ml of hydrogen peroxide (100 volumes, i.e. about 30%; BDH Ltd. UK) was added. Some samples generated many small bubbles on addition of the hydrogen peroxide. After 24 h the hydrogen peroxide was decanted and a fresh aliquot of hydrogen peroxide added. If initial bubbling was minor then the samples were placed in an ultrasonic bath for 5 min. This procedure was repeated twice more giving a total of 4 hydrogen peroxide treatments over 5 days. Some samples partially decompose to a ®ne clayey sludge with sandy grains during the hydrogen peroxide treatment. This sludge was removed after the ®nal hydrogen peroxide treatment when the samples were washed (6) with distilled water. The remaining rock particles were then air dried at 40 C for 48 h. The solvent extracted and hydrogen peroxide treated dry rock particles were then further Soxhlet extracted twice. The ®nal Soxhlet extract was cleaned-up on a mini-column as described above, concentrated to 100 ml and analysed by GC and GC±MS. If the chromatograms still showed the presence of surrogate standards, then the hydrogen peroxide and subsequent Soxhlet extraction procedure was repeated. If the chromatogram showed no or negligible amounts of surrogate standards and other hydrocarbons, the rock particles were then crushed under 30 ml of DCM:methanol (9: 1) using a chromic acid cleaned pestle and mortar to release the hydrocarbons from any petroleum ¯uid inclusions in the rock particles. This was termed the crush-leach extraction. The resulting slurry was transferred to a glass centrifuge tube and centrifuged at 3000 rpm for 5 min. The supernatant was transferred to a round bottomed ¯ask and combined with the supernatants from two further DCM washes of the sludge in the centrifuge tube. Since the supernatants often still contained suspended clayey material they were carefully rotary evaporated just to dryness so that the suspended material remained stuck to the walls of the ¯ask. The ¯ask was then carefully rinsed (3) with 2 ml aliquots of DCM. This DCM extract was then reduced in volume to 0.5 ml and cleaned-up on a minicolumn as described above, concentrated to 100 ml and analysed by GC and GC±MS. For quantitative analysis, internal standards of n-heptadecylcyclohexane (ICN Biochemicals Ltd), p-terphenyl (Fluka Ltd), n-dodecylperhydroanthracene (a gift from BP) and D4 cholestane (synthesised in this laboratory) for quanti®cation of the saturated hydrocarbons, aromatic hydrocarbons, triterpanes and steranes, respectively, were added to the
vial containing the included hydrocarbons, just prior to GC and GC±MS analysis. These internal standards were chosen on the same basis as the surrogate standards i.e. that they do not generally coelute with other abundant oil components and they are unlikely to occur naturally in petroleums in signi®cant quantities (though caution is required since, for example, some oils can contain squalane). 2.2. GC and GC±MS Gas chromatographic analyses on the free hydrocarbon fractions were carried out on a Hewlett Packard 5890A-II instrument ®tted with a Hewlett Packard 7673 autosampler. A Hewlett Packard HP-1 polymethylsilicone coated (0.25 mm ®lm thickness) fused silica capillary column (25 m0.25 mm i.d.) was employed, using hydrogen as carrier gas and a ¯ame ionisation detector. Splitless injection was used and the oven was programmed from 50 C (2 min) to 300 C at 4 C minÿ1. The gas chromatographic analyses of the included oils were carried out using cold on-column injection onto a Carlo Erba Mega series 5360 gas chromatograph, ®tted with a Hewlett Packard HP-5 phenylmethylsilicone-coated (0.25 mm ®lm thickness) fused silica capillary column (25 m0.25 mm i.d.). Hydrogen was used as carrier gas and ¯ame ionisation detection was used. The GC oven program was as follows: 50 C for 2 min; 50±300 C at 6 C minÿ1; 300 C for 20 min. Data were acquired and processed using a VG Multichrom chromatography data system. Gas chromatography±mass spectrometry (GC±MS) analyses were carried out on a Hewlett Packard 5890-5972 MSD quadrupole instrument ®tted with a HP-1 coated (0.25 mm ®lm thickness) fused silica column (25 m0.25 mm i.d.). Splitless (1 min) injection was used and the GC oven temperature programmed from 40 C (held for 5 min) to 300 C at 4 C minÿ1 where it was held for 20 min. Data were mainly acquired in the selected ion monitoring (SIM) mode, though some full scan data were also acquired. Some analyses were also carried out, under similar analytical conditions, on a VG/Fisons Trio 1000 GC±MS system. 3. Results and discussion Some examples of the results of this included hydrocarbon analysis procedure are given below. The samples analysed were commercial cuttings from carbonate rocks from four wells in a South American basin on which background data is not available, but they are merely used as examples to demonstrate the data that can be produced using the method described. Microscopical screening analysis showed that the samples did not contain unusually abundant numbers of petroleum
D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
¯uid inclusions. These carbonate rocks samples contained stylolytes with abundant insoluble organic matter which was dicult to remove. However, since these samples were carbonates, the hydrogen peroxide oxidation procedure was necessary for the sample clean-up since an aggressive acidic oxidising agent (such as chromic acid) could not be used, though the latter would clearly be a very eective agent for the clean-up of quartz grains (e.g. Karlsen et al., 1993; George et al., 1998b). Furthermore, in samples where rock disaggregation and separation into single grains of individual minerals is possible (e.g. Karlsen et al., 1993; George et al., 1998b), then in addition to increasing the selectivity of the inclusions analysed this can also reduce the number of clean-up steps required and improve the clean-up eciency, since it is possible that standards added to composite grains may not easily penetrate microfractures or voids in mineral cements.
Fig. 2. Gas chromatograms of GA1 (a) and GA2 (b) S1 free total hydrocarbons. n-Alkanes are numbered, Pr is pristane, Ph is phytane, SPCZ, SBN and SSQ are the surrogate standard spikes n-phenylcarbazole, 1,10 -binaphthyl and squalane, respectively.
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3.1. Saturated hydrocarbons Examples of the gas chromatograms of the free hydrocarbons from the ®rst solvent extraction (S1) of the samples GA1 and GA2 are shown in Fig. 2, while Fig. 3 shows those of the included hydrocarbons released by the crush-leach procedure after exhaustive clean-up of these samples. Comparison of Figs. 2 and 3 clearly shows the major dierences in the saturated hydrocarbon distributions between the free (extractable) and the included fractions released by the crush-leach procedure. While the free saturated hydrocarbons are dominated by narrow ranges of n-alkanes (centered around C19 and C15 in samples GA1 and GA2, respectively) and contain signi®cant unresolved complex mixtures (UCMs) in their chromatograms, similar to those often seen in oil-based-mud contaminated samples, the distributions seen in the crush-leach fractions are much more like those found in crude oils. Although the chromatograms from the crush-leach hydrocarbon fractions also tend to show a ``humpy'' baseline, this is exaggerated
Fig. 3. Gas chromatograms of GA1 (a) and GA2 (b) crushleach (included petroleum) total hydrocarbon fractions. nAlkanes are numbered, Pr is pristane, Ph is phytane.
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D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
by the very low signal intensities and the increased visibility of the baseline hump due to column bleed that occurs after about 55 min in the chromatograms. It is also clear from the chromatograms of the crush-leach hydrocarbons that the saturated distribution from the GA2 sample was signi®cantly dierent from the others that were analysed from this region, being much more waxy and containing much lower relative abundances of pristane and phytane. Quanti®cation of the individual hydrocarbons released by the crush-leach procedure (see Table 1) showed that they were generally in extremely low abundance in these samples, with many being found in the low ng/g (ppb) levels. In these particular samples, the individual included n-alkane concentrations ranged from below the detection limit of around 1 ng/g in some samples to over 100 ng/g for certain n-alkanes in the GA2 sample. In this latter sample, the ¯uid inclusion C15±C35 hydrocarbons were about an order of magnitude more abundant than some of the other samples analysed from this region. Assuming that the n-alkanes comprise about 10% of a typical petroleum, then from the summed nC15-to n-C35 concentrations in Table 1, the amounts of petroleum released by the crush-leach procedure was
calculated to be approximately 3000 and 14,000 ng/g rock for the GA1 and GA2 samples, respectively. Since approximately 10 g of each rock sample was crushed and we previously estimated that a typical petroleum ¯uid inclusion may contain 0.4 ng of petroleum, then it would appear that the number of inclusions analysed from the GA1 and GA2 samples were approximately 75,000 and 350,000, respectively. Clearly, with these numbers of inclusions being crushed, the presence of more than a single generation of inclusions would result in mixtures of hydrocarbons being analysed. 3.2. Aromatic hydrocarbons Alkylated naphthalenes, phenanthrenes and dibenzothiophenes were detected by GC±MS in the included hydrocarbon crush-leach fractions and ratios (see Table 2) commonly used for maturity assessment, such as those based on methylphenanthrenes and methyldibenzothiophenes were measured (e.g. Radke, 1987). Such aromatic hydrocarbon parameters have previously been reported from petroleum ¯uid inclusions (e.g. George et al., 1997, 1998a). Interestingly, anthracene and methylanthracenes were observed in many, but not all, of the included
Table 1 Included petroleum individual alkane concentrations (ng/g rock) from crush-leach total hydrocarbon fractions Analyte
GA1(ng/g)
GA2Ab(ng/g)
GA2Bb(ng/g)
GA3(ng/g)
nC15 nC16 nC17 nC18 nC19 nC20 nC21 nC22 nC23 nC24 nC25 nC26 nC27 nC28 nC29 nC30 nC31 nC32 nC33 nC34 nC35
10.8 14.9 21.8 26.7 25.4 28.0 25.5 21.7 18.4 17.4 15.0 13.6 11.4 10.1 10.1 9.0 8.0 7.4 5.4 5.4 5.8
110.2 106.2 103.0 91.3 82.5 87.0 82.9 78.4 69.9 69.2 65.8 67.3 58.5 57.0 48.0 55.9 46.6 47.8 31.0 25.4 25.6
120.2 108.9 102.6 88.2 79.5 81.9 78.1 73.1 69.5 64.4 61.0 63.3 54.2 53.4 44.1 52.0 43.8 40.9 28.2 23.6 19.3
11.6 8.1 14.3 21.5 25.3 29.1 27.3 26.8 22.2 19.9 17.3 16.4 15.3 11.7 15.3 12.0 10.1 8.1 6.5 5.6 5.0
Pristane Phytane
8.5 9.9
9.5 20.2
10.8 20.6
5.7 10.9
C29ab hopane C30ab hopane
0.97 1.29
24.59 11.96
25.75 11.61
a b
1.18 2.03
nm : Not measurable. GA2A and GA2B are two aliquots of the same sample analysed separately.
GA4(ng/g)
GA5(ng/g)
GA6(ng/g)
2.4 1.7 2.1 3.7 4.8 7.8 7.8 7.0 6.0 5.3 5.1 4.1 4.2 3.2 3.5 2.2 2.5 1.7 1.3 nma nm
13.5 21.4 25.0 26.9 23.9 24.1 22.8 23.2 20.1 21.1 20.0 21.4 18.5 19.0 14.5 18.3 12.9 15.0 9.7 8.3 7.7
12.1 14.4 15.5 15.6 16.1 16.1 14.8 13.6 11.7 10.7 9.8 9.1 10.8 11.1 12.4 10.4 9.1 6.6 4.7 3.2 3.0
0.8 1.4
2.7 6.0
9.3 5.8
11.94 10.52
0.16 0.21
nm nm
D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
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Table 2 Geochemical parameters from included petroleums Sample
Pr/n-C17 Pr/Phc Ts/(Tm+Ts)d C29H/C30Hd C31 22S/(S+R) homohopane
C29 Sterane MPI-1f MPI-3f Rc(%)f 4-/1-MDBTg (bb/bb+aa)e
GA1 GA2A*b GA2B*b GA3 GA4 GA5 GA6
0.39 0.09 0.11 0.39 0.37 0.11 0.60
nm 0.56 0.59 nm nm nm nm
a b c d e f g
0.85 0.47 0.52 0.52 0.55 0.46 1.60
0.40 0.28 0.27 0.36 nma 0.13 0.55
0.75 2.06 2.22 0.58 nm 1.13 0.75
0.56 0.51 0.51 0.50 nm 0.54 nm
0.51 0.60 0.62 0.64 0.84 0.68 0.92
0.78 0.65 0.66 0.75 0.90 1.01 1.18
0.71 0.76 0.77 0.78 0.90 0.81 0.95
2.89 1.83 1.96 2.44 2.24 1.05 3.00
m=Not measurable. GA2A* and GA2B* are two aliquots of the same sample analysed separately. Pr and Ph are pristane and phytane, respectively. H denotes hopane, Ts and Tm are de®ned in Table 3. bb and aa refer to the isomeric positions at C-14 and C-17 of the C29 sterane. MPI-1, MPI-3 and Rc (%) are methylphenanthrene based maturity parameters de®ned in Radke (1987). 4- and 1-MDBT are methyldibenzothiophenes.
petroleum crush-leach extracts that were analysed in this and other ¯uid inclusion studies in this laboratory (e.g. see Fig. 4). They have also been reported in petroleum ¯uid inclusion studies by others (e.g. George et al., 1995, 1997, 1998a). Anthracenes are rarely associated with conventional crude oils but they have recently been noted to occur in unusual crude oils from the Canadian Williston Basin, where they were thought to have been generated by short term, high temperature pyrolysis reactions (Li et al., 1998). Anthracene and methylanthracenes have previously been detected in coaly sediments, especially those with vitrinite re¯ectance values below about 1.0% (Radke et al., 1982; Garrigues et al., 1988) and methylanthracenes were apparent (D. Karlsen, 2000, personal communication) in the aromatic hydrocarbon fraction of some of the more terrestriallyin¯uenced crude oils from the Haltenbanken region of the Norwegian continental shelf shown by Karlsen et al., (1993). The presence of anthracenes requires caution when using methylphenanthrene maturity parameters on included petroleum released by crush-leach techniques because of the coelution of 1-methylanthracene with the 9- or 1-methylphenanthrene, depending on the gas chromatographic stationary phase. Although present in a minority of included oils, typically less than 20% (S. George, 2000, personal communication), the relatively common occurrence of anthracenes in included petroleums compared to crude oils also raises a question of whether they could be analytical artefacts. For example, are they formed by some kind of very localised heating which occurs when the mineral grains are crushed to release the ¯uids from the inclusions, even though this was done under solvent and, in our case, by relatively gentle hand crushing using a pestle and mortar? It is possible, for example, that local temperatures near
Fig. 4. m/z 178 and m/z 192 mass chromatograms of GA1 crush-leach (included petroleum) total hydrocarbon fraction. P, A and 2-MA are phenanthrene, anthracene and 2-methylanthracene, respectively. The methylphenanthrenes in the m/z 192 mass chromatogram are numbered according to their methylation position.
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shearing grains may be very high. An alternative explanation may be that these aromatic hydrocarbons could have been generated from terrigenous organic matter, and were adsorbed onto mineral surfaces then preserved in inclusions. 3.3. Biomarkers Examples of the m/z 191 and 218 mass chromatograms of the included hydrocarbons released by the crush-leach procedure from samples GA1 and GA2 are shown in Figs. 5 and 6. The concentrations of the hopanes in many of these samples were very low (see Table 1), and were close to the detection limits of the GC±MS instrument used, resulting in poor signal to noise ratios in the mass chromatograms from some samples (e.g. GA1). Sterane concentrations in the crushleach fractions were even lower, with individual C29 sterane isomers being between 4 and about 70 times lower in abundance than the C3017a(H),21b(H)-hopane peak, and generally were too low to accurately quantify. They were thus of limited use for correlation purposes, though a higher sensitivity mass spectrometer may widen the useful range of concentrations of these compounds. However, even with the instrumentation used, the m/z 191 mass chromatogram showing the hopane distribution of the included petroleum from the GA2 sample
displayed a clear and distinctive distribution which was useful for correlation purposes. The C29 17a (H),21b(H)norhopane to C3017a(H),21b(H)-hopane ratio in this sample was also signi®cantly dierent from those of the other samples in this region and was consistent with the clear dierences seen in the gas chromatograms of these crush-leach extracts. 3.4. Free hydrocarbon input to crush-leach extracts An assessment of potential contamination by surface hydrocarbons was made by using mass chromatograms
Fig. 6. m/z 191 and m/z 218 mass chromatograms of GA2A crush-leach (included petroleum) total hydrocarbon fraction showing the presence of hopane and sterane distributions. Peak assignments are as for Fig. 5.
Table 3 Hopane peak assignments in m/z 191 mass chromatograms
Fig. 5. m/z 191 and m/z 218 mass chromatograms of GA1 crush-leach (included petroleum) total hydrocarbon fraction showing the presence of hopanes and steranes in abundances close to the detection limits of the GC±MS system used. Hopane peak assignments are given in Table 3, the retention time positions of the C27, C28 and C29 14b(H),17b(H) steranes in the m/z 218 mass chromatograms are marked.
Peak
Assignment
H1 H2 H3 H4 H5 H6
C27 18a(H)-trisnorneohopane (Ts) C27 17a(H)-trisnorhopane (Tm) C29 17a(H),21b(H)-norhopane C30 17a(H),21b(H)-hopane C31 17a(H),21b(H)-homohopane [22S] C31 17a(H),21b(H)-homohopane [22R]
D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
to monitor for the presence of surrogate standards which were used to spike the rock before analysis. The abundant peaks due to the n-phenylcarbazole (100 mg), 1,10 -binaphthyl (200 mg) and the squalane (500 mg) which were added to a 10 g aliquots of the sample GA2 prior to clean-up are clearly seen in the respective m/z 243, 253 and 57 mass chromatograms of the ®rst solvent extract of the free hydrocarbons of this sample which are shown in Fig. 7a. These peaks are no longer seen in the corresponding crush-leach hydrocarbon fraction mass chromatograms (Fig. 7b). A peak eluting around 55 min in the m/z 253 mass chromatogram of the crushleach hydrocarbon fraction has a similar retention time as that of 1,10 -binaphthyl, but is probably an unknown coelutant. A quantitative assessment of the likely amount of cross contamination of the included hydrocarbons from the crush-leach extraction by remaining free hydrocarbons in the samples was made by analysing the hydrocarbons in the ®nal Soxhlet extraction after the ®nal hydrogen peroxide treatment. The results of these are given in Table 4. They show that although none of the samples in this set were rich in petroleum ¯uid inclusions, and some were very lean, the concentrations of individual alkanes in the crush leach extracts in these samples were between about 100 (in the richest) and 3 (in the leanest) times greater than those in their corresponding ®nal Soxhlet cleanup extract. This
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Table 4 Individual alkane concentrations (ng/g rock) of free total hydrocarbons in the ®nal soxhlet extract after peroxide treatment Analyte GA1 GA2A*b GA2B*b GA3 GA4 GA5 GA6 (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) (ng/g) nC15 nC16 nC17 nC18 nC19 nC20 nC21 nC22 nC23 nC24 nC25 nC26 nC27 nC28 nC29 nC30 nC31 nC32 nC33 nC34 nC35 Pristane Phytane
2.4 2.2 4.6 3.1 1.4 2.1 2.3 2.6 2.3 2.3 1.6 2.3 1.3 1.4 1.5 1.1 nm nm nm nm nm 3.0 1.9
2.5 2.2 0.7 1.0 1.0 1.0 1.0 1.2 1.2 1.3 1.3 1.6 1.1 1.0 0.9 1.0 nm nm nm nm nm 0.5 0.4
0.9 1.8 1.4 2.0 1.2 1.0 1.4 1.1 1.0 1.2 1.4 1.5 2.5 3.5 4.2 4.3 4.0 3.2 2.5 1.7 1.1 1.4 1.0
0.8 2.7 2.2 2.2 1.4 nma 0.8 1.4 1.2 1.4 1.3 1.1 1.2 1.5 1.1 0.0 nm nm nm nm nm 2.2 1.2
0.0 0.2 0.2 0.8 2.2 1.4 1.5 1.4 1.3 1.1 0.8 1.1 1.5 1.0 2.7 0.9 nm nm nm nm nm nm nm
0.0 0.5 0.4 0.3 0.5 0.4 0.3 0.3 0.2 0.0 0.3 0.2 0.2 0.2 0.2 0.2 nm nm nm nm nm nm nm
0.6 0.7 1.1 0.5 0.6 0.4 0.4 0.4 0.4 0.4 0.5 0.2 0.3 0.2 0.3 0.2 nm nm nm nm nm nm nm
a
nm: Not measurable. *GA2A and *GA2B are two aliquots of the same sample analysed separately. b
Fig. 7. Comparison of mass chromatograms used to show the presence of the surrogate standard spike compounds in the GA2A free (a) and corresponding (b) crush-leach (included petroleum) total hydrocarbon fractions. Peak assignments are as given in Fig. 2.
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D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
shows that the reliability of the included petroleum analyses is vastly improved in samples that contain more abundant ¯uid inclusions. 3.5. Reproducibility After initial disaggregation, sample GA2 was divided into two aliquots A and B, which were then cleaned-up and analysed separately as though they were two dierent samples. The quantitative analysis results given in Tables 1 and 2 show excellent reproducibility for this duplicate analysis both in terms of the quantities of hydrocarbons released and also the composition of them as shown by the various geochemical parameters and ratios measured. 4. Conclusions A method, based on spiking samples with a mixture of standard hydrocarbons, has been developed allowing the analysis of biomarker and other hydrocarbons in petroleum ¯uid inclusions in reservoir rocks and carrier systems to be made with more con®dence that the hydrocarbons extracted are free from extraneous hydrocarbons derived from the surface of the mineral. The method, which ultimately involves crushing the cleanedup mineral grains under solvent in order to extract the hydrocarbons released from the ¯uid inclusions (termed crush-leach), is very time-consuming but necessary to ensure a satisfactory clean-up. However, meaningful results from this method are also critically dependent on there being a sucient abundance of petroleum ¯uid inclusions in the sample and also that these inclusions are representative of a single period of geological time (i.e. a single generation of inclusions). This requires that suitable samples can only be selected after a careful petrographic and microthermometric study has been conducted. In some circumstances, the method therefore generates data which can be combined with microthermometry and paleo-PVT data in order to give a detailed picture of the source, maturity and physical properties of palaeo-petroleum. Acknowledgements We are grateful to M. Chen for her technical assistance in the method development and quantitation work. We also thank Steve Larter and Andy Aplin for their useful discussions and P. Donohoe, K. Noke, R. Hunter and I. Harrison for their technical support. We acknowledge the reviewers D. Karlsen and T. Ruble and associate editor S. George for their constructive criticism and helpful comments which considerably improved this paper. Associate EditorÐS.C. George
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