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Organic Geochemistry 31 (2000) 1163±1173
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: martin.jones@ncl.ac.uk (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 Ho€man, 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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D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173

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 diculties. 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

1165

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

1166

D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173

(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 dicult 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 e€ective 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 eciency, 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.

1167

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 di€erences 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.

1168

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 di€erent 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

1169

D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173
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.

1170

D.M. Jones, G. Macleod / Organic Geochemistry 31 (2000) 1163±1173

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 di€erent from those of the
other samples in this region and was consistent with the
clear di€erences 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]

1171

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

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.

1172

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 di€erent 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 sucient 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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