Petroleum tricyclic terpanes: predicted physicochemical
behavior from molecular mechanics calculations
Kenneth E. Peters
ExxonMobil Upstream Research Company, P.O. Box 2189, Houston, TX 77252-2189, USA
Received 31 July 1999; accepted 9 March 2000 (returned to author for revision 10 December 1999)
Abstract
Petroleum contains a diastereomeric doublet for each of the C25to C29tricyclic terpanes due to stereoisomerization at
C-22, where the elution order of the 22S and 22R epimers is unknown. Geometry-optimized molecular mechanics models for each pair of epimers show similar calculated total energies, indicating similar thermal stability. Similar stability explains the nearly equivalent size of the 22S and 22R chromatographic peaks for each doublet in nonbiodegraded pet-roleum. Molecular mechanics MM+ and COMPASS force-®eld calculations indicate an abrupt conformational change between the C28and C29tricyclic terpanes, corresponding to a discontinuity on plots of molecular mass versus log of gas
chromatographic retention time. The second-eluting peak in each C26to C29doublet is more readily biodegraded, with
(Alberdi, M., Moldowan, J.M., Peters, K.E., Dahl, J.E., 2000. Stereoselective biodegradation of tricyclic terpanes in heavy oils from Bolivar Coastal Fields, Venezuela. Submitted to Organic Geochemistry) or without microbial de-methylation to form 17-nor-tricyclic terpanes. Factors controlling the chromatographic elution order of epimers are not fully understood. However, elution order can be inferred if one assumes that epimers with greater calculated surface areas are more susceptible to microbial attack, as for the extended hopanes where C-22 epimer elution order is known. Surface areas of 22R epimers exceed 22S for C25to C29tricyclic terpanes, suggesting that the 22R epimers elute after
22S. Proof of elution order will require co-injection of authentic standards. Four epimers are possible for each of the C30and C31tricyclic terpanes. Molecular mechanics and high-resolution chromatography suggest that all four peaks
occur in petroleum, but only two are normally observed due to co-elution. Complete resolution of these epimers will require improved chromatographic methods.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Tricyclic terpanes; Cheilanthane; Molecular mechanics; QSAR; Biodegradation
1. Introduction
Tricyclic terpanes or cheilanthanes were ®rst observed in extracts from the Green River Formation (Anders and Robinson, 1971; Gallegos, 1971). The most prominent tricyclic terpanes (14-alkyl, 13-methylpodocarpanes, Fig. 1) range from C19 to at least C54 and are important
components in the saturated hydrocarbon fractions of petroleum (Moldowan et al., 1983; De Grande et al., 1993). Tricyclic terpanes are used to correlate crude oils
and source-rock extracts, predict source-rock character-istics, and evaluate the extent of thermal maturity and biodegradation (Seifert et al., 1980; Seifert and Moldo-wan, 1981; Zumberge, 1987; Peters and MoldoMoldo-wan, 1993). Extended tricyclic terpanes (>C24) contain a regular
isoprenoid side chain at C-14 (Aquino Neto et al., 1982) as evidenced by lower abundance of the C22, C27, C32,
C37, and C42homologs (Moldowan et al., 1983), which
require cleavage of two carbon±carbon bonds to form from higher homologs (Fig. 1). Based on their results for the C20and C21tricyclic terpanes, Chicarelli et al.,
(1988) infer that higher homologs in thermally mature
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petroleum show 13b,14(H)-stereochemistry. The C25to
C29 tricyclic terpanes occur as diasteromeric doublet
peaks onm/z191 mass chromatograms, which represent epimers resulting from stereochemical dierences at C-22 in the side chain (C25is poorly resolved in Fig. 1).
Split-ting of these two peaks into four peaks should occur for the C30to C34homologs because of the asymmetric
cen-ter at C-27 (however, see below). Under the chromato-graphic conditions used by Moldowan et al. (1983) splitting of the ®rst- and second-eluting peaks in each doublet was not observed until C35and C38, respectively.
Regular polyisoprenols, such as C30tricyclohexaprenol
in bacterial membranes or malabaricatrienes from algae or bacteria, likely account for many tricyclic terpanes in petroleum (Ourisson et al., 1982; Aquino Neto et al., 1983; Heissler et al., 1984; Behrens et al., 1999). Higher homo-logs may originate from C40tricyclooctaprenol (Azevedo
et al., 1998) or larger precursors. High concentrations of tricyclic terpanes and their aromatic analogs commonly correlate with high paleolatitude Tasmanite-rich rocks, suggesting an origin from these algae (Aquino Neto et al., 1989, Azevedo et al., 1992; Peters et al., 1997). However, stable carbon isotopic data suggest that other
algal or bacterial sources are possible (Revill et al., 1994). Kruge et al. (1990) and De Grande et al. (1993) note prominent tricyclic terpanes in saline lacustrine and marine carbonate environments, suggesting to them that the precursor organisms lived in moderate salinity con-ditions. However, caution must be exercised in these interpretations because tricyclic terpanes are thermally more stable than many other terpanes. Thus, highly mature petroleum commonly contains abundant tricyclic terpanes, regardless of source-rock organic matter input (Peters and Moldowan, 1993).
The purpose of this work is to investigate four unex-plained aspects of tricyclic terpane distributions in petro-leum (e.g., Figs. 1 and 2) and to promote further research to better de®ne their occurrence, stereochemistry, and chromatographic properties.
1. The 22S and 22R epimer peaks in each C25to C29
13b,14(H)-tricyclic terpane chromatographic
doublet show similar abundance in nonbiodegraded oils and source-rock extracts (Fig. 2, top), unlike the 22S and 22R peaks for the extended 17,21b
(H)-hopanes (C31to C35hopanes, Peters et al., 1996).
Fig. 1. Mass chromatogram (m/z191, solid trace) shows C19±C30tricyclic terpanes in the saturated hydrocarbon fraction of heavily
biodegraded crude oil from Rubiales Field, Well 12 (2603±2608 feet), Llanos Basin, Colombia. C30tricyclohexaprenane (inset) is a
widespread higher homolog in petroleun. Doublets eluting after C24are stereoisomers resulting from the asymmetric center at C-22.
In-reservoir biodegradation resulted in C-10 demethylation of tricyclic terpanes and formation of the corresponding 17-nor-tricyclic terpanes (m/z177, dotted trace), where examples are indicated by arrows. Demethylation slightly favors the second-eluting tricyclic terpane peak, especially for the C26, C28, and C29homologs (e.g., Alberdi et al., 2000). Samples in Figs. 1 and 2 were treated with
molecular sieves to remove n-parans. C22DM TC=C22 13b,14a(H)-17-nor-tricyclic terpane, C27
DM=l7a,21b(H)-25,28,30-tri-norhopane, C28DM=17a,21b(H)-25,30-dinorhopane (inferred from retention times ofm/z177 peaks compared to probable precursor
2. Chromatographic elution order for the 22S and 22R epimers is unknown and plots of homolog number or mass versus log retention time are non-linear (Fig. 3).
3. Heavy biodegradation of crude oils in some reser-voirs favors C-10 demethylation of the second-elut-ing epimer in each doublet to form the correspondsecond-elut-ing 17-nor-tricyclic terpane (e.g., Alberdi et al., 2000). 4. Routine gas chromatography±mass spectrometry
(GC±MS) resolves only two peaks for the C30and
C31tricyclic terpanes, whereas asymmetric centers
at C-22 and C-27 allow four peaks for each homolog (22S27S, 22S27R, 22R27S, 22R27R).
2. Methods
Molecular mechanics calculations were completed on structural models of the C21 to C31 tricyclic terpanes
using the MM+ force ®eld and default options (bond dipoles, no cutos) in HyperChem 5.1/ChemPlus 2.01
(Professional Version, Molecular Visualization and Simulation Program Package, Hypercube Inc., Gainesville, Florida, 1998). Stereochemistry was assumed to be 5(H),
8b(CH3), 9b(H), 10b(CH3), l3b(H), l4b(H) (Aquino Neto
et al., 1982, Chicarelli et al., 1988). Geometry optimization was completed using the Polak-Ribiere conjugate gra-dient in vacuo with a termination root mean square (RMS) gradient of 0.01 kcal/AÊmol.
The conformational search option in HyperChem was applied to each geometry-optimized structure to improve the optimized energy (likely local minimum) and approach the global energy minimum. Calculations on large molecules, such as hopanes (Peters et al., 1996), diahopanes (Dasgupta et al., 1995) and tricyclic ter-panes, require long computing times. Conformational search was completed in usage directed mode (Monte Carlo multiple minimum approach; Chang et al., 1989) by varying the torsion angles for key dihedral bonds in the side chain of each compound, leaving the ring sys-tem in the conformation established by the stereo-chemistry and original geometry optimization. Dihedral angles in the side chain were varied about C-13-21 for the C21 to C24tricyclic terpanes, C-13-21 and C-21-25
for the C25to C28tricyclic terpanes, and C-13-21,
C-2l-25, and C-25-29 for the C29to C31tricyclic terpanes. In
some cases for epimers A and B, the local energy mini-mum for epimer A was further reduced by inverting the stereochemistry of the key asymmetric carbon atom in the geometry-optimized conformation of epimer B and repeating the conformational search.
Quantitative structure±activity relationships (QSAR) were calculated using the minimum-energy structures determined by conformational search in HyperChem. QSAR is widely used to predict the physicochemical behavior of compounds, including molecular volume and surface area (e.g. Famini and Wilson, 1996 and references therein). For example, solvent-accessible or Van der Waals grid surface areas for geometry-opti-mized tricyclic terpanes were computed in ChemPlus 2.0 using the method of Bodor et al. (1989) and atomic radii of Gavezotti (1983). The solvent probe radius was 1.4 AÊ. The method to determine grid surface area requires more computation time, but is more accurate than other methods for a given set of atomic radii (Hasel et al., 1988; Still et al., 1990).
Conformations of the tricyclic terpanes were also determined by COMPASS force-®eld calculations using Cerius21 software (Molecular Simulations Inc., San
Diego, California, 1999). COMPASS (Condensed-phase Optimized Molecular Potentials for Atomistic Simulation
Fig 2. Mass chromatogram (m/z191, solid trace) shows C19±
C30tricyclic terpanes in the saturated hydrocarbon fraction of
nonbiodegraded (top) and heavily biodegraded (bottom) crude oils from Jusepin Field, Well 110 and Quiriquire Field, Well 449 (unknown depths), Maturin Basin, Venezuela. Relative peak-heights show that biodegradation favors the second-elut-ing peak for the C28, C29, and C30tricyclic terpanes (top versus
bottom) without formation of 17-nor-tricyclic terpanes (DM,
m/z 177, dotted trace). Although four C30 tricyclic terpane
Studies) is a force ®eld calibrated empirically from ab initio data to yield structural, conformational, vibra-tional, and thermophysical properties in good agree-ment with experiagree-mental data (Sun, 1998). As a further test, the lowest energy C28 and C2922S and 22R
con-formers were optimized by the rigorous semi-empirical AM1 method (Dewar et al., 1985) in Cerius2to con®rm
the molecular mechanics conformational results. Saturated hydrocarbon fractions from crude oils were analyzed for tricyclic terpanes using multiple ion detec-tion-gas chromatography±mass spectrometry (MID-GC±MS). The Hewlett Packard (HP) 5890 Series H gas chromatograph was equipped with a 50 m HP Ultra 2 crosslinked 5% phenylmethylsilicone column (0.2 mm I.D., 0.25mm ®lm thickness) coupled to a HP 5970 mass spectrometer with helium as carrier gas at 40 psi. After injection at 270C, volatile compounds were refocused
at 50C for 4 min. The oven temperature was increased
to 150C at 4C/min, held for 5 min, and then increased
to 325C at 2C/min and held for 5 min. The mass
spectrometer was run in electron impact mode (70 eV) and data were acquired using dwell times of 100 ms/
ion for processing on the HP ChemStation.
For the 6 h slow-programmed high-resolution experi-ment, a HP 5890 Series II gas chromatograph was cou-pled to a Finnigan TSQ-70 mass spectrometer used in MID mode.
The 60 m HP-5 Ultra column (0.2 mm I.D., 0.1mm ®lm thickness) was heated to 50C for 4 min and then to
325C at 0.1C/min with split mode injection and 50 psi
helium carrier gas pressure. The injector and transfer line temperatures were 270C.
3. Results and discussion
Calculated total energies using the MM+ force ®eld decrease with carbon number for geometry-optimized conformations of the C25to C28tricyclic terpanes, but
increase for the C29homolog (Fig. 4, Table 1). The 22S
and 22R epimers show similar energies, indicating similar thermal stability throughout the range from C25to C29.
For simplicity, Figs. 4 and 5 include data for only the C25
to C29tricyclic terpanes, although complete data for the
range C21±C31are in Table 1. The C24tricyclic terpane
lacks an asymmetric center at C-22 and thus shows no 22S or 22R epimers, while the C30and C31tricyclic
ter-panes have asymmetric centers at both C-22 and C-27, resulting in four possible epimers (Fig. 1, inset).
The calculated energies explain the similar size of the ®rst- and second-eluting chromatographic peaks for each tricyclic terpane doublet in the range C25±C29 in
saturate fractions of oils and source-rock extracts (top Fig. 2, Table 1). Although the C25±C2822S tricyclic
ter-panes show slightly higher energies than the corre-sponding 22R epimers (Fig. 4), these energy dierences are less than those for the 22S and 22R epimers of the extended 17a,2lb(H)-hopanes. The 22S and 22R epimer energies dier by less than 0.3 kcal/mol for C25±C29
tri-cyclic terpanes (Table 1), but range from 0.5 to 2.4 kcal/
Fig. 3. Molecular mass versus log of chromatographic retention time for tricyclic terpanes in a nonbiodegraded West African oil (30.5
API) shows at least two distinct breaks in the linear trend at C24and C29. The C24and C29tricyclic terpanes contain terminal isopropyl
groups where addition of a methyl group to form the next homolog activates asymmetric carbon atoms at C-22 and C-27, respectively. Data points for the 22S and 22R diastereomers of the C25±C28tricyclic terpanes plot in nearly identical locations due to the log retention
time scale. The HP-5 Ultra chromatographic column was slow-programmed to 325C at 0.1C/min as described in methods (e.g. Fig. 7).
mol for C31±C35hopanes (Peters et al., 1996). The larger
energy dierences for the hopane epimers result in measured 22S/22R ratios near 1.2 to 1.5 in non-biodegraded petroleum (Peters et al., 1996), while the tricyclic terpane epimers generally show 22S/22R ratios near 1.0 (e.g., Fig. 2, top). Most chromatographic separations of C25 tricyclic terpanes into 22S and 22R
doublets are incomplete and C27 tricyclic terpanes are
low in crude oils or source-rock extracts (Figs. 1 and 2), as discussed above.
Using a modi®ed Gibbs free energy equation and assumptions described in Peters et al. (1996), the theo-retical 22S/22R ratios for the C25±C29tricyclic terpanes
in mature nonbiodegraded petroleum are 1.0, 1.0, 1.0, 0.9, and 0.8, respectively. Nearly all oil samples have measured tricyclic terpane 22S/22R ratios near 1.0 when the saturate fraction is treated with molecular sieves to removen-parans prior to analysis (Fig. 2, top). Excep-tions include certain heavily biodegraded oils (Fig. 2, bottom), highly mature oils where tricyclic terpanes are low compared to contaminant peaks (e.g., Fig. 4 in Sei-fert and Moldowan, 1981), or where they were con-centrated and possibly fractionated using an aluminum oxide column (Fig. 1 in Moldowan et al., 1983).
The isoprene rule for the C29tricyclic terpane requires
a terminal isopropyl group at C-26, which breaks the conformational trend established by the lower homologs from C25to C28(Fig. 1, inset). For this reason, the
geo-metry-optimized shapes of the C29and higher tricyclic
terpanes dier from the lower homologs (Fig. 6) and they show dierent optimized dihedral angles, calculated
energies, volumes, and surface areas than might be expected from the data for the lower homologs (Figs. 4 and 5, Table 1).
Chromatographic retention time increases dis-continuously with mass for the tricyclic terpanes (Table 2, Fig. 3). For example, because the C25±C28 tricyclic
terpanes show a similar conformational style (Fig. 6) with an asymmetric center at C-22, log retention time versus mass is highly linear (R2=0.997). However, this
linear trend is broken at C24and C29, where the terminal
carbons consist of an isopropyl group as discussed above (Fig. 3, insets). One additional carbon attached to these isopropyl groups activates either C-22 or C-27 as an asymmetric center for C25and C29tricyclic terpanes,
respectively, which initiates a new conformational style for higher homologs. For example, the transition from C28to C29tricyclic terpanes results in a more compact
molecular shape. For geometry-optimized C28 tricyclic
terpanes, the side-chain is directed away from the ring system, while the side-chain for the C29tricyclic terpanes
curves back toward the ring system in a boomerang shape (Fig. 6). The boomerang shape allows greater mass without a corresponding increase in retention time (dierence between solid and dashed line above C28 in
Fig. 3).
The abrupt change in conformational style between the C28 and the C29 tricyclic terpanes is surprising
because the side-chain simply increases from 9 to 10 carbons (Fig. 6). To test whether this remarkable con-formational shift as determined by molecular mechanics (MM+) is reproducible, more rigorous calculations
Fig. 4. Relative energies of geometry-optimized C25±C2922S and 22R tricyclic terpanes from molecular mechanics force-®eld
calcu-lations suggest that each 22S shows similar thermal stability to the corresponding 22R epimer. Inset shows abbreviated structure of the C29tricyclic terpane, with an asymmetric carbon at C-22, but not at C-27. C-27 becomes asymmetric for the C30tricyclic terpane (Fig.
were completed using the COMPASS force ®eld and Cerius2 software (see methods). The conformational
shapes for the C24±C29 tricyclic terpanes determined
using the MM+ and COMPASS force ®elds are essen-tially the same, including the abrupt conformational change from C28to C29(Fig. 6). Table 3 compares the
global-energy minima for the C28and C2922S tricyclic
terpanes with the energies of the nine nearest local-energy minimum conformations determined using the COMPASS force ®eld. Dierences in absolute energies of conformers calculated by dierent methods (Tables 1 and 3) are common and result from the methods of cal-culation. The critical values for interpretation are the relative dierences in energies between conformations calculated by the same method. Table 3 shows that the dierences in energy between the global energy mini-mum and the next higher energy conformation are about 0.04 and 1.34 kcal/mol for the C28and C2922S
tricyclic terpanes, respectively. The small energy dier-ence for the two lowest energy C28 22S conformers is
consistent with a ¯exible side chain directed away from the ring system. The energy dierence between the two
lowest energy C29 22S conformers is signi®cant and
appears to be caused by increased intramolecular Van der Waals interaction between the side chain and ring system for the lowest energy conformer. This conclusion is based on calculated intramolecular hydrogen±hydro-gen distances in the range of 2.2±2.9 AÊ, which are similar to intermolecular hydrogen±hydrogen distances among hydrocarbons.
The lowest energy C28S and C29S conformers were also
optimized using the semi-empirical (AM1) method, resulting in the same stable conformations. Semi-empiri-cal Semi-empiri-calculations account for electronic interactions and yield a more rigorous solution to conformation than molecular mechanics. The calculated total energies for 22S and 22R epimers were similar for each tricyclic ter-pane homolog, supporting the results from the MM+ force-®eld calculations.
The stereochemistry of the C30±C32tricyclic terpanes
is more complex than lower homologs due to an asym-metric center at C-27, which results in 22S27S, 22R27R, 22S27R, and 22R22S con®gurations (Table 1). How-ever, petroleum with high tricyclic terpanes commonly
Table 1
Properties of geometry-optimized tricyclic terpanes from molecular mechanics and QSAR
Energyb Volumec Aread Torsion Angle ()
Carbona C-22a C-27a Mass (kcal/mol) (AÊ3) (AÊ2) 13±21 21±25 25
ÿ29
21 m m 290.53 ÿ49.35 939.47 516.06 ÿ98.93 M M
22 m m 304.56 ÿ49.81 991.97 544.06 ÿ99.64 M M
23 m m 318.59 ÿ50.36 1045.98 577.47 ÿ99.72 M M
24 m m 332.61 ÿ51.56 1086.41 594.73 ÿ99.64 M M
30 S S 416.77 ÿ56.09 1332.09 677.63 ÿ100.09 52.97 ÿ65.26
30 R R 416.77 ÿ56.59 1334.19 685.12 ÿ101.14 53.14 ÿ65.50
30 S R 416.77 ÿ56.36 1331.28 680.40 ÿ100.67 53.81 ÿ65.20
31 R S 430.80 ÿ57.64 1360.36 692.09 51.13 ÿ53.90 64.74
31 S S 430.80 ÿ57.70 1363.32 693.00 50.67 ÿ52.93 65.46
31 R R 430.80 ÿ57.47 1365.72 696.68 51.18 ÿ53.29 65.71
31 S R 430.80 ÿ57.52 1367.11 695.93 51.07 ÿ52.77 66.70
a Tricyclic terpane carbon number is followed by stereochemistry at C-22 and C-27, e.g. 31SR=C
3113b, 14(H)-tricyclic terpane
22S27R; M=missing.
b Starting structures based on molecular mechanics energy minimization were subjected to a Monte Carlo conformational-search
routine in torsional coordinates to determine total energy as described in the text.
c Molecular volume and grid surface area determined using quantitative structure±activity relationships (QSAR).
d Only two peaks each are resolved for the C
shows only two of the four expected chromatographic peaks for each of the C30and C31tricyclic terpanes (e.g.
C30 in Fig. 1). These peaks are small and dicult to
identify on the m/z191 mass chromatograms of many petroleum samples. As discussed above, the C32tricyclic
terpanes are low in all samples because they require cleavage of two carbon±carbon bonds to form from higher homologs.
Molecular modeling shows that the four epimers for each of the C30 tricyclic terpanes have similar energies
(Table 1) and are thus likely to occur in similar abun-dance with no relatively high-energy, unfavorable con-®gurations. This suggests that co-elution rather than stability dierences accounts for two rather than four
Fig. 5. Molecular volumes and surface areas of geometry-opti-mized tricyclic terpanes from quantitative structure±activity relationships (QSAR, bottom) increase from C25±C28, but the
trend is broken at C29, consistent with a change in
conforma-tional style (Fig. 6). The 22R epimers for C25±C29homologs
show greater volumes and surface areas than the corresponding 22S epimers. Biodegradation of hopanes by C-10 demethyla-tion favors epimers with greater surface areas (arrows at top, Peters et al., 1996). The 22S hopane epimers elute prior to the corresponding 22R epimers (Armanios, 1995). Assuming that biodegradation also favors tricyclic terpane epimers with greater surface areas, the C26and C2922R epimers will be
pre-ferentially biodegraded (bottom). Because the second-eluting peak in each the C26±C29tricyclic terpane doublet undergoes
preferential biodegradation (Figs. 1 and 2), it is inferred be the 22R epimer.
Fig. 6. The C25±C28tricyclic terpanes show similar
conforma-tional styles based on molecular mechanics MM+ force-®eld calculations (only the C25 and C28 22S and 22R
geometry-optimized conformations are shown). An abrupt change in confomational style occurs from the C28±C29 and higher
tri-cyclic terpanes, resulting in a more compact molecular shape. These MM+ force-®eld conformations are the same as those determined by COMPASS force-®eld and semi-empirical (AM1) calculations as discussed in the text.
Table 2
Masses and chromatographic retention times of tricyclic ter-pane homologs in a nonbiodegraded oil from West Africa
Mass RT(1)a RT(2)a
Carbon (amu) (min) logRT(1) (min) LogRT(2)
21 290.53 53.23 1.726 230.75 2.363
22 304.56 56.99 1.756 244.60 2.388
23 318.59 61.33 1.788 261.92 2.418
24 332.61 63.60 1.803 271.12 2.433
25 346.64 68.16 1.834 289.42 2.462
25 346.64 68.28 1.834 289.54 2.462
26 360.67 71.57 1.855 302.66 2.481
26 360.67 71.83 1.856 303.63 2.482
27 374.69 75.64 1.879 317.88 2.502
27 374.69 75.90 1.880 318.85 2.504
28 388.72 79.00 1.898 332.07 2.521
28 388.72 79.49 1.900 333.93 2.524
29 402.75 81.07 1.909 340.22 2.532
29 402.75 81.65 1.912 342.57 2.535
30 416.77 85.03 1.933 355.74 2.551
30 416.77 85.70 1.930 358.36 2.554
31 430.80 87.97 1.944 367.37 2.565
31 430.80 88.70 1.948 367.48 2.565
31 430.80 88.85 1.949 370.31 2.569
31 430.80 89.49 1.952 370.31 2.569
a RT(1)=retention time using 50-m HP Ultra 2 column and
2C/min heating rate, RT(2) = retention time using 60-m HP-5
chromatographic peaks, where each peak represents two epimers. Slow-ramp temperature-programmed chroma-tography supports these modeling results. A 6 h high-resolution chromatography experiment failed to resolve the two C30tricyclic terpane peaks. However, the ®rst of
the two C31 tricyclic terpane peaks to elute shows
broadening, reduced peak height, and initial separation into two poorly resolved components (Fig. 7). Routine
GC±MS on the same sample yields two C30 tricyclic
terpane peaks of approximately equal size. Chiral-phase chromatography (Schurig, 1994; Tang et al., 1994) using a 10 m CP Chirasil-Dex CB fused silica column did not improve resolution of the C30and C31tricyclic terpanes
because the bonded phase became unstable at the required temperatures.
Geometry-optimized C25±C28 tricyclic terpane
epi-mers show systematic increases in molecular volume and surface area based on QSAR (Fig. 5, bottom). However, the C29 epimers do not conform to this trend. For
example, calculated surface areas for the C29epimers are
less than the corresponding C28 epimers due to the
change in conformational style discussed above (Fig. 6). Despite having identical masses, each 22R tricyclic ter-pane epimer has greater surface area than 22S in the range from C25to C29.
QSAR properties are widely used to predict the physi-cochemical behavior of compounds. For example, chro-matographic elution times for dierent compounds can be described using various QSAR parameters, including molecular volume, polarizability, covalent basicity or acidity, and electrostatic basicity or acidity (Famini and Wilson, 1996 and references therein). However, the author is not aware of publications that use QSAR to predict elution times for epimers. Correlation of abso-lute con®guration with elution order among epimers in
Fig. 7. Slow-programmed high-resolution chromatography of a nonbiodegraded West African oil (same as in Fig. 3) resolves two peaks for C30, but suggests at least three peaks for C31tricyclic terpanes. The ¯rst-eluting C31peak is broader and shorter than that from routine
GC±MS (where it is slightly taller than the second peak) and initial separation of two co-eluting peaks is evident. Tricyclic terpanes were enriched by high-performance liquid chromatography to minimize hopanes. Geometry-optimized models show that the four epimers for each of the C30and C31tricyclic terpane epimers have similar energies, suggesting similar stability and abundance (Table 1).
Table 3
COMPASS force-®eld conformer search results for ten lowest energy conformations of the C28and C2922S tricyclic terpanes
C2822S C2922S
energy energy
Conformer (kcal/mol) (kcal/mol)
1 ÿ67.21 ÿ73.18
2 ÿ67.17 ÿ71.84
3 ÿ67.11 ÿ71.16
4 ÿ66.75 ÿ69.50
5 ÿ66.28 ÿ67.41
6 ÿ65.05 ÿ67.41
7 ÿ64.92 ÿ67.33
8 ÿ64.59 ÿ67.33
9 ÿ64.59 ÿ67.25
homologous series of compounds is problematic because many phenomena can aect their separation, including hydrogen bonding, dipole-dipole interactions, and other forces (Schurig, 1994). Unequivocal proof of absolute con®guration requires co-injection of authentic standards with known stereochemistry.
The extended hopanes may serve as analogs to predict the elution order of the tricyclic terpane epimers. Like the tricyclic terpanes, the C31±C35hopanes are characterized
by 22S and 22R epimers (Fig. 5, top). However, unlike the tricyclic terpanes where elution order of the epimers is unknown, the 22S epimer for each hopane homolog elutes prior to 22R, as con®rmed by co-elution experi-ments (Armanios, 1995). Hopane homologs of increasing mass, volume, and surface area elute later during gas chromatography. However, elution orders of hopane epimers do not depend directly on mass (epimers have the same mass), volume, or area, but rather on shape. The hopane 22S epimers show a ``scorpion'' conforma-tion and elute prior to their corresponding 22R epimers, which show a ``rail'' conformation (Peters et al., 1996).
Data for the extended hopanes suggest that epimers with larger volumes and surface areas are more suscep-tible to microbial attack. For each 22S and 22R hopane doublet in the range C31±C35, the epimer with greater
volume and surface area is preferentially demethylated at C-l0 to form 25-norhopanes during biodegradation (Fig. 5, top, Peters et al., 1996). The volumes and sur-face areas of the 22S hopanes exceed the 22R epimers for the C31and C32homologs, but are less than the C34
and C3522R epimers. The 22S epimers of the C31and
C32hopanes show preferential conversion to
25-norho-panes compared to 22R, while the opposite applies to the C34and C35epimers. The C33hopane 22S and 22R
epimers show similar susceptibilities to biodegradation. A study of Venezuelan crude oils shows that the sec-ond-eluting tricyclic terpane peak for each homolog in the range C26±C29undergoes preferential demethylation
at C-10 during biodegradation in some reservoirs (Alberdi et al., 2000). The microbial demethylation of tricyclic terpanes at C-10 to form 17-nor-tricyclic ter-panes is analogous to C-10 demethylation of hoter-panes to form 25-norhopanes in petroleum reservoirs (Peters et al., 1996). Fig. 2 shows that the second-eluting tricyclic terpane peak is also preferentially attacked in heavily biodegraded oils that lack C-10 demethylation.
The QSAR results show that the C25±C29 22R
tri-cyclic terpanes have larger surface areas and similar or larger volumes than the corresponding 22S epimers. If the analogy with the hopanes is correct, the 22R tri-cyclic terpane epimers are predicted to be preferentially biodegraded compared to the corresponding 22S epi-mers. Because GC±MS shows that the second-eluting peak for these compounds is preferentially biodegraded (Figs. 1 and 2), it is inferred that 22S elutes prior to 22R for the C25±C29tricyclic terpanes.
4. Conclusions
Molecular mechanics and observations of tricyclic terpanes in petroleum using routine and slow-pro-grammed, high-resolution chromatography are used to conclude the following:
1. 22S/22R epimer ratios for the C25±C29 tricyclic
terpanes approach 1.0 for nonbiodegraded petro-leum because total energies of the favored con-formations are similar (Fig. 4).
2. The second-eluting peak for each of the C26±C29
tricyclic terpanes is more readily biodegraded, with or without microbial demethylation to form l7-nor-tricyclic terpanes (Figs. 1 and 2).
3. A change in the preferred conformational style occurs between geometry-optimized C28 and C29
tricyclic terpanes due to the branch point at C-27. This change in conformational style results in dis-continuous linear trends on plots of tricyclic terpane mass versus log retention time (Fig. 3).
4. All four possible epimers for each of the C30and
C31 tricyclic terpanes show optimized
conforma-tions with similar energies, suggesting that they occur in similar abundance, but only two peaks are observed for each by routine GC±MS. A 6 hour high-resolution chromatography experiment indicates that at least one of the two peaks in the C31 doublet consists of two co-eluting epimers
(Fig. 7). Improved chromatographic methods will be required to separate these peaks.
5. 22S epimers are inferred to elute prior to 22R for C25±C29tricyclic terpanes. This prediction is based
on (1) the observation that the second-eluting peak in each tricyclic terpane doublet undergoes pre-ferential biodegradation and (2) the assumption that epimers with larger exposed surface areas are more readily biodegraded, as supported by analogy with the 17(H)-hopanes (Fig. 5).
It is hoped that this study will stimulate further research on tricyclic terpanes, especially in regard to items 4 and 5 above. Proof of the chromatographic elu-tion order for tricyclic terpane 22S and 22R epimers will require co-elution experiments using synthesized authentic standards. However, the predicted elution order for tricyclic terpane epimers is consistent with data for the hopanes, which contain an analogous asymmetric center at C-22.
Acknowledgements
publish this work. John Zumberge (Geomark Research, Inc.) kindly provided the chromatograms for Figs. 1 and 2. Robert Carlson, Francisco de Aquino Neto, Kirk Schmitt, and Simon George provided useful review com-ments. Special thanks are due Yitian Xiao and John Longo (ExxonMobil Upstream Research Company) for assistance with the COMPASS force-®eld and semi-empirical AM1 calculations and related discussions.
Associate EditorÐS. George
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