molecules

Review
Stable Isotope Geochemistry of the Organic Elements within
Shales and Crude Oils: A Comprehensive Review
Abiodun Busuyi Ogbesejana 1,2,3 , Bo Liu 1,2, * and Mehdi Ostadhassan 1
1 State Key Laboratory of Shale Oil and Gas Enrichment Mechanism and Effective Development, Beijing 100101,
China; [email protected] (A.B.O.); [email protected] (M.O.)
2 Institute of Unconventional Oil & Gas, Northeast Petroleum University, Daqing 163318, China
3 Department of Applied Chemistry, Federal University Dutsin-Ma, Dutsin-Ma P.M.B. 5001, Nigeria
* Correspondence: [email protected]

Abstract: Over time, stable isotopes have proven to be a useful tool in petroleum geochemistry.
However, there is currently insufficient literature on stable isotope geochemistry of the organic
elements within shales and crude oils in many petroleum systems around the world. As a result,
this paper critically reviews the early and recent trends in stable isotope geochemistry of organic
elements in shales and crude oils. The bulk and compound-specific stable isotopes of H, C, and S,
as well as their uses as source facies, depositional environments, thermal maturity, geological age,
and oil–oil and oil–source rock correlation studies, are all taken into account. The applications of
the stable isotopes of H and C in gas exploration are also discussed. Then, the experimental and
instrumental approaches to the stable isotopes of H, C, and S, are discussed.

Keywords: stable isotopes; shales; crude oils; organic elements; hydrocarbon exploration






Citation: Ogbesejana, A.B.; Liu, B.;
Ostadhassan, M. Stable Isotope
1. Introduction
Geochemistry of the Organic
Elements within Shales and Crude
The analysis of stable isotopes of various elements present in the organic matter
Oils: A Comprehensive Review. (OM) enables us to understand diverse geochemical processes that take place during
Molecules 2022, 27, 34. https:// geological periods [1]. Stable isotopes have been used in gas and petroleum exploration for
doi.org/10.3390/molecules27010034 correlation [1–8], maturation [9–12], and OM evolution studies [1–19], as well as assessing
the depositional environment of the source rocks [13–19]. Because OM is made up mostly
Academic Editors: Antonio
of carbon (C) and hydrogen (H), with other heteroatoms, i.e., nitrogen (N), oxygen (O),
V. Herrera-Herrera and
and sulfur (S), knowing the stable isotopes of these elements is crucial for petroleum
Margarita Jambrina-Enríquez
exploration [1]. However, the analysis of isotopic compositions of oxygen and nitrogen
Received: 27 November 2021 has been rarely employed in petroleum exploration, mainly due to analytical challenges in
Accepted: 17 December 2021 quantitatively converting organic oxygen and nitrogen to CO2 and N2 [1].
Published: 22 December 2021 To understand geochemical and geological processes, stable isotope geochemistry
Publisher’s Note: MDPI stays neutral
uses the ratios of isotopes between distinct phases or chemical species in a compound [20].
with regard to jurisdictional claims in Isotopes of an element are atoms with the same number of protons but a different number
published maps and institutional affil- of neutrons, resulting in a different atomic mass [20]. In contrast to unstable or radioactive
iations. isotopes, stable isotopes have nuclei that do not decay over time. Thus, except for physical,
chemical, and biological processes that lead to their fractionation, stable isotope abundances
in geologic materials remain constant across time [20]. The ratio of two stable isotopes
of the same element can be used to calculate the relative amounts of light and heavy
Copyright: © 2021 by the authors. isotopes in a phase or chemical species. The degree to which the light or heavy isotope is
Licensee MDPI, Basel, Switzerland. preferentially integrated into a substance during a phase transition or chemical reaction
This article is an open access article can be determined by comparing isotope ratios of materials [1,20]. Although, temperature,
distributed under the terms and
reaction kinetics, and mass can all influence the degree of this preferential incorporation,
conditions of the Creative Commons
knowing such ratios can provide us with insight into reaction mechanisms, formation
Attribution (CC BY) license (https://
temperatures, and other information regarding the evolving earth [1,20,21].
creativecommons.org/licenses/by/
4.0/).

Molecules 2022, 27, 34. https://doi.org/10.3390/molecules27010034 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 34 2 of 28

C and H stable isotopes, since they are the most abundant elements, are important for
determining the processes that sedimentary organic matter has undergone. Carbon and hy-
drogen, for example, both have two stable isotopes: 12 C and 13 C and 1 H and 2 D. 12 C makes
up to 98.899 wt.% of the whole carbon pool, while 13 C makes up only 1.111 wt.% [22]. The
complete hydrogen pool is made up of 99.985 wt% of 1 H and 0.0105 wt% of 2 D [22]. Silver-
man et al. [2], conducted the first in-depth isotopic investigation of petroleum. The initial
basic understanding of petroleum isotope geochemistry was developed by [3–5,23,24], in
the United States, [25,26] in East Germany, [27] in Italy, [28] in West Germany, and [6,29,30],
in the Soviet Union. In addition, in the 1960s, the Galimov group at Gubkin’s Institute con-
ducted theoretical and practical research of isotope fractionation in hydrocarbon systems,
as well as oil and gas studies in the Volga–Ural, Sakhalin, and Kaspian regions, which are
some of the most pioneering studies in this field reported in the literature [31–40].
Sulfur is a common element in nature that plays a key role in a variety of natural
processes, and understanding sulfur isotopic composition sheds light on a wide range of
geological events throughout history [41–43]. Sulfate is an important link between the
carbon, sulfur, and iron cycles, particularly in sedimentary basins, through geochemical
processes such as bacterial sulfate reduction (BSR), iron sulfide production, and organic
matter sulfation [7,8,44,45]. Bacterial sulfate reduction (BSR) is an anaerobic process that
occurs in anoxic conditions with abundant organic matter as a reductant and results in a
depletion of 34 S in sulfide products (such as pyrite) compared to coeval carbonate-associated
sulfate if no obvious alteration occurs in the burial process [46–48].
Even though there are many papers on the stable isotope geochemistry of C, H, and S
and their applications to hydrocarbon explorations, the literature is still insufficient when
considering the importance of C, H, and S isotopes in oil and gas explorations. As a result,
the early and modern uses of the stable isotope geochemistry of organic elements (C, H, S)
within shales and crude oils are further explored in this review paper.

2. Standards and Notation

The stable isotope delta (δ) notation, which is expressed by the following equation, is
the measured values relative to an internationally accepted standard.

δ(‰) = [(Rsample − Rstandard )/Rstandard ] × 1000 (1)

R depicts the isotope abundance ratio, such as 13 C/12 C, 18 O/16 O, 34 S/32 S, 15 N/14 N,
or D/H (2 H/1 H). For example, the δ value for carbon is a convenient way to characterize
tiny differences in the relative abundance of 13 C in biological matter. In comparison to
the standard, a negative value indicates that the sample is depleted in the heavy isotope,
whereas the positive value indicates that the sample is enriched in the heavy isotope. To
express relative isotope composition, the terms “light” and “heavy” are replaced for carbon,
and instead “13 C-depleted” and “13 C-enriched” are used, respectively. The isotopic ratios
of stable isotopes and their corresponding reference standard are shown in Table 1 [21].

Table 1. Isotopic ratios of stable isotopes [21].

Element Notation Ratio Standard Absolute Ratio

Hydrogen δD D/H(2 H/1 H) SMOW 1.557 × 10−4
>Carbon δ13 C 13 C/12 C PDB 1.122 × 10−2
Nitrogen δ15 N 15 N/14 N Atmosphere 3.613 × 10−3
Oxygen δ18 O 18 O/16 O SMOW, PDB 2.0052 × 10−3
Chlorine δ37 Cl 37 Cl/35 Cl seawater −0.31978
Sulfur δ34 S 34 S/32 S CDT 4.43 × 10−2
SMOW = standard mean ocean water; PDB = Pee Dee belemnite; CDT = Cañon Diablo troilite.

3. Stable Isotope Fractionation

Isotope fractionation is a physical phenomenon that occurs when mass differences
between isotopes produce variations in relative abundance [49]. Isotopic fractionation
Molecules 2022, 27, 34 3 of 28

occurs in nature as a result of binding strength during chemical, biological, and physical
processes [20]. Isotopes that are “heavy” make stronger bonds than isotopes that are
“light”. As a result of isotopic fractionation associated with these processes, the isotopic
composition of crude oils and sediments, as well as their constituents, can be affected by
isotopic source signatures and processes such as maturation and biodegradation [50–52].
Kinetics and equilibrium are the two fractionation processes that cause these isotopic shifts.
While both processes may be active, the kinetic isotope impact is the most relevant in most
hydrocarbon exploration investigations, such as in natural gas exploration studies [52–54].

Kinetic Isotope Effect (KIE)

Different reaction rates for heavy vs. light isotopes would cause KIE related to degra-
dation and maturation processes (e.g., 13 C vs. 12 C). The presence of a “heavy” species
(e.g., 13 C) in the degradation-targeted bond raises the bond cleavage activation energy
and, as a result, slows the rate of degradation. The leftover substrate becomes increasingly
enriched in “heavier” isotope species as degradation progresses (i.e., 13 C/12 C increases).
Isotope effects associated with degradation are significant for the atoms present when the
chemical bond is broken (primary isotope effects), whereas secondary isotope effects are
moderate for the remaining atoms. Considering hydrogen, only secondary isotope effects
are significant at the normal precision of CSIA (compound-specific isotopic analysis). When
a biodegradation process exclusively targets a single atomic position (or positions) (e.g.,
oxidation of a methyl group in toluene), the overall expression of isotope fractionation,
as assessed by CSIA, is “diluted”. The intrinsic role of an isotope that affects the atoms
directly involved in the reaction is unaffected by molecule size and can be determined
using CSIA data [20,21,37,38,55]. The initial rate-limiting phase of the reaction cycle is
reflected by isotope effects on biodegradation. In other circumstances, the delayed step
may not be a bond cleavage but rather a non-isotope fractionating process, such as the
development of the substrate–enzyme complex. There will be no isotope fractionation if
the latter is entirely rate limiting. The rule of thumb is that biodegradation isotope effects
are restricted to tiny compounds that can easily pass through degraders’ cell membranes.
This would include chlorinated ethenes and ethanes, methyl tertiary butyl ether (MTBE),
and mono-aromatic compounds that have been extensively researched and where isotope
fractionation has been seen [55,56]. Fractionation has been seen in the lighter hydrocarbons
in the C5 to C10 range in exploration studies [57]. Semi-volatile environmental pollutants
including long-chain n-alkanes, multi-ring polycyclic aromatic hydrocarbons (PAHs), and
polychlorinated biphenyls (PCBs) often do not produce measurable isotopic fractionation
during biodegradation. It is also worth noting that in larger compounds with more than
10–12 carbon atoms, the isotopic alterations that occur to the one carbon atom that is being
attacked are diluted by the presence of the other carbon atoms that are not impacted. As
a result, overall changes in the isotopic makeup of these bigger molecules are usually
undetectable when isotope values are measured [21]. Isotope fractionation can also be
caused by physical processes such as phase transitions, sorption, and diffusion [58–60].
Different kinetic energies of gaseous phase molecules result from the mass difference
between isotope species, resulting in differential rates of vapor migration and different
bond energies of light isotope- vs. heavy isotope-substituted molecules, affecting phase
partitioning equilibria and evaporation–condensation [20,21]

4. Experimental and Instrumental Methods in Stable Isotopes (C, H, and S)

The analytical methods applied in the analyses of stable carbon, hydrogen, and sulfur
isotopes are reported below.

4.1. Stable Isotopic Analyses

4.1.1. Fractionation of Sediment Extracts and Crude Oils
Column chromatography with silica gel–alumina as the stationary phase is generally
used to fractionate extracts and crude oils. A typical glass column is 50 cm long and has
Molecules 2022, 27, 34 4 of 28

an internal diameter of 0.5 cm. The column is rinsed twice, with DCM first and then light
petroleum spirit (petroleum ether). After that, the column is filled with n-hexane and
cotton wool is used as a resting pad for the stationary phase, silica gel (SiO2 ). After that,
the stationary phase (SiO2 ) is added. Two (2 g) of Alumina (Al2 O3 ) is used to stabilize the
surface. A total of 70 mL of n-hexane, 70 mL of dichloromethane–n-hexane (2:1, v/v), and
70 mL of dichloromethane–methanol (1:1, v/v) are used to elute the saturated, aromatic
hydrocarbon, and polar fractions, respectively. Each fraction is recovered by carefully
evaporating solvents on a rotary evaporator, followed by the removal of the leftover solvent
under a nitrogen gas stream (modified from [61]). The recovered saturated hydrocarbon,
aromatic hydrocarbon, and polar fractions can now be analyzed using elemental analysis–
isotope ratio mass spectrometry (EA–irMS) (bulk isotope analysis).

4.1.2. Molecular Sieving

Straight chain hydrocarbons are commonly separated from branched and cyclic hydro-
carbons by treating saturated fractions with activated (250 ◦ C, 8 h) 5A molecular sieve [62]
in cyclohexane. The n-alkanes are recovered by dissolving the filter in HF (2 mL, 50%
w/v) and neutralizing the solution with saturated sodium bicarbonate [9]. n-pentane is
used to extract the aqueous phase (1 mL × 5). This fraction has n-alkanes that could be
analyzed using gas chromatography–isotope ratio mass spectrometry (GC–irMS). Using
a ZSM-5 molecular sieve, the branched and cyclic (5A excluded) hydrocarbon fractions
from crude oils and sediments might be further simplified by removing methyl alkanes and
alkyl cyclohexanes. The branched or cyclic (5A excluded) fraction is transferred to a small
column (7–8 cm × 0.5 cm i.d.) of activated (250 ◦ C) ZSM-5 molecular sieve (pre-rinsed with
pentane) and allowed to stand (1–5 min). The branched or cyclic fraction is obtained after
rinsing the filter with pentane (2 mL) (ZSM-5 excluded) [10].

4.1.3. Elemental Analysis–Isotope Ratio Mass Spectrometry (Bulk δ13 C Analysis)

The principle behind determining the isotopic composition of individual molecules
or the bulk stable isotope values of complicated mixtures is fairly similar. Regardless of
the sample’s origin, each process necessitates complete combustion and conversion to CO2
and water [21]. For bulk determinations, the sample can be converted to CO2 using an
elemental analyzer interfaced directly to an isotope ratio mass spectrometer in a sealed
tube in the presence of CuO, or the sample can be converted to CO2 using an elemental
analyzer interfaced directly to an isotope ratio mass spectrometer. A stable isotope ratio
mass spectrometer is used to transfer and analyze CO2 , which is made up of both 13 CO2
and 12 CO2 . The relative amounts of 13 CO2 and 12 CO2 are measured in comparison to a
standard material, Vienna Pee Dee belemnite (VPDB), which has a carbon isotope value of
0, given in the notation (see Table 1). Almost all samples will have lower 13 C levels than
the standard and will be depleted in 13 C, resulting in negative 13 C results. Stable carbon
isotope ratios (R = 13 C/12 C) are expressed relative to the VPDB standard using the “delta”
notation, where: δ 13 C = (Rsample /Rstandard − 1) × 1000 (units are‰ or per mil or parts per
thousand) [21]. The sample is weighed inside a small silver capsule, which is then folded
and dropped into a pyrolysis reactor containing glassy carbon chips kept at 1260 ◦ C for
bulk D analysis. The sample is pyrolyzed to produce H2 and CO, as well as N2 if necessary.
The pyrolysis products are separated on a 1 m chromatographic column packed with 5A
molecular sieve and kept at 80 ◦ C in an oven before being passed via a thermal conductivity
detector (TCD) and into the irMS instrument. The D values are calculated and expressed
in delta notation to VSMOW (Vienna standard mean ocean water) [10]. By precipitating
barium sulfate, which is then combusted to produce sulfur dioxide, a custom-built stable
isotope ratio mass spectrometer [63] can perform stable sulfur isotope ratio analysis. The
sulfur isotopes are expressed in terms of the CDT standard. The ratios of the other elements,
such as O, N, or Cl, are represented in the same way with their unique standard. Table 1
shows the natural abundance of the stable isotopes typically employed in geochemistry, as
well as the international standards used to calculate their values.
Molecules 2022, 27, 34 5 of 28

4.1.4. Gas Chromatography–Isotope Ratio Mass Spectrometry (GC-IRMS)

A gas chromatograph coupled to the combustion system can allow the carbon isotope
ratios to be determined on individual organic compounds (GC/combustion/IRMS, also
called compound-specific isotope analysis (CSIA). After being eluted from the GC column,
the components pass through a combustion reactor and are combusted to CO2 and water
to determine the isotopic composition of individual compounds using GC-IRMS (Figure 1).
The water is removed, and the CO2 is transferred to an isotope ratio mass spectrometer,
which determines the relative amounts of 13 CO2 and 12 CO2 and calculates 13 C values for
each molecule. A Micromass IsoPrime isotope ratio mass spectrometer interfaced to a gas
chromatograph for determining compound-specific stable carbon isotopic composition
(13 C values) or a chromatograph for determining stable hydrogen isotopic compositions
(D values) is commonly used in GC–irMS [10]. For routine analysis, the oven is set to a
temperature range of 40 to 310 ◦ C at a rate of 3 ◦ C/min, with initial and final hold times
of 1 and 30 min, respectively. Samples are injected into an autosampler utilizing a split
or splitless injector in pulsed splitless mode. Ultra-high purity (UHP) He is commonly
utilized as carrier gas with a flow rate of 1.1 mL/min and a constant flow injector. The
aromatic fractions of the samples are separated by a GC coupled to an inductively coupled
plasma mass spectrometer to determine the sulfur isotope ratios of individual dibenzothio-
phenes. The operating conditions are described in detail in [64–66]. The samples are run in
duplicates to ensure accuracy and the standard deviation between the two duplicates is
noted accordingly [11]. The schematic diagram of GC-IRMS is shown in Figure 1 [21].

Figure 1. Schematic diagram of GC-IRMS system (modified from [21]).

4.1.5. Methods of Stable Sulfur Isotope Analysis

SO2 is the gas of choice for gas–source mass spectrometric measurements. With the
emergence of online combustion technologies [67], multistep offline preparations have
been simplified to a single step, namely combustion in an elemental analyzer. Sample
preparations have become less time-consuming and less reliant on potentially fractionating
wet chemical extraction procedures, resulting in a minimum sample gas of less than 1 mg.
Puchel et al. [68] and Rees [69] initially described a method that uses SF6 instead of SO2 ,
Molecules 2022, 27, 34 6 of 28

which has several advantages, including no mass spectrometer memory effect and no
need for raw data corrections of recorded isotope ratios because fluorine is monoisotopic.
The reliability of the SO2 correction for oxygen isobaric interferences has been called into
doubt by a comparison of 34 S-values produced using the traditional SO2 and the laser SF6
techniques [70]. As a result, despite the high toxicity of the gases, the SF6 method has
been revived [71], revealing that SF6 is an appropriate gas for detecting 33 S/32 S, 34 S/32 S,
and 36 S/32 S ratios. Sulfur isotope ratios can now be determined using microanalytical
techniques such as laser microprobe [71–74] and ion microprobe [75–79]. Hauri et al. [80]
used NanoSIMS to collect data on several sulfur isotopes.

Bulk Stable Sulfur Isotope Analysis

Cai et al. [12] described the procedures for separating kerogen and analyzing sulfur
isotopes in kerogen and oil. Pyrite is usually removed from kerogen by introducing a
mixture of hot 6N HCl and CrCl2 to the ground dry kerogen under a nitrogen flow, with the
H2 S being carried to a trap and recovered as Ag2 S. Water washing removes excess acids and
acid-soluble ions from residual kerogen. The residual kerogen is collected and reground 2 h
later to expose additional pyrite surfaces, and the process is repeated. Following the two
treatments, the leftover kerogen is examined using X-ray diffraction (XRD) to see if pyrite
levels are below detection limits (60.5% depending on conditions). If not, more treatments
would be necessary. To oxidize organically linked sulfides to sulfate, a known weight
(between 350 mg and 900 mg) of kerogen isolate or 1–4 g of oil is combusted in a Parr bomb
apparatus at 25 atm oxygen. Total residual kerogen sulfur is calculated by precipitating
dissolved sulfate as BaSO4 and weighing it. The maximum residual pyrite content in the
kerogen after the chromium reduction is determined by measuring dissolved iron at pH
< 2 with an atomic absorption spectrometer (assuming that all Fe occurs as pyrite in the
kerogen). The produced BaSO4 is only evaluated for δ34 S when leftover kerogen samples
have contaminated pyrite sulfur and total sulfur < 0.08 to ensure low levels of errors (based
on the differences in 34 S value between kerogen and the related pyrite).

Individual Sulfur Compounds Isotope Analysis

Bendall et al. [81], Craddock et al. [82], and Parris et al. [83] have reported the use of
multi-collector–inductively coupled plasma mass spectrometer (MC-ICP-MS) techniques.
Amrani et al. [64] proposed a method for analyzing individual sulfur organic compounds
using a gas chromatography–multi-collector–inductively coupled mass spectrometer (GC-
MC-ICP-MS). Sample sizes are at orders of magnitude smaller than what is commonly used
for SO2 and SF6 [84] due to poor detection limits. The GC-MC-ICP-MS method does not
require any chemical preparation and allows for simultaneous collection of all four sulfur
isotopes; however, it has lesser precision than other methods. The aromatic fractions of
the oils and sediment extracts are separated by GC-MC-ICPMS to determine the sulfur
isotope ratios of specific sulfur compounds. Amrani et al. [64,65] and Said-Ahmad and
Amrani [66] described the system and its working conditions in detail. Duplicates for some
of the oils and sediment extracts are measured and the standard deviation between the two
duplicates is usually better than 1‰.

5. Applications of Stable Isotopes (C, H, S) in Shales and Crude Oils

Hydrogen isotopes are used occasionally in petroleum exploration to support biomark-
ers data but are widely used in gas exploration, while sulfur isotopes are rarely used in
petroleum exploration. However, carbon isotopes are by far the most frequently used in
gas and petroleum exploration. The following sections describe the uses of the isotopic
compositions of carbon, hydrogen, and sulfur in petroleum and gas exploration.

5.1. Stable Carbon Isotope

The organic materials in a source rock must be characterized to determine if a forma-
tion will generate oil, gas, or both. Visual characterization, extraction, and identification
Molecules 2022, 27, 34 7 of 28

of specific biomarkers and stable isotopes are all techniques that can be used to define
organic matter [85]. Craig [86] and Silverman [2,87] discovered that oils from terrigenous
sources were more depleted in 13 C than oils from marine sources in the early studies of
stable isotopes in hydrocarbon exploration. Land plants were generally deficient in 13 C
compared to aquatic plants and marine heterotrophs, which appeared to mirror the trend
seen in living systems. Organic matter in recent sediments has also been found to become
depleted in 13 C as the depositional facies shifts from marine to terrigenous [88]. Carbon
isotopes are frequently utilized to distinguish between oils originating from marine and
non-marine organic matter. The relationships between the isotopic composition of the
saturate and aromatic fractions [1] reveal differences between marine and non-marine oils.
The effects of the source on stable carbon isotope ratios have been examined [89] and it was
found that as the concentration of terrestrial input increases, the δ13 C values become less
negative. In west Texas, isotopic age trends were employed successfully to correlate and
differentiate crude oils on a geographical basis [4]. Moreover, a general trend of lighter
isotope enrichment with the increasing geological age of oils was observed, possibly caused
by variations in photosynthesis intensity and changes in the isotopic composition of atmo-
spheric CO2 [50,90]. This observation formed the basis for isotopic age-dating methods
developed later [88]. By fractionating the oil into saturates, aromatics, NSO (nitrogen,
sulfur, oxygen), and asphaltene fractions, the usage of bulk isotopes can be taken a step
further to improve the precision. Individual fraction isotope data can then be plotted in a
variety of ways. As shown in Figure 2, a plot of saturates vs. aromatics can be very useful
for grouping oils formed from comparable source materials or distinguishing oils from
distinct depositional settings [91].

Figure 2. A plot of δ13 C saturates versus δ13 C aromatics (Reproduced from [91] with permission
of Elsevier).
Molecules 2022, 27, 34 8 of 28

The Stahl-type curves [92] can also be constructed using the isotope values. The stable
isotope value for the kerogen can be integrated into this graph if the suspected source rock is
available [21]. If the correct source rock is chosen, the individual fractions will have a direct
link, as shown in Figure 3. The advantage of this method is that 13 C values distinguish
the carbon in different fractions and provide a more powerful correlation tool, though
caution must be exercised in distinguishing the effects of increasing maturity [93], phase
effects, and subsequent reservoir changes such as water washing [94] or biodegradation [95].
Understanding and quantifying these effects is therefore critical for the reliability of isotope
correlation [96,97]. As a result, the factors that can alter the isotopic composition of crude
oils and source rocks will be discussed in the following sections.

Figure 3. The isotopic relationship between the carbon isotope values of the various fractions and
the source rock extracts. SAT: saturate hydrocarbons; OIL: whole oil; AROM: aromatic hydrocar-
bons; NSO’s: nitrogen, sulfur, oxygen compounds; ASPH: asphaltenes (Reproduced from [50] with
permission from Elsevier).

5.1.1. Source and Depositional Facies

Crude oil source rocks are generated from a variety of source materials that are
deposited in a variety of depositional conditions [98]. Many factors will impact the isotopic
compositions of these source materials, such as plants, phytoplankton, and bacteria, when
they are first generated through photosynthesis. All of the original isotopic source signals
will be homogenized and change slightly as this organic material is buried, degraded, and
eventually heated to generate crude oils or natural gas [21]. However, because different
crude oils are generated from diverse mixtures of source materials, some variations in the
eventual isotopic composition of the producing crude oils are reasonable to expect. As a
result, the isotopic composition of various crude oils from various sources is used.
Molecules 2022, 27, 34 9 of 28

As stated by the basic reaction below, the starting point in the generation of crude
oil in atmospheric CO2 begins through the process of photosynthesis, which leads to
the incorporation of that C into the cell wall components, providing energy to promote
plant growth.
6CO2 + 12H2 O −→ C6 H12 O6 + 6H2 O + 6O2
The organic material from living plants and aquatic organisms is deposited in a variety
of environments after they die. A substantial portion of this material will deteriorate
depending on the environment, but a small part will be absorbed into the sedimentary
record, go through diagenetic processes, and then thermally transform the residual organic
matter to generate oil, gas, or both [21,98]. The level of fractionation between atmospheric
13 CO and 12 CO during photosynthesis varies depending on whether the source material
2 2
is a land plant or a marine organism, and the extent of fractionation within land plants is
further determined by distinct photosynthetic cycles.
The isotopic compositions of plants that were initially determined [86] showed that
most of the plant materials investigated at the time had a rather stable δ13 C value, about—
27‰ in a thorough investigation on the carbon isotopic content of natural materials. It
was noted that there did not appear to be any significant species or geographic effects;
however, one grass with a δ13C value of 12‰ was later identified as a C4 plant. C3 plants, as
defined by [86,99], incorporate CO2 from the atmosphere through carboxylation of ribulose
bisphosphate. CO2 is absorbed by C4 plants by the carboxylation of phosphoenolpyruvate.
The carboxylation product is transferred from photosynthetic cells’ outer layer (mesophyll
cells) to the inner layer (the bundle sheath), where ribulose bisphosphate carboxylase
decarboxylates and refixes it. C4 plants have lower negative δ13 C values than C3 plants,
according to isotope research. Plants with the C3 cycle, such as grasses, corn, and maize,
grow in cooler, wetter climates and have isotope values in the 26 to 30 per mil range. Plants
with the C4 cycle, such as grasses, corn, and maize, grow in dryer, hotter climates and have
isotope values in the 10 to 14 range. The difference in the isotopic composition of C3 and C4
plants has become one of the primary methods for distinguishing these plant kinds [100],
and it also serves as a useful diagnostic tool for distinguishing specific source materials.
Due to variations in the isotopic composition of carbon sources, numerous inves-
tigations of marine and terrestrial plants have demonstrated a depletion of 13 C in land
plants compared to marine plants [32,86]. Isotopic differences similar to this have been
observed in recent sediments [13,53,54,101]. In comparison to local marine fauna and flora,
organic matter in recent marine sediments is often depleted in 13 C [102–104]. Diagenetic
factors may be the cause of the 13 C depletion. The 13 C depletion has been attributed
to the diagenetic loss of 13 C-enriched carboxyl carbon [105,106]. Alternatively, it was
proposed that the maturation of 13 C-depleted lipids in marine sediments results in the
formation of 13 C-depleted organic matter [104]. However, selective preservation, in which
13 C-depleted refractory components of algae are selectively accumulated in sediment and

the 13 C-enriched components are microbially degraded and lost, could explain the 13 C
depletion of marine sedimentary carbon [107]. Because source rocks are heterogeneous
mixtures of many different types of organic material, the resulting isotopic composition of
the source rock will be a weighted average of the isotopic compositions of all the different
preserved and residual source materials, and thus the situation becomes far more compli-
cated and less specific. As a result, depending on the initial mixture of source materials,
there will be changes in the bulk isotopic compositions of the source rocks, and hence the
oils. The significant contribution of terrestrial organic debris to the organic precursors of
marine organic matter has been widely thought to explain the relative depletion in 13 C of
several marine sediment samples [2,5,108]. One explanation for differences in the resulting
crude oils is isotopic variations in the source materials.
Moreover, there exists a comprehensive geochemical analysis of oils from offshore
Brazil, using a variety of characteristics to distinguish oils from distinct depositional
settings [109]. Based on a variety of criteria, including δ13 C values, the oils were categorized
into five groups (I to V). Table 2 summarizes some of the bulk parameters for each category
Molecules 2022, 27, 34 10 of 28

of oils, including bulk isotope analyses, elemental analyses, and alkane distributions.
Figure 4 shows how the oils were grouped into five types using δ13 C values of the saturated
and aromatic hydrocarbons. Group II oils are isotopically heavy (δ13 C values about 25‰),
which could be explained by increased salinity. Carbonate complexes are preferred as a
carbon source for photosynthesis by plants from saline environments. These have a higher
13 C content than atmospheric carbon dioxide, which has a higher 12 C content [110].

Table 2. Bulk and elemental properties of Brazilian offshore oils (Reproduced from [109] with the
permission of Elsevier).

Property Group I Group II Group III Group IV Group V

δ13 C‰ < −28.0 −23.0 to −25.6 −25.4 to −26.6 −26.8 to −27.6 −24.4, −25.1
% Sulphur Low Medium High High Medium
V/Ni Low High/medium Medium High High
%Saturates High High Low/medium Low/medium High
n-Alkanes’ dominance C23 -C25 C17 -C21 C17 -C19 C18 -C20 C20 -C22
Odd/even High High Low Low Low
Pristane/Phytane High High Low Low Low
Inferred depositional Lacustrine/ Lacustrine/ Marine Marine
Marine deltaic
environment freshwater saline water evaporitic carbonate

Figure 4. Saturate and aromatic carbon isotopic data of the crude oils from Brazil. (Reproduced
from [109] with permission from Elsevier).
Molecules 2022, 27, 34 11 of 28

5.1.2. Biodegradation
Biodegradation has a well-defined set of changes that occur during the degradation
process [85]. The primary changes in isotopic composition, on the other hand, are due to
bulk changes in the oil rather than changes in individual molecules. The biodegradation
of crude oil in the reservoir is a significant secondary alteration process with significant
economic implications. Although the actual mechanisms involved in the in-reservoir
biodegradation of crude oil are still being studied (e.g., site and rate of degradation, nutri-
ent availability, and nature of by-products), their impact on the composition and physical
qualities is well documented [111]. In most cases, the components of crude oils are removed
sequentially in the order of n-alkanes > monocyclic alkanes > alkyl benzenes > isoprenoid
alkanes > alkyl naphthalenes > bicyclic alkanes > steranes > hopanes. Many compound-
related markers for determining the level of crude oil biodegradation have been proposed
based on this sequence [112]. Biodegradation’s effects on the isotopic compositions of
individual compounds have been explored for prospective use in petroleum and environ-
mental research [57,113–121]. The effects of slight and moderate biodegradation on light
hydrocarbons (C5 –C9 ) consistently lead to 13 C enrichment for each surviving molecule,
according to studies by [57,119]. However, slight and moderate biodegradation resulted
in insignificant enrichment in residual C10 –C14 n-alkanes, with a maximum δ13 C increase
of 0.5‰, according to [111,114]. The stable carbon isotope compositions of natural gas
components (C2 –C5 ) are significantly fractionated during biodegradation, as has long been
observed [122]. During biodegradation, lower carbon number molecules are removed first,
followed by higher carbon number ones. Except in extreme cases of biodegradation, the
amount of change in the aromatic and polar fractions is negligible [1,13]. The impact on
the isotopic composition of the saturate fraction, on the other hand, can be much greater
because n-alkanes make up a large portion of the saturate fraction, and their removal alters
the isotopic composition, causing the saturate fraction to become isotopically heavier in
general, as shown in the Stahl diagram in Figure 3.

5.1.3. Maturity
Unlike biodegradation, maturity can have an impact on the isotopic composition of
all fractions, as maturity causes carbon–carbon bonds to break in all fractions, resulting in
increased proportions of lighter hydrocarbons. Crude oil is formed by burial and maturity,
and as a result of thermal maturation, there will be some variation in isotope composition.
The effects of maturation on the 13 C values of kerogens in the natural system have been
thoroughly studied [123,124]. The kinetic isotope effect dominates during the maturation
phase, resulting in the preferential breaking of bonds containing the lighter isotope. As
a result, the heavier isotope will be enhanced in the residual fractions, and the overall
trend will be one of isotopic enrichment as maturity increases. The degree of enrichment
will differ from one oil to the next and will also be determined by the level of maturation.
Oil-to-gas cracking degrades oil by turning it into low molecular weight molecules in
a reservoir. Isotopic effects are more likely to represent kinetic isotope fractionation in
this scenario, as 12 C–12 C bonds are preferentially cleaved over 13 C–12 C bonds, leaving
the residual oil enriched in 13 C [96,97]. The residual kerogen will become isotopically
heavier with increasing maturity of source rocks, and the cracking of the kerogen within
the source rock to generate oil will have similar effects. The products formed from kerogen
will initially be isotopically light, but as maturity levels rise and more isotopically enriched
kerogen is thermally degraded, the resulting products will become isotopically heavier. In
general, kinetic isotopic fractionation in processes involving compounds with six or more
carbon atoms is insignificant [96,97]. An additional isotopic fractionation step must be
considered for condensates or light oils. As the subsurface gas phase (“gas condensate”)
is raised to the surface and temperature and pressure decrease, condensates (usually C6+
compounds) condense from the subsurface gas phase (“gas condensate”). Because we are
dealing with a transition from a liquid state (“oil”) to a vapor state (“condensate”), isotope
effects will be limited during this process. Any isotope effect will be due to equilibrium
Molecules 2022, 27, 34 12 of 28

isotope effects rather than the kinetic isotope effect in this case. However, in comparison
to the oil from which they emerged, condensates will preferentially accumulate smaller
molecular weight molecules. Differences in bulk δ13 C between low and high molecular
weight molecules will be reflected in bulk δ13 C values for oil and condensate [96,97]. There
are two ways to look into isotope effects that occur during the maturation and generation
process. First, accumulated oils derived from a single source rock but maturing at distinct
rates based on biomarker maturity characteristics can be isotopically identified, with
discrepancies in the δ13 C values attributed to maturation. This also presupposes that no
further in-reservoir changes have happened. Laboratory maturation investigations, such as
hydrous pyrolysis experiments [125], where an immature source rock sample is heated in
the presence of liquid water at subcritical temperatures (<374 ◦ C), provide an alternative
option. Although these tests use relatively high temperatures (300–365 ◦ C) to compensate
for the long period required to generate oil in the natural system (106–109 years), the
pyrolysates released from potential source rocks are similar to natural crude oil [125].
The vitrinite reflectance and atomic H/to C ratio of pyrolysates, which were measured
in the matured source rocks, revealed similar alterations to those found in the natural
system [124,126]. Immature source rock samples from the Woodford Shale were artificially
matured to see how thermal maturation affected the δ13 C values of kerogen, bitumen, and
crude oil. It was discovered that as thermal maturation increased, the expelled oil became
more enriched in 13 C. (Figure 5). As stated above, the increased cleavage of 13 C–12 C bands
in the bitumen as it continues to decompose into expelled oil and preferential cleavage of
12 C–12 C bonds in the expelled pyrolysate as it decomposes into gases and pyrobitumen can

be attributed to the kinetic isotope effect and increased cleavage of 13 C–12 C bands in the
bitumen as it continues to decompose [21].

Figure 5. Changes in the isotopic compositions of crude oils and kerogen with maturation. (Repro-
duced from [124] with permission from Elsevier).
Molecules 2022, 27, 34 13 of 28

5.1.4. Age Dating

To age date oil samples, exploration studies have used several biomarker parameters
that can be linked to certain evolutionary processes [127]. The interest in age dating stems
from the fact that in many exploratory cases, only oil samples are recovered, and being able
to anticipate the age of the source rock from crude oil properties is extremely important
in determining the source formation responsible for the development of a certain oil.
Andrusevich et al. [88] studied the possibility of age dating crude oils using stable carbon
isotope compositions of different fractions of oil samples and found that the oils became
more enriched in 13 C as geologic age decreased (Figure 6). Changes in δ13 C, in particular,
are linked to global fluctuations in 13 C carbonate carbon [88]. Previous studies of the stable
carbon isotopic composition of oils (δ13 C) over geologic time have found a general tendency
of 13 C enrichment with decreasing age [20,128]. The δ13 C value of oil is often determined by
the δ13 C value of the kerogen in the source rock from which it was derived. The δ13 C value
of kerogen is determined by the sorts of organisms maintained as well as the δ13 C values
of its substrate, which leads back to the original photosynthetic conditions as stated above.
Assuming that the present is the key to the past, variables known to be essential in the
modern biosphere, such as temperature, pCO2 , and depositional settings, are likely to have
influenced the stable isotope composition of carbon in ancient biospheres. Organic carbon
and carbonate carbon production, as well as their corresponding δ13 C values, would have
been influenced by changes in these settings [129]. The geologic record includes changes
in the relative quantities of atmospheric gases such as CO2 and O2 as well as fluctuations
in the δ13 C values of organic and carbonate carbon [21,129]. Variations in carbon isotope
values over time can be explained very readily. The first CO2 in the atmosphere came from
the outgassing of the core and had a fixed isotopic composition [21,128]. The CO2 content
of the atmosphere declined over time as more complex photosynthetic creatures and plants
evolved, and the atmosphere became isotopically heavier in general [20,21,128]. There were
isotopic excursions where CO2 became even heavier as a result of considerable productivity
increases, and the principal photosynthesizers took on a heavier isotopic signature as a
result. Because most fossil fuels and their combustion products have isotopic values in the
range of 25 to 35 per mil [21], the isotopic composition of atmospheric CO2 is becoming
lighter as the burning of fossil fuels increases.

Figure 6. Stable carbon isotope composition of crude oils versus geologic age (Reproduced. from [88]
with permission from Elsevier).
Molecules 2022, 27, 34 14 of 28

5.1.5. Carbon CSIA

It took over two decades to develop the ability to determine the isotopic composi-
tion of individual compounds. Continuous flow determination of carbon isotope values
of individual compounds in complex mixtures of geochemical interest is possible with
gas chromatography–isotope ratio mass spectrometry (GC–IRMS) [130–133]. Only bulk
isotopic data were available before the introduction of GC–IRMS [102], which lack a lot
of specificity due to the heterogeneous character of the source material [134]. As a result,
isotopic data from crude oils and source rock extracts must be interpreted in conjunction
with other geochemical information [135]. The use of GC-IRMS now allows the isotopic
composition of individual compounds in crude oils and source rock extracts to be deter-
mined. The n-alkanes and isoprenoids have received the greatest attention since they are
often well resolved in chromatograms [136]. Individual n-alkane δ13 C values have been
widely employed in oil–oil and oil–source rock correlations [137–139] as well as recon-
structions of the paleoenvironment and paleoclimate [132,140]. The isotopes of individual
compounds provide an additional correlation parameter, as well as the ability to determine
whether or not many compounds are derived from the same precursor molecule. This
is especially true for the pristane to phytane ratio [141], which is one of the most exten-
sively used geochemical parameters in geochemical investigations. This ratio is commonly
utilized for correlation as well as indicating the depositional environments and redox
conditions. However, this latter application assumes that both compounds are derived
from the side chain of chlorophyll, and if they are not, the ratio is invalid. The only way
to determine whether they are from the same source is by measuring the carbon isotope
value of these substances. A carbon isotopic investigation of crude oils and the Messel
shale was conducted [131], which demonstrated that the isotopic values in the oils were
quite comparable, indicating that they came from the same source, but the values in the
Messel extract were significantly different, indicating that the pristane and phytane came
from distinct origins. Isotopic compositions of individual compounds have also been
successfully used to characterize specific components in the saturated hydrocarbon fraction
and as a parameter to establish oil–oil and oil–source rock correlations [53,54,137,142,143].
This technique has been used by several researchers to assess the presence of uncommon
polycyclic alkanes in extracts of source rocks and crude oils where hopanoids and steranes
were either absent or present in extremely low quantity [144–148]. The assumption behind
the oil–source rock correlation is that the oil and the corresponding source rock have similar
δ13 C values. To use isotopic compositions from crude oils and source rock extracts for
direct oil–oil and oil–source correlations, two pre-requisites must be met: first, the maturity
level of the oil and corresponding source rock must be similar; and second, secondary
alterations such as hydrocarbon expulsion, migration, biodegradation, and water washing,
among others, must have had no significant effect on the isotopic composition of either the
oil or potential source rock [149,150]. Regardless of whether bulk or compound-specific
isotopes are being identified, these requirements must be met. Carbon isotope profiles of
n-alkanes can easily distinguish source rock samples from different areas within a basin,
as demonstrated by [151], who analyzed δ13 C values of specific n-alkanes for different
formations throughout the Liaohe Basin in China. The n-alkanes of one of the Es4 mem-
bers have a relatively constant isotopic profile, with δ13 C values ranging from 28 to 32‰,
whereas the n-alkane δ13 C values for the other Es3 source rocks range from 23 to 28‰, with
a trend toward isotopically lighter values with increasing n-alkane chain length, typically
2–5‰ [152,153]. The n-alkanes found in the samples have similar characteristics to the Es4
source rocks, implying a saline lacustrine environment that is rather closed (Figure 7; [151]).
Molecules 2022, 27, 34 15 of 28

Figure 7. Carbon isotopic compositions of individual n-alkanes in source rocks of the.Western

depression of the Liaohe Basin (Reproduced from [151] with permission from Elsevier).

5.1.6. Application of Stable Carbon Isotopes to Gas Exploration

Natural gases, unlike crude oils, are generally simple mixtures, limiting the number of
factors that can be used to differentiate gases from different sources [21]. Hydrocarbons in
the C1 to C5 range will predominate in most natural gas samples, with varying quantities
of CO2 , H2 S, and N2 [21]. The maturity of the source rock or oil from which the gas
was formed will, to a significant extent, affect the proportions of individual components.
At high levels of thermal maturity, methane-dominated gases are generated, whereas at
low levels of thermal maturity, biogenic activity can result in methane-dominated gases.
Differentiating biogenic from thermogenic methane is one of the more common uses of
stable isotopes. Biogenic gas has a very light isotope, often around 70 per mil, whereas
thermogenic methane has an isotope of 30 to 50 per mil [21,154]. This was realized many
years ago, which led to one of the first correlation or source discrimination diagrams [154].
Essentially, this is a cross plot of methane δ13 C vs. C1 /C2+ ratio, and as seen below, biogenic
samples are characterized by isotopically light methane and a high C1 to C2+ ratio (Figure 8).
Thermogenic gas samples will be heavier isotopically and have lower compositional ratio
values. There is also a mixed gas area were assigning a specific source to the gases in that
region is a little more challenging. The kinetic isotope effect is responsible for the variance in
these isotopic ratios [21,154]. Bacteria preferentially cleave 12 C–12 C bonds, resulting in the
formation of isotopically light biogenic gas. From a thermogenic standpoint, these bonds
take less energy to break at low levels of maturity than a 12 C–13 C bond. As the maturity
level rises, more bonds containing the heavier isotope will be broken, causing the methane
produced to become heavier isotopically. Natural gas samples have fewer alternatives
for correlation or discrimination than crude oil samples, as previously stated [21,154].
The isotopic composition of the individual compounds in the gas samples, as well as
variations in the relative quantities of the individual compounds, must be used to make
these associations [21,155]. The GC-IRMS technique has been shown to be quite beneficial,
making it comparatively simple to determine the isotopic compositions of individual
compounds in complex mixtures. Before the invention of the GC-IRMS, each compound
had to be physically isolated to determine its isotopic composition.
Molecules 2022, 27, 34 16 of 28

Figure 8. The relationship between carbon isotope values in methane and the C1 /(C2 +C3 ). composi-
tional ratios of natural gas samples. (Reproduced from [154] with permission from Elsevier).

With the GC-IRMS system, it is of course possible to simply introduce the sample
into the GC and obtain the isotopic composition of each component in a relatively short
period. It is also worth noting that these individual compounds can have both carbon and
hydrogen isotope values, and a combination of the two isotopes offers an extra correlation
parameter [21,154,155]. Simply plotting the isotope levels of specific compounds against
their carbon number is the most useful application of isotopes for correlation purposes.
Then, with a sufficient number of gas samples from the same basin, those that are connected
will plot together, but those from other sources or created at different levels of maturity
will plot independently, as illustrated in Figure 9 [21].

Figure 9. Diagram establishing the relationships between families of gases within a specific. Basin
(after [21]).
Molecules 2022, 27, 34 17 of 28

This graph has many interesting extensions that can reveal a significant amount of ad-
ditional information. As previously stated, we are constrained in our ability to distinguish
between natural gas sources. However, after examining the isotopic composition of the
individual compounds in the condensates, it has been proven that for wells that produce
both gas and condensates, the plots presented above can be extended into the condensate
range. The isotope values for the individual compound in the condensate will simply be an
extension of the relevant gas sample, as illustrated if the condensate and gas are generated
from the same source and at the same maturity level (Figure 10) [155]. The benefit of col-
lecting these extended plots is that we can utilize the isotopes to establish the relationship
between gas and condensate, but there are also biomarkers, such as diamondoids, in the
condensates that may be used to correlate or discriminate between samples [21,155].

Figure 10. Plot showing the relationships between gases and condensates produced from the same
formation (Reproduced from [155] with permission from Elsevier).

5.2. Stable Hydrogen Isotope

Hydrogen isotope has been applied to oil–oil and oil–source rock correlations in
petroleum geochemistry [18,156]. The δD signature of the precursor must be conserved
throughout sedimentation, burial, diagenesis, and catagenesis for the stable hydrogen iso-
topic composition (δD) of sedimentary hydrocarbons to resemble that of their biosynthetic
precursors [10]. Over long periods of geological time (millions of years), diagenetic and
catagenetic effects are thought to induce considerable hydrogen (H/D) exchange between
organic hydrogen and hydrogen species in the surrounding environment [9,14,19,157–163].
Thermal maturation, in particular, has been discovered to play an important role in the
change of indigenous δD signatures, with increasing maturity resulting in a general en-
richment of D in hydrocarbon fractions and individual hydrocarbons [14,19,162,163]. The
hydrocarbons are expected to be exchanging hydrogen with the comparatively D-rich
formation fluids [158,164]. The influence of maturation on the δD values of sedimentary
hydrocarbons (n-alkanes, pristane, and phytane) in a sequence of marine source rocks from
the Perth Basin was demonstrated by [162] (Western Australia). Immature source rocks
(%R0 = 0.53) have distinct δD signatures, with pristane and phytane considerably reduced
Molecules 2022, 27, 34 18 of 28

in deuterium (D) compared to the n-alkanes. The discrepancy between the δD values of
n-alkanes and isoprenoids gradually reduces as maturity increases (up to %R0 = 1.13). Pris-
tane and phytane become increasingly D-enriched, while then-alkanes maintain a steady
isotopic composition until late maturity when D-enrichment in n-alkanes becomes consid-
erable. Isotopic exchange processes linked with thermal maturation have been attributed
to the D enrichment.
A similar trend of D enrichment in n-alkanes and isoprenoids was observed in other
studies [14,163]. The former study presented the effects of thermal maturity on n-alkane and
isoprenoids δD levels in two sediment sections in Poland (Kupferschiefer, KS) and Germany
(Posidonia Shale). With increasing age, all n-alkanes and isoprenoids were enriched in
D, with isoprenoids enriching at a faster rate than n-alkanes. The latter investigated a
450-meter core of Early Cretaceous lacustrine sediments from West Africa, which ranged
in maturity from immature to early mature (%R0 0.55–0.7). The effects of maturation on
the δD values of individual sedimentary hydrocarbons from sedimentary sequences with
a wider maturity range (%R0 0.6–1.6), such as Paqualin-1 and Vulcan-1B from the Vulcan
Sub-basin offshore northern Australia has been analyzed [10]. The enrichment in D in
isoprenoids was found to be connected to the epimerization of pristane and phytane and
to correspond substantially with traditional maturity criteria. D enrichment was seen in
pristane and phytane isolated from post-mature Paqualin-1 sediment, demonstrating that
D enrichment was maintained at extremely high maturity, more so for regular isoprenoids
than n-alkanes. This corroborated the theory that hydrogen (H/D) exchange, rather than
initiating free radical hydrogen transfer, causes the observed shift in δD values. In the
sedimentary environment, a mechanism was postulated to account for both H/D exchange
and the epimerization of pristane and phytane. Pristane was found to be more enriched
in D than phytane across the Vulcan Sub-basin sequences, indicating that they exchanged
hydrogen at equal rates during maturation. This was attributed to a reduced relative algal
input to the isoprenoids [10].

5.2.1. Hydrogen CSIA

δD measurements of bulk OM or whole fractions from crude oils or sediment extracts
were the only way to analyze the distribution of stable hydrogen isotopes in sedimentary
OM. Because aliphatic C-bound hydrogen is probably the most isotopically conservative,
isotopic analysis of individual aliphatic compounds with solely C-bound hydrogen (e.g.,
n-alkanes) is appealing. Compound-specific hydrogen isotope analysis [18,165] has made
it possible to determine the D/H composition of individual compounds in complex mix-
tures. Because it has the highest mass difference between its two stable isotopes (D and
H), and hence the greatest natural variability in stable isotope ratios of all elements, hy-
drogen compound-specific isotope analysis has shown remarkable promise in petroleum
geochemistry. The existing literature has looked into the relationship between δD val-
ues of whole crude oils and bitumen sand and their origins (including organic matter
type and depositional circumstances), thermal maturity, and secondary processes such
as biodegradation [147], mixing [10], and migration [147]. In a different study, it was
explained how different organisms fractionate hydrogen isotopes in the production of
lipids [166] while another one reported individual n-alkanes and isoprenoids δD values
as evidence of large and rapid climate change [9]. Furthermore, the efficacy of hydrogen
CSIA in petroleum correlation studies using a large number of crude oil samples from
the Western Canada Sedimentary Basin was illustrated [10]. Additionally, it was shown
that sedimentary aliphatic hydrocarbon D values can be utilized to determine the age
of source rocks and crude oils from the Perth Basin (WA) and Vulcan Sub-basin (Timor
Sea) [9,147,166,167]. Pristane (Pr) and phytane (Ph) have substantially lower δD values
than n-alkanes; however, this difference diminishes with age due to thermal hydrogen
isotope exchange. These studies suggested that the δD measurements of sedimentary
hydrocarbons are a good source of the effects-aware maturity metric. For Devonian source
rocks from the Western Canada Sedimentary Basin (WCSB), the D/H of biomarkers has
Molecules 2022, 27, 34 19 of 28

been employed as a maturity proxy [147]. Additionally, how various organisms fractionate
hydrogen isotopes in lipid production was investigated [168]. The δD values of compound-
specific lipid biomarkers extracted from peat deposits [15] and sediments [16] have been
utilized as a proxy for palaeoenvironmental and palaeoclimatic conditions. During the
Messinian salinity crisis, δD values of individual n-alkanes and isoprenoids were reported
as evidence of substantial and rapid climatic fluctuation [147]. Using crude oil samples
from the Western Canada Sedimentary Basin, the utility of hydrogen CSIA in petroleum
correlation studies and palaeoenvironmental reconstructions was evaluated [169]. Next, the
ability of lipid δD values to be preserved in Miocene lacustrine sediments and plant fossils
from Clarkia, Idaho, USA was demonstrated [170]. The Clarkia sediments they looked at
are the oldest samples known (15–20 million years) with original δD values that appear
to have been retained. There is concern that long-term diagenetic effects can result in
considerable hydrogen isotope exchange between organic hydrogen and the surrounding
environment [157–161,171]. Thus, the extent to which hydrogen CSIA can be employed
for palaeoenvironmental investigations, particularly when applied to older sedimentary
organic matter, is of interest. Organic geochemists and palaeoclimatologists are interested
in the δD values of organic compounds preserved in sediments because they can represent
the isotopic composition of water in ancient environments. Because water mobility and
the energy transferred as it changes physical states are major elements in weather and
climate, hydrogen isotopic fractionations are assumed to be linked to a range of naturally
occurring events in the hydrological cycle. ‘Meteoric’ waters are those that have gone
through the hydrological cycle. Temperature, altitude, latitude, closeness to the ocean, and
other factors affect the isotopic values of meteoric waters [50,172]. Water is the primary
hydrogen source for photosynthetic organisms, and the deuterium concentration in the
source water is mirrored in the deuterium composition of the organism [168]. The remains
of species are eventually absorbed into sediments and added to petroleum’s organic matter.
Similarly, CSIA of hydrogen to investigate Late Carboniferous to Late Permian torbanites
from Torbane Hill, Scotland, and the southern hemisphere, eastern Australia, was used [17],
where it was discovered that the δD values of n-alkanes and isoprenoids were similar
to those found in modern biological samples, which implies that their indigenous δD
signatures may have been preserved for at least 260–280 million years.

5.2.2. Application of Hydrogen Isotope to Gas Exploration

The hydrogen isotopes of alkane gases, as well as other saturated and aromatic hy-
drocarbons combined with carbon isotopes, play a critical role in the identification of
natural gas genetic-type analysis, parent material source, maturity, mixing, biodegradation,
and (TSR) [147,173–188]. Apart from the parent material type, maturity, biodegradation,
and TSR, the water environment during the deposition and diagenesis of source rock
(such as salinity) also plays a crucial effect [147,189]. Many studies have been conducted
on the hydrogen isotopic characteristics of natural gases from the Ordos Basin Permian
and the Upper Triassic Xujiahe Formation of the Sichuan Basin; the origin and source of
natural gases have also been analyzed based on the carbon and hydrogen isotopic and
chemical composition characteristics of the alkane gas. The natural gases in the above
two places have been proven to be coal-formed gas [190–200], and hydrogen isotopic
indices for detecting natural gas genesis and vitrinite reflectance (Ro) values have been
proposed [147,197]. Some studies on factors affecting hydrogen isotopes of alkane gas
have been conducted [167,197], but most of them focused on the composition of hydrogen
isotopes and affecting factors of Permian natural gases (methane and its homologs) and
the Upper Triassic Xujiahe Formation in the two basins from a single basin perspective,
and a few made comparisons [197], but only for methane hydrogen isotopes. The composi-
tion, influencing factors, maturity, and natural gas genetic identity indicators of hydrogen
isotopes of heavy alkane gases, such as ethane and propane, received little attention. In
a recent study [201] the hydrogen isotopic compositions of coal-formed Permian gas in
the Ordos Basin and Upper Triassic gas in the Sichuan Basin, and the hydrogen isotopic
Molecules 2022, 27, 34 20 of 28

compositions of alkane gas in various areas were also studied. To develop and improve
the coal-formed gas theory and genetic identification theory of natural gas, as well as to
guide natural gas exploration, the influencing factors and their degree of influence on the
hydrogen isotopic composition of alkane gases were investigated, and hydrogen isotope
indexes of alkane gas capable of identifying the natural gas genetic type and Ro value of
gas were proposed.

5.3. Stable Sulfur Isotopes

Sulfur isotopes have been utilized to correlate source rocks and oils in a rapidly buried
basin that have not been altered by thermochemical sulfate reduction (TSR) [11,12,202–204].
Hydrocarbons are produced quickly in such a basin, and peak oil is anticipated to occur
under semi-closed to closed circumstances. In case studies and experimental simulations,
this property, together with strong H2 S solubility and quick sulfur isotope homogenization,
is thought to result in modest discrepancies (up to 2) in 34 S values between mature kerogens
and their generated oils [12]. With the δ34 S values reported in closed range in both the
oils and the source rocks, sulfur isotopes have been effectively used to correlate Cambrian-
derived oils with Cambrian source rocks [11,12]. To identify the source of oils and oil-source
rock correlations in oils and source rocks from the Tazhong area, Tarim Basin, China, bulk
and individual n-alkane δ13 C and individual alkyldibenzothiophene δ34 S values were
used [205]. The majority of the oils from the Tazhong area were most likely generated
from Cambrian source rocks, and δ13 C and δ34 S values can be utilized to indicate the
oil–source rock relationship in the Tazhong uplift, Tarim Basin, China. Sulfur isotopes have
also been successfully used for oil–oil correlations in the oils from the Williston Basin of
North Dakota and Saskatchewan [202]. The sulfur isotopic composition can be utilized to
reconstruct changes in the sedimentary environment since large cycles between sulfate and
sulfide are associated with organic and inorganic carbon and iron cycles [205–207]. A series
of geological and geochemical studies, including a pyrite sulfur isotopic investigation of
lacustrine black shales from the Yanchang Formation in Ordos Basin, China was recently
carried out [208]. The paleoredox chemical conditions were confirmed to be the trigger
for the anomalous sulfur isotopic compositions based on the analysis of sulfur isotope
fractionation during the sulfur cycle.

6. Conclusions
This paper critically reviewed the early and recent trends in stable isotope geochem-
istry of organic elements in shales and crude oils. The bulk and compound-specific stable
isotopes of H, C, and S, as well as their uses as source facies, depositional environments,
biodegradation, thermal maturity, geological age, and oil–oil and oil–source rock corre-
lation studies, were discussed. The applications of the stable isotopes of H and C to gas
exploration were also discussed. Then, the experimental and instrumental approaches
in the stable isotopes of H, C, and S were discussed. This review showed that the stable
isotopes of H, C, and S could be utilized to establish the origin, depositional conditions,
and thermal maturity of organic matter of shales, as well as oil–oil and oil–source rock
correlation studies, which are critical to hydrocarbon exploration. However, there is limited
literature that discussed the sulfur isotopic compositions of individual biomarkers in crude
oils, while papers on compound-specific isotopic analysis of individual biomarkers in
shales are very rare in the literature. The reason is that these compounds are present in
relatively low concentrations and generally cannot be seen on a regular gas chromatogram;
hence, it is not possible to obtain isotopic compositions of individual biomarkers on a
routine basis, especially sulfur. The need here is to develop more routine molecular sieving
methods that can be used to isolate concentrates of the biomarkers and then determine
the isotopic composition of these compounds and provide another fingerprinting tool for
correlation purposes. Conclusively, emerging isotopic techniques such as laser microprobe,
ion microprobe, and NanoSIMs, which can collect data on the surface, are recommended
for further stable isotopic studies on shales and crude oils.
Molecules 2022, 27, 34 21 of 28

Author Contributions: Conceptualization, A.B.O., B.L. and M.O.; writing—original draft preparation,
A.B.O.; writing—review and editing, A.B.O., B.L. and M.O.; supervision, B.L.; project administra-
tion, B.L.; funding acquisition, B.L. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was supported by the Science and Technology Project of Heilongjiang
Province (2020ZX05A01).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors appreciate the anonymous reviewers and editors, whose construc-
tive comments have greatly improved the quality of this paper.
Conflicts of Interest: No conflicts of interest among the authors.

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