Fuel 182 (2016) 788797

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Fuel
journal homepage: www.elsevier.com/locate/fuel

Analysis of crude oils using gas purge microsyringe extraction coupled

to comprehensive two dimensional gas chromatography-time-of-flight
mass spectrometry
Xuanbo Gao a, Shukui Zhu a,b,, Wanfeng Zhang a,c, Donghao Li d, Wei Dai a, Sheng He a
a

Key Laboratory of Tectonics and Petroleum Resources of Ministry of Education, China University of Geosciences, Wuhan 430074, China
State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, China
Key Laboratory of Isotope Geochronology and Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China
d
Key Laboratory of Natural Resource of the Changbai Mountain and Functional Molecular of Ministry of Education, Yanbian University, Yanji 133002, China
b
c

a r t i c l e

i n f o

Article history:
Received 14 March 2016
Received in revised form 7 June 2016
Accepted 8 June 2016
Available online 15 June 2016
Keywords:
Gas purge microsyringe extraction
Comprehensive two-dimensional gas
chromatography
Biomarkers
Diamondoids
Carbazoles
Crude oils

a b s t r a c t
As a complex mixture, the analysis of individual compounds in crude oil is a great challenge. The traditional sample preparation method such as column chromatography is time-consuming, solvent-wasting,
and very possible to cause the loss of light hydrocarbons. In addition, coelution of compounds with similar boiling points or polarities often occurs in one dimensional gas chromatographymass spectrometry
(1D GCMS). To overcome these disadvantages, a homemade gas purge microsyringe extraction
(GP-MSE) device was prepared and coupled to comprehensive two-dimensional gas chromatographytime-of-flight mass spectrometry (GC  GC-TOFMS) for the analysis of crude oils from different regions.
The extraction conditions of GP-MSE were optimized in detail. The whole process of GP-MSE extraction
only takes 7 min, only 20 lL of organic solvent is needed, and almost complete extraction of analytes is
achieved. Compared with the common used methods, light hydrocarbons in all the samples were well
retained and more compounds were separated and identified, including diamondoid series, pyrrolic
nitrogen compounds and other important biomarkers such as terpane and sterane series. Coelution of
compounds in 1D GCMS was eliminated in GC  GC-TOFMS. Based on the developed GP-MSEGC  GC-TOFMS, the distribution of trace compounds in crude oils and effect of biodegradation were
discussed in detail.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
Crude oil is a very complex mixture. The large number of individual components present in each class is the main reason of this
complexity [1]. The complexity increases substantially with
increasing molecular weight (MW) of the compounds in the sample. It has been reported that more than 10,000 different compounds were identified in crude oils [2]. Among the various
classes of compounds present in crude oil, biomarkers provide
important geochemical parameters used to determine the characteristics of petroleum system such as thermal maturity [3], depositional environment [4], biodegradation level (BL) [57], oil
migration [8,9], as well as oil-oil and oil-source correlations
[10,11]. Apparently, all this information is very useful to petroleum

Corresponding author at: 388 Lumo Road, China University of Geosciences,

Wuhan 430074, China.
E-mail address: shukuizhu@126.com (S. Zhu).
http://dx.doi.org/10.1016/j.fuel.2016.06.050
0016-2361/ 2016 Elsevier Ltd. All rights reserved.

exploration and development. Therefore, the analysis of individual

compounds in crude oil is absolutely important.
Before instrumental analysis, crude oils generally need to be
pretreated to decrease the complexity of samples. Currently, the
widely used method is column chromatography [10,12], which is
efficient for the analysis of biomarkers with relatively high boiling
points. However, this method could result in the loss of volatile
components e.g. light hydrocarbons and parts of diamondoids. Furthermore, column chromatography is time consuming and needs a
large quantity of solvent. In order to avoid these problems, some
researchers analyzed oil samples using direct injection without
prior sample pretreatment and achieved satisfactory results
[13,14]. However, it is only suitable for the analysis of light crude
oils, and is not recommended for normal or heavy crude oils, due
to the existence of many non-volatile components.
The most common techniques used to analyze petroleum is
one-dimensional gas chromatography mass spectrometry (1D
GCMS) [6,12] and tandem MS (MS-MS) [15] owing to the vapour
pressure or final boiling point being compatible with the maxi-

X. Gao et al. / Fuel 182 (2016) 788797

mum temperatures of gas chromatograph (GC) columns [16,17].

However, peaks overlapped seriously when analyzing complex
samples due to the insufficient peak capacity of 1D GC, even with
the enhanced separation capabilities of the multiple reaction monitoring (MRM) or MS-MS techniques. Therefore, it is necessary to
develop some new techniques to improve the separation efficiency
of crude oil. Comprehensive two dimensional gas chromatography
(GC  GC) is one of the most powerful analytical tools, since it has
two capillary columns of different polarity to separate substances
[18]. The GC  GC organizes the sample components into chemical
classes, thus forming ordered chromatograms or distinct patterns
that simplify the process of characterization and quantification.
When coupled with time-of-flight mass spectrometer (TOFMS),
the deconvolution of some overlapping peaks and identification
of compounds becomes possible. Therefore, GC  GC-TOFMS system shows not only a superb separation ability, but also authentic
data for identification and has already been applied for biomarker
analysis [19,20]. In addition, the discovery of new compounds in
complex samples is possible because of the high peak capacity
[21].
To overcome the disadvantages of traditional sample preparation and analysis, GP-MSE of crude oils was developed based on
a homemade device, and coupled with GC  GC-TOFMS for analyzing oils from two different regions. Based on the developed
method, a wider range of compounds, including biomarkers as well
as non-hydrocarbons were revealed.

2. Samples and experimental

2.1. Samples and chemicals
Oil samples for the analysis of light hydrocarbon, diamondoids
and biomarkers were collected from Pearl River Mouth Basin in
China. Oil samples for the analysis of demethylated terpanes and
pyrrolic nitrogen compounds were collected from the northwest
of Junggar Basin in China, which show different biodegradation
levels. Silica gel (100200 mesh, activated at 150 C for 8 h before
use), alumina (50100 mesh, activated at 400 C for 4 h before use)
and silicic acid (60100 mesh, activated at 150 C for 4 h before
use) were purchased from Nahui (Shanghai, China). Toluene, nhexane, methanol and chloroform were of chromatographic grade
quality and purchased from Tedia (Fairfield, OH, USA). Medical
absorbent cotton was rinsed by chloroform until there was no
apparent fluorescence. The standard of carbazole was purchased
from J&K scientific Co., Ltd., USA. The standard of adamantane
was purchased from Tokyo Chemical Industry Co., Ltd., Japan.
The standard of 17b(H),21b(H)-hopane was purchased from Chiron
(Trondheim, Norway). The standard of 5a-androstane was purchased from Dr. Ehrenstorfer (Augsburg, Germany).

2.2. Sample preparation

2.2.1. Column chromatography
Firstly, the oil samples were de-asphalted by precipitation with
n-hexane followed by filtration. Then the de-asphalted samples
were fractionated by column chromatography with alumina and
silica gel. Saturated hydrocarbons, aromatic hydrocarbons and
non-hydrocarbons were obtained by sequentially eluting with nhexane, toluene and a chloroform and methanol solution (98:2,
v:v). Pyrrolic nitrogen compounds were isolated from the nonhydrocarbon fraction by column chromatography with 4 g of silicic
acid and elution of an n-hexane and toluene solution (1:1, v:v,
50 ml) [22]. All of the filtrates were collected and carefully concentrated under nitrogen flow to 0.5 ml for analysis.

789

2.2.2. GP-MSE
Oil samples were pretreated by a homemade GP-MSE apparatus
which mainly consists of a microsyringe, sample vial, gas flow system, condenser and heater. The schematic diagram and more
details of the GP-MSE were described in our earlier research [23].
The extraction procedures were: (1) the standard or oil sample
was added on the glass wool, then put the glass wool into a small
glass tube which was positioned in the heater and sealed with a
PTFE silicone septum pad; (2) a 500 ll volume microsyringe was
installed through the condenser, and a stainless steel wire was
inserted into the syringe barrel to prevent solvent bumping; (3)
the syringe needle was carefully inserted into the glass tube
through the septum cap and did not contact the glass wool; (4)
to start the extraction, n-hexane was added into the syringe barrel,
meanwhile, the heating power and the gas valve were opened; (5)
the gas flow (high purity nitrogen) carried target chemicals
through the syringe needle into the syringe barrel, and the evaporated compounds were trapped by the solvent (n-hexane); (6) after
the preset time, the syringe was removed from the condenser, and
the extracting solvent was transferred into a sample vial with the
volume of 0.5 ml for GC  GC-TOFMS analysis.
2.3. Instrumental analysis
The GCMS system consisted of an Agilent 7890A gas chromatograph and an Agilent 5975C mass spectrometer (Agilent Technologies, Wilmington, DE, USA). The GC  GC system consisted of a
GC (7890A model, Agilent Technologies, Wilmington, DE, USA)
equipped with a flame ionization detector (FID) and a time-offlight mass spectrometer (Pegasus 4D, Leco Corp., St. Joseph, MI,
USA). The GC oven contained two capillary columns that were connected serially by a quad-jet dual-stage modulator. Nitrogen and
air were used as the cold and hot gases, respectively.
In GCMS analysis, a DB-5MS column 60 m  0.25 mm
 0.25 lm (J&W Scientific, Folsom, CA, USA) was used. The carrier
gas was helium (purity P 99.9995%) with a flow rate of
1.0 ml min 1. The injector temperature was 300 C. The injection
volume was 1.0 ll. All injections were done with a 7683B series
autosampler and in the same temperature program. The oven temperature was programmed from 50 C (1 min hold) to 100 C at
20 C min 1, and then to 315 C (15 min hold) at 3 C min 1. The
mass spectrometer was operated in the electron impact mode
(70 eV). The ion source temperature was held at 220 C. The
transfer-line was maintained at 300 C. The scanned mass range
was from 50 to 550 u.
In GC  GC-TOFMS analysis, a DB-Petro column 50 m 
0.20 mm  0.50 lm and a DB-17HT column 1.5 m  0.25 mm
 0.15 lm (J&W Scientific, Folsom, CA, USA) were used as the
primary and secondary dimensional columns, respectively. The
carrier gas was helium (purity P 99.9995%) with a flow rate of
1.0 ml min 1. The injector temperature was 300 C. The injection
volume was 1 ll. All injections were done with a 7683B series
autosampler with the same temperature program. The temperature program for analyzing general biomarkers is: the 1st oven
temperature was initially held at 60 C for 1 min, programmed to
100 C at 10 C min 1, and to 300 C (30 min hold) at 2 C min 1.
Additionally, the temperature program for analyzing light hydrocarbons and diamondoids is: the 1st oven temperature was initially held at 50 C for 3 min, programmed to 300 C (30 min
hold) at 2 C min 1. The 2nd oven temperature was 10 C higher
than the 1st oven. The modulation period was 6 s with 1.5 s of
hot and cold jet pulse time for biomarkers and carbazoles analysis,
and 5 s with 1.25 s of hot and cold jet pulse time for diamondoids
analysis. The mass spectrometer was operated at an acquisition
rate of 100 spectra per second for a mass range of 50550 u, using
70 eV electron impact ionization and 1500 V multi-channel plate

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X. Gao et al. / Fuel 182 (2016) 788797

voltage. The ion source temperature was 230 C and the transferline temperature was 280 C. The pressure inside the flight tube
was 1.1  10 7 torr. Data acquisition and processing were performed using the ChromaTOF software version 4.33.
The identification of the general biomarkers, light hydrocarbons, diamondoids and carbazoles were based on authentic standard injection, NIST08 library of the TOFMS software, the ordered
GC  GC chromatogram and the comparison with literatures
[3,6,13,22,2426].

3. Results and discussion

3.1. Optimization of GP-MSE condition
During GP-MSE extraction process, oil samples were heated in a
temperature program. Analytes were evaporated from the sample
matrix and then flowed into the extraction solvent by carrier gas.
The condenser guarantees the target compounds were completely
trapped into the extraction solvent at low temperatures. Therefore,
extraction temperature, gas flow rate, extraction time and condensing temperature are the key conditions to increase extraction
efficiency during GP-MSE extraction process. Therefore, the four
experimental parameters were systematically optimized by using
standards of adamantane, 17b(H),21b(H)-hopane, 5a-androstane
and carbazole in the following discussion.

3.1.1. Effect of extraction temperature on the extraction efficiency

Like chromatographic injection temperature condition, the
extraction temperatures were set to 200, 230, 260, 280, 300, 320,
340 and 350 C. The results of the different temperatures were
shown in Fig. 1a. During the low temperature stage, the low boiling
point compounds (e.g. adamantane) have relative high recoveries,
but high boiling point compounds such as hopane, androstane and
carbazole have very low recoveries. When the temperature exceed
300 C, the recoveries of both high and low boiling point compounds reached 90% and showed no obvious increase after
300 C. Considering too high temperature leads to quick evaporation of the extracted solvent, 300 C was selected as a proper
extraction temperature for oil pretreatment in this study. In addition, in this temperature, very heavy compounds in asphaltene
would not be evaporated and enter the extraction solvent. Therefore, GC column would not be contaminated or damaged using this
method, because no non-volatile components move through the
GC column.
3.1.2. Effect of gas flow rate on the extraction efficiency
Gas flow rate is another important parameter which affects
extraction efficiency. Too high or too low gas flow rate is not good
to increase the extraction efficiency. To determine how gas flow
rate affects extraction efficiency, the gas flow rates were set to 1,
2, 3, 4 and 5 ml min 1 respectively. As shown in Fig. 1b, in the
gas flow rate of 1 ml min 1, the recoveries of all the target

Fig. 1. The recoveries of adamantane, 17b(H),21b(H)-hopane, 5a-androstane and carbazole varying with the (a) extraction temperature, (b) gas flow rate, (c) extraction time,
(d) condensing temperature.

X. Gao et al. / Fuel 182 (2016) 788797

791

Fig. 2. Total ion chromatogram (TIC) and m/z 85 extracted ion chromatogram (EIC) of GC  GC-TOFMS of Pearl River Mouth Basin oil prepared by different methods:
(a) GP-MSE, (b) column chromatography. C8 to C11, C8 to C11 n-alkanes.

compounds dont reach 90%, because volatiles are easily condensed

to occlude the needle in the too low gas flow rate condition. When
the gas flow rate increased to 2 ml min 1, the recoveries of
adamantane, hopane and androstane reach the maximums except
carbazole. The optimum recovery of the carbazole was 3 ml min 1.
When the gas flow rate exceeded 3 ml min 1, the recoveries of the
target compounds generally decreased with the increasing gas flow
rate. That may because that too high gas flow rate leads to the
incomplete trapping of volatiles. Taking account of the optimum
recoveries of the most compounds and all the data of different
compounds in one sample should be acquired in one single experiment in order to increase the efficiency, 2 ml min 1 was selected
as a proper gas flow rate in this experiment.
3.1.3. Effect of extraction time on the extraction efficiency
Extraction times were set to 2, 4, 7, 10, 13, 16 and 19 min
respectively. As shown in Fig. 1c, the recovery of adamantane
increases sharply from 2 min to 4 min. During the time between
4 min and 10 min, the recovery of adamantane has no big change.
After 10 min, the recovery of adamantane began to decrease
slowly. Moreover, the recoveries of other 3 compounds have similar variation trends: increase sharply from 2 min to 7 min and
reach the maximums at 7 min, then decrease slowly with the
increasing time. The experimental data indicate that increase
appropriately extraction time contributing to get higher recovery.
However, too long extraction time leads to the lower recovery
because of the evaporation of volatile compounds and extraction
solvent. Therefore, 7 min was selected as the proper extraction
time in the following experiments.
3.1.4. Effect of condensing temperature on the extraction efficiency
In GP-MSE process, gas phase extracts were extracted by
organic solvent with a high temperature. The organic solvent will
be quickly evaporated if without cooling. Therefore, the condenser
is an important unit in GP-MSE device which protects organic
solvent and target compounds from evaporating. The condensing
temperatures were set to 0, 2, 4, 6 and 8 C respectively.
As shown in Fig. 1d, the recovery of adamantane increased from
0 C to 6 C, and reached the maximum at 6 C, then decreased
with the decreasing temperature. The recoveries of hopane and
carbazole have the similar variation trend: increased from 0 C to
4 C and reached the maximum at 4 C, then decreased slightly

from 4 C to 8 C. Though, the recovery of androstane reaches

the maximum at 8 C, it has no obvious variation from 2 C to
8 C. Considering 95% is already a high recovery and to increase
the experimental efficiency, 6 C was selected as the proper
extraction time in the following experiments.
3.2. Application to the analysis of compounds in real oil samples
3.2.1. Light hydrocarbons
The same oil sample was pretreated by GP-MSE (Fig. 2a) and
column chromatography (Fig. 2b) respectively. As shown in
Fig. 2, compared with column chromatography, low boiling points
alkanes from C8 to C12 and some low MW aromatics were well preserved by the method of GP-MSE. Additionally, there was no loss of
the compounds of high boiling points in GP-MSE analysis. The
complete information of light hydrocarbons is benefit to reveal
the true properties of the crude oils.

Fig. 3. GC  GC-TOFMS m/z 85 + 135 + 136 + 149 + 163 + 177 EIC and TIC of the
typical Pearl River Mouth Basin oil, showing adamantanes and n-alkanes. The
identification of adamantanes is based on authentic standard injection, NIST08
library of the TOFMS software, the ordered GC  GC chromatogram and the
comparison with literatures and listed in Table 1. C11 to C13, C11 to C13 n-alkanes;
TMA, trimethyladamantane.

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X. Gao et al. / Fuel 182 (2016) 788797

Fig. 4. GC  GC-TOFMS m/z 85 + 187 + 188 + 201 + 215 EIC and TIC of the typical
Pearl River Mouth Basin oil, showing diamantanes and n-alkanes. The identification
of diamantanes is based on authentic standard injection, NIST08 library of the
TOFMS software, the ordered GC  GC chromatogram and the comparison with
literatures and listed in Table 1. C15 to C17, C15 to C17 n-alkanes; Pr, pristine; TeMTeHN, 1,4,6,7-tetramethyl-1,2,3,4-tetrahydronaphthalene.

3.2.2. Diamondoids
Diamondoids are rigid, three-dimensional cyclohexane-ring
alkanes with a diamond-like cage structure. They are resistant to
thermal cracking and biodegradation [27]. Therefore, diamondoids
are important geochemical indicators of maturity and depositional
environments in petroleum exploration [25,28,29]. However, it is
difficult to identify diamondoids of crude oils using conventional
sample preparation coupled with GCMS due to their low concentration and low boiling point. GP-MSE prevents the evaporation of
diamondoids during sample pretreatment. When coupled with
GC  GC-TOFMS, diamondoids are good identified and quantitatively analyzed.
More than 100 diamondoids were detected by GC  GC-TOFMS.
Adamantanes and diamantanes of a typical oil sample from Pearl
River Mouth Basin were assigned from GC  GC-TOFMS by monitoring m/z 85 + 135 + 136 + 149 + 163 + 177 extracted ion chromatogram (EIC, Fig. 3) and m/z 85 + 187 + 188 + 201 + 215 EIC
(Fig. 4). Twenty-six adamantanes and seventeen diamantanes were
identified and presented in Table 1. A roof tile effect was observed,
where each tile represents adamantanes and diamantanes with the
same number of carbons. The elution orders of adamantanes were
between C11-alkane and C13-alkane (Fig. 3) and the elution orders
of diamantanes were between C15-alkane and C17-alkane (Fig. 4).

Table 1
Diamondoids identified in crude oil samples by GP-MSE coupled with GC  GC-TOFMS analysis.
Peak number

Compound

Molecular formula

m/z

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43

Adamantane
1-Methyladamantane
2-Methyladamantane
1,3-Dimethyladamantane
1,4-Dimethyladamantane
2,4-Dimethyladamantane
1,2-Dimethyladamantane
1-Ethyladamantane
2-Ethyladamantane
1,3,5-Trimethyladamantane
1,3,6-Trimethyladamantane
1,3,4-Trimethyladamantane
1,2,4-Trimethyladamantane
1-Ethyl-3-Methyladamantane
1,3,5-Trimethyladamantane
1,3,6-Trimethyladamantane
1,3,4-Trimethyladamantane
1,2,4-Trimethyladamantane
1,2,3-Trimethyladamantane
1,3,5,7-Tetramethyladamantane
1,2,5,7-Tetramethyladamantane
1-Ethyl-3,5-Dimethyladamantane
1,2,5,6-Tetramethyladamantane
1,3,5,6-Tetramethyladamantane
1,2,3,5-Tetramethyladamantane
1-Ethyl-3,5,7-trimethyladamantane
Diamantane
4-Methyldiamantane
1-Methyldiamantane
3-Methyldiamantane
4,9-Dimethyldiamantane
1,4-Dimethyldiamantane
2,4-Dimethyldiamantane
1,3-Dimethyldiamantane
1,5-Dimethyldiamantane
4,8-Dimethyldiamantane
2,3-Dimethyldiamantane
3,4-Dimethyldiamantane
1,4,9-Trimethyldiamantane
3,4,9-Trimethyldiamantane
1,2,5-Trimethyldiamantane
1,3,4-Trimethyldiamantane
1,2,4-Trimethyldiamantane

C10H16
C11H18
C11H18
C12H20
C12H20
C12H20
C12H20
C12H20
C12H20
C13H22
C13H22
C13H22
C13H22
C13H22
C13H22
C13H22
C13H22
C13H22
C13H22
C14H24
C14H24
C14H24
C14H24
C14H24
C14H24
C15H26
C14H20
C15H22
C15H22
C15H22
C16H24
C16H24
C16H24
C16H24
C16H24
C16H24
C16H24
C16H24
C17H26
C17H26
C17H26
C17H26
C17H26

136
135
135
149
149
149
149
135
135
163
163
163
163
149
163
163
163
163
163
177
177
163
177
177
177
177
188
187
187
187
201
201
201
201
201
201
201
201
215
215
215
215
215

28.6667
30.0000
34.6667
31.0000
35.3333
35.7500
37.5833
39.3333
41.4167
31.7500
36.2500
38.0833
38.4167
40.1667
40.5000
41.9167
42.3333
42.7500
43.1667
32.4167
38.6667
40.7500
41.0000
41.0833
42.6667
43.0000
57.6667
58.4167
60.7500
62.2500
59.0833
60.9167
61.2500
62.9167
64.5833
65.0833
66.4167
66.5833
61.3333
65.1667
66.5833
66.8333
67.0833

1.910
1.750
1.950
1.720
1.800
1.820
1.880
1.880
1.940
1.640
1.710
1.770
1.780
1.780
1.800
1.810
1.830
1.870
1.870
1.580
1.680
1.690
1.740
1.610
1.730
1.730
2.330
2.190
2.290
2.310
2.040
2.130
2.140
2.160
2.230
2.250
2.300
2.290
2.000
2.100
2.120
2.180
2.180

D retention time (min)

D retention time (s)

X. Gao et al. / Fuel 182 (2016) 788797

Fig. 3 illustrates in m/z 163 EIC coelution between 1,2,3trimethyladamantane

(peak
15),
1-ethyl-3,5dimethyladamantane (peak 22) and unidentified trimethyladamantane, often observed in GCMS, was totally solved using
GC  GC-TOFMS. In addition, a new compound was detected and
tentatively assigned as 1,4,6,7-tetramethyl-1,2,3,4-tetrahydronaph
thalene, which has never been detected using GCMS (Fig. 4). It
coeluted with diamantane (peak 27) in m/z 188 EIC of 1D GC, also
well separated in two dimensional plane of GC  GC (Fig. 4).
3.2.3. Tri-, tetra- and pentacyclic terpanes
Tricyclic terpanes, tetracyclic terpanes and pentacyclic terpanes
of the typical oil sample of Pearl River Mouth Basin were identified
in the m/z 191 EIC by GC  GC-TOFMS (Fig. 5). As shown in Fig. 5,
the roof tile effect of these compounds was obviously observed.
Therefore, the identification of different compounds families are
easily achieved by the ordered nature of GC  GC. The distribution
and characterization of these compounds are similar to the observations in the previous literatures [19,26,30].
C24 to C27 tetracyclic terpane series were fully detected, attribute to the high peak capacity and sensitivity of GC  GC-TOFMS.
They were usually coeluted with C26 to C29 tricyclic terpane series
in 1D GC, but were well separated in GC  GC chromatogram
(Fig. 5). The origins of tetracyclic terpanes are regarded as thermal
or microbial rupture of the E-ring in hopanes or precursor hopanoids [6]. Compared with C25-C27 tetracyclic terpanes, C24 tetracyclic terpane generally has more widespread occurrence (Fig. 5)
[6]. The high abundance of C24 tetracyclic terpane indicates that
the oil appear to be originated from carbonate and evaporite
source rock [31].
Tricyclic terpanes are generally only identified up to the C30
homologues by traditional analytical method (e.g. GCMS). The
high MW tricyclic terpanes often coeluted with hopanes in the
m/z 191 EIC (Fig. 5). By GC  GC-TOFMS, tricyclic terpanes up to
C36 were detected and the problem of coelution between the high

793

MW tricyclic terpane and hopane series was fully resolved (Fig. 5).
In addition, compared with GCMS, it was only possible to identify
the four stereoisomeric forms of tricyclic terpanes from C19 to C24
by using GC  GC-TOFMS because of the sensitivity and high peak
capacity (Fig. 6). In these four stereoisomers of tricyclic terpanes,
14a was the most abundant stereoisomer in C19 tricyclic terpanes.
13a, 14a was the most abundant stereoisomer in C20 tricyclic terpanes. In addition, 13a, 14b was the most abundant stereoisomer
in C21 tricyclic terpanes. However, 13b, 14a was the most abundant stereoisomer in C23 tricyclic terpanes and C24 tricyclic terpanes. The distributions of the four stereoisomers of tricyclic
terpanes of the oils of Pearl River Mouth Basin were consistent
with the research of the oils of Potiguar Basin [19]. In addition, a
stereoisomer of C22 tricyclic terpanes was coeluted with C24 13b(
H),14b(H)-tricyclic terpane in 1D GC while separated in the 2nd
dimension of GC  GC chromatogram (Fig. 6).
As shown in Fig. 5, hopane series compounds were completely
identified by monitoring the m/z 191 EIC including the C27 to C34 1
7a(H),21b(H)-hopane series (ab-hopanes) and the C29 to C33 17b(H
),21a(H)-hopane isomers (moretanes). C30 17a(H),21b(H)-hopane
was the most abundant compound of the series in nonbiodegraded oils. Between C29 hopane and C30 hopane, six rearranged hopanes were identified, which included 18a(H),21b(H)-30norneohopane (C29Ts), C30 17a(H)-diahopane (DiaH30), C29 28-norspergulane (28NS), C30 tetracyclic polyprenoid (TPP), C29 moretane
and oleanane (Ole). The problems of the coelutions for these compounds were completely resolved by GC  GC-TOFMS (Fig. 5). The
high oleanane concentration may indicate that the oils of Pearl River
Mouth Basin were originated from higher-plant of Cretaceous or
younger age [32]. Additionally, the occurrence of TPP and 28NS generally indicate the samples derived from lacustrine depositional
environment [33,34]. Gammacerane was also identified in the oils
of Pearl River Mouth Basin by monitoring the m/z 191 EIC (Fig. 5)
and it indicate water-column stratification which is generally resulting from hypersalinity in the process of deposition [35].

Fig. 5. m/z 191 EIC of the typical Pearl River Mouth Basin oil analyzed by GCMS and GC  GC-TOFMS. Tr, tricyclic terpanes and carbon number; TeT, tetracyclic terpanes and carbon
number; H, hopanes and carbon number; M, moretanes and carbon number; DiaH, 17a(H)-diahopanes and carbon number; Ts, 18a(H),21b(H)-22,29,30-trisnorhopane; Tm, 17a
(H),21b(H)-22,29,30-trisnorhopane; Gam, gammacerane; Ole, oleanane; C29Ts, 18a(H),21b(H)-30-norneohopane; 28NS, C29 28-norspergulane; TPP, C30 tetracyclic polyprenoid.

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X. Gao et al. / Fuel 182 (2016) 788797

Fig. 6. m/z 191 EIC of the typical Pearl River Mouth Basin oil analyzed by GCMS and GC  GC-TOFMS. Tr, tricyclic terpanes and carbon number.

3.2.4. Demethylated terpanes

The oils of Junggar Basin were used to analyze the demethylated terpanes. The biodegradation levels of these oil samples were
mainly eight (Peters-Moldowan scale [6]). Terpanes can be
demethylated during the process of biodegradation. Tricyclic terpanes lose the C17 methyl group and hopanes lose the C25 methyl
[36]. The complete series of 25-norhopanes from C26 to C31 and
demethylated tricyclic terpanes from C20 to C25 were identified
by monitoring the m/z 177 and compared to the normal terpanes
by monitoring the m/z 191 in one chromatogram (Fig. 7). In addition, C23 demethylated tetracyclic terpane and C24 des-A-hopane
were also detected in the oils of Junggar Basin. The problem of
coelution between C24 des-A-hopane and C25 demethylated tricyclic terpane in GCMS was resolved by GC  GC-TOFMS
(Fig. 7). The occurrence of demethylated tricyclic terpanes and
demethylated tetracyclic terpanes was reported for the first time
in the oils of Junggar Basin. As shown in Fig. 7, the relative abundance of tricyclic terpanes was higher than hopanes because of
the higher biodegradable resistance of tricyclic terpanes than
hopanes.

3.2.5. Steranes
In the analysis of steranes in Pearl River Mouth Basin oils, the
four stereoisomers of C27-C29 steranes (aaaS, abbR, abbS and

aaaR), C27 diasteranes (baS, baR, abS and abR) and C30 methylsteranes (aaaS, abbR, abbS and aaaR) were identified by monitoring the m/z 217 EIC (Fig. 8). The occurrence of hopanes was also
detected in m/z 217 EIC, which may cause the problem of coelution
with regular steranes and methylsteranes in 1D GC. However, this
problem was totally resolved in the 2nd dimension of GC  GC
chromatogram (Fig. 8), allowing for more accurate identification
and quantification of sterane series. The similar results were also
described by Oliveira et al. [19] and Aguiar et al. [26].

3.2.6. Pyrrolic nitrogen compounds

The oils of Junggar Basin were used to analyze the pyrrolic
nitrogen compounds. The analysis of pyrrolic nitrogen compounds
was both by GCMS and GC  GC-TOFMS. Carbazole series and
benzocarbazole series were fully detected by GC  GC-TOFMS
because of the unique roof-tile effect (Fig. 9 and Table 2). The problem of coelution in GCMS was also completely resolved by
GC  GC-TOFMS, which makes more accurate identification and
quantification of the pyrrolic nitrogen compounds.
In this study, the variation of the relative abundance of carbazoles with biodegradation was observed. The distributions of
C0-C5 carbazoles varied according to the biodegradation level,
which were displayed in m/z 167 + 181 + 195 + 209 + 223 + 237
EIC (Fig. 10). The relative abundance of carbazole homologous

Fig. 7. m/z 191 + 177 EIC of the typical Pearl River Mouth Basin oil analyzed by GC  GC-TOFMS. Tr, tricyclic terpanes and carbon number; TeT, tetracyclic terpanes and
carbon number; DTr, demethylated tricyclic terpanes and carbon number; H, hopanes and carbon number; NH, 25-norhopanes and carbon number; Ts, 18a(H),21b(H)22,29,30-trisnorhopane; Tm, 17a(H),21b(H)-22,29,30-trisnorhopane; DTm, 17a(H),21b(H)-22,25,29,30-tetranorhopane; M29, 17b(H),21a(H)-30-norhopane; 28NM, C28
normoretane; Gam, gammacerane; DTeT23, C23 demethylated tetracyclic terpane; Des-A-H24, C24 des-A-hopane.

X. Gao et al. / Fuel 182 (2016) 788797

series exhibits strong differences in different BL oils. As shown in

Fig. 10, the relative abundance of C0-C2 carbazoles decreased with
increasing degrees of biodegradation: carbazole was from 5% to
0.2%, C1 carbazoles were from 12% to 0.7% and C2 carbazoles were
from 27% to 2%. In contrast, the relative abundance of C4-C5

795

carbazoles increased with an increasing degree of biodegradation:

C4 carbazoles were from 18% to 42% and C5 carbazoles were from
8% to 41%. Furthermore, the relative abundance of C3 carbazoles
initially increased and then decreased with the increasing degrees
of biodegradation. This result was also consistent with the

Fig. 8. m/z 217 EIC of the typical Pearl River Mouth Basin oil analyzed by GC  GC-TOFMS. Ts, 18a(H),21b(H)-22,29,30-trisnorhopane; Tm, 17a(H),21b(H)-22,29,30trisnorhopane; H29, 17a(H),21b(H)-30-norhopane; C29Ts, 18a(H),21b(H)-30-norneohopane; H30, 17a(H),21b(H)-hopane.

Fig. 9. (a) TIC of the typical Junggar Basin oil obtained via GC  GC-TOFMS analysis. (b) m/z 167 + 181 + 195 + 209 + 223 + 237 EIC obtained via GCMS and GC  GC-TOFMS
analysis. (c) m/z 217 + 231 + 245 + 259 EIC obtained via GCMS and GC  GC-TOFMS analysis. The identification of carbazoles and benzocarbazoles is listed in Table 2.

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X. Gao et al. / Fuel 182 (2016) 788797

Table 2
Carbazoles and benzocarbazoles identified in crude oil samples by GC  GC-TOFMS
analysis.
Peak
number

Compound

m/z

D retention time
(min)

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

Carbazole
1-Methylcarbazole
3-Methylcarbazole
2-Methylcarbazole
4-Methylcarbazole
1,8-Dimethylcarbazole
1-Ethylcarbazole
1,3-Dimethylcarbazole
1,6-Dimethylcarbazole
1,7-Dimethylcarbazole
1,4-Dimethylcarbazole
1,5-Dimethylcarbazole
3-Ethylcarbazole
2,6-Dimethylcarbazole
2,7-Dimethylcarbazole
2,4-Dimethylcarbazole
2,5-Dimethylcarbazole
Benzo[a]carbazole
Benzo[b]carbazole
Benzo[c]carbazole

167
181
181
181
181
195
195
195
195
195
195
195
195
195
195
195
195
217
217
217

52.5
56.3
57.8
58.0
58.7
59.3
59.8
61.3
61.5
61.8
62.2
62.4
62.9
63.1
63.5
63.8
64.0
80.2
82.2
82.9

3.480
3.310
3.370
3.340
3.550
3.040
3.200
3.200
3.210
3.150
3.310
3.360
3.280
3.250
3.340
3.410
3.390
4.070
4.280
4.510

D retention
time (s)

observation by Huang et al. [37], which means the relative abundance of lower alkylated carbazoles decreased with the increasing
degrees of biodegradation and the relative abundance of higher
alkylated carbazoles increased with the increasing degrees of
biodegradation. Moreover, not only biodegradation but also water
washing may result in the variation of the relative abundance of
alkylated carbazoles. Water washing typically accompanies
biodegradation of petroleum and results in selective loss of light
hydrocarbons, especially benzene, toluene, and other aromatics
[6,38,39]. In the study of Bartha et al. [40], the similar variation
occur in alkyl naphthalene: the methyl naphthalenes are in low
abundance compared to higher-alkylated analogues due to the
water solubilities of light aromatics decrease with increasing alkyl
substitution, suggesting that the parent compounds (naphthalene
and phenanthrene) have been removed principally by water washing. As discussed above, the higher alkylated carbazoles are more
resistant to biodegradation than the lower alkylated carbazoles
and the water solubilities of carbazoles may decrease with increasing alkyl substitution. It is also important to note that though carbazoles were more resistant to biodegradation than biomarkers
[6,37], they were hardly detected in high BL oils.

4. Conclusions
GP-MSE is a very suitable sample preparation method for crude
oils. Compared to the traditional method, GP-MSE is easy to operate, time and solvent saving. After optimization of GP-MSE analytical conditions and coupled to the GC  GC-TOFMS, complete
extraction of analytes is achieved. Compared with the commonly
used methods, light hydrocarbons in all the samples were well
retained and more compounds were separated and identified,
including diamondoid series, pyrrolic nitrogen compounds and
other important biomarkers such as terpane and sterane series.
Coelutions among these compounds were eliminated in 2nd
dimension of GC  GC-TOFMS chromatograms. The variation of
the relative abundance of alkylated carbazoles influenced by
biodegradation and water washing was observed, which may indicate that the higher alkylated carbazoles are more resistant to
biodegradation than the lower alkylated carbazoles and the water
solubilities of carbazoles decrease with increasing alkyl
substitution.
Acknowledgements
The authors would like to greatly thank two anonymous
reviewers for their comments and constructive suggestions which
significantly improved the quality of this paper. The study was
supported by grants from the National Natural Science Foundation
of China (No. 21077039), the Fundamental Research Funds for the
Central Universities (No. 26420130179) and the Programme of
Introducing Talents of Discipline to Universities (No. B14031).
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Fig. 10. (a) m/z 167 + 181 + 195 + 209 + 223 + 237 EIC of typical Junggar Basin oils
in different BL levels displaying the distributions of C0-C5 carbazoles obtained via
GCMS and GC  GC-TOFMS analysis. The percentage represents the relative
concentration of different carbazole homologous series.

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