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Isolation of individual hydrocarbons from the unresolved complex hydrocarbon mixture of a biodegraded crude oil using preparative capillary gas chromatography
Organic Geochemistry Organic Geochemistry 36 (2005) 963–970 www.elsevier.com/locate/orggeochem
Isolation of individual hydrocarbons from the unresolved complex hydrocarbon mixture of a biodegraded crude oil using preparative capillary gas chromatography P.A. Sutton, C.A. Lewis *, S.J. Rowland Petroleum and Environmental Geochemistry Group, School of Earth, Ocean and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK Received 28 June 2004; accepted 22 November 2004 (returned to author for revision 27 September 2004) Available online 31 March 2005
Abstract We describe the isolation, using preparative capillary gas chromatography, of hydrocarbon fractions from an unresolved complex mixture (UCM) of hydrocarbons isolated from a biodegraded crude oil. Some of the individual hydrocarbons in these fractions were then resolved by gas chromatography (GC) and identified using GC–mass spectrometry (GC–MS). The mass spectra contained distinct molecular ions. As a preliminary example, after preparative open column chromatography, preparative HPLC (three columns in series) and preparative GC on two stationary phases, one of the compounds from the UCM was tentatively identified using electron impact ionisation GC–MS as a novel C26 17desmethyl triaromatic steroid. If the concentration of this compound (ca. 4 lg g1 whole oil) is typical of other individual compounds in the UCM, the proportion of UCM represented by each compound is obviously very small (<0.0004%) and suggests that the whole oil UCM contains in the order of 250,000 mostly unidentified compounds. This suggests that oil UCMs are amongst the most complex mixtures of organic compounds on Earth and explains the difficulty of UCM component identification. Nevertheless, it also indicates that multi-step chromatography can lead to the identification of individual compounds, some of which have geochemical, petrochemical and environmental significance. Acquisition of such knowledge is fundamental if we are to improve our understanding of petroleum. 2005 Elsevier Ltd. All rights reserved.
1. Introduction Despite the reliance of the economies and lifestyles of the industrialised world on crude oil, the chemical
* Corresponding author. Tel.: +44 1752 232988; fax: +44 1752 233035. E-mail address: [email protected] (C.A. Lewis).
composition of quite a large proportion of liquid petroleum remains a mystery (Altgelt and Boduszynski, 1993; Rowland and Revill, 1995). Although many of the hydrocarbons can be made volatile enough for gas chromatography (GC) analysis, relatively few are separable into individual, identifiable compounds using routine one-dimensional gas chromatography. Rather, most compounds appear as unresolved complex mixtures (UCMs) or GC ÔhumpsÕ (e.g. Gough and
0146-6380/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2004.11.007
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Rowland, 1990). These UCMs are particularly obvious when biodegraded oils are examined using GC. A lack of detailed knowledge of UCM composition is a hindrance to the development of theories of the origin of petroleum. It is also a restriction on the prediction of the effects of refinery processing of crude oils and may be an important limitation to the assessment of the effects of unresolved oil residues in the environment (e.g. Rowland et al., 2001; Reddy et al., 2002; Donkin et al., 2003). GC–MS techniques have been routinely used to distinguish the presence of particular compounds within complex petroleum mixtures but use of GC–MS is usually confined to detection of those compounds which are resolved and have characteristic mass spectra (e.g. reviewed by Peters and Moldowan, 1993). Such methods leave most of the UCM hydrocarbons unidentified. However, a variety of techniques has been applied to try to identify some of the unresolved components within oils, including, for example, studies of oxidised UCMs (Gough and Rowland, 1990; Killops and Al-Juboori, 1990; Revill, 1992; Warton, 1999; Warton et al., 1999), isolation of individual or groups of compounds following treatment of oil UCMs with molecular seive (e.g.
Armanios et al., 1994; Ellis et al., 1994; Fazeelat et al., 1994), statistical deconvolution methods (Pool et al., 1997; Dagan, 2000; Demir et al., 2000), and multidimensional GC (Blomberg et al., 1997; Phillips and Beens, 1999; van Deursen et al., 2000; Reddy et al., 2002; Frysinger et al., 2003). These have led to proposed structures for some of the non-aromatic (Gough and Rowland, 1990; Warton et al., 1997) and aromatic (Gough and Rowland, 1990; Warton et al., 1999) hydrocarbons. Synthetic compounds, which exhibit the properties of some UCM components, have also been made. Thus, Gough et al. (1992) showed that simple ÔTÕ branched alkanes were relatively resistant to biodegradation and were oxidised to compounds similar to those obtained from oxidation of some UCMs. Later, Warton et al. (1997) showed that 3% of the alkanes from a crude oil were ÔTÕ branched. Similarly, Thomas (1995) suggested that some aromatic UCM components were substituted alkyl tetralins on the basis of oxidation stud-
(a)
97 % UCM (whole oil)
Whole oil
(b)
Open column chromatography (non-aromatic, aromatic-1, aromatic-2, polar, residual)
RIC
97 % UCM (non-aromatic)
Normal phase (HPLC) (cut each 1 min)
(c) 92 % UCM (aromatic-1)
Preparative (GC) (apolar phase) (ca 10 s cuts)
81 % UCM (aromatic-2)
(d)
Preparative (GC) (polar phase) (cut peaks)
5
15
25
35
45
55
retention time (min)
GC – MS Fig. 1. Flow diagram of sequential preparative chromatographic approach to resolving components from the UCM of a biodegraded oil.
Fig. 2. Total ion chromatograms of Tia Juana Pesado, Venezuela, oil (a) crude oil (b) non-aromatic fraction (c) aromatic-1 fraction (d) aromatic-2 fraction. GC–MS conditions: Agilent Ultra-1 column (12.5 m · 0.20 mm id · 0.33 lm) programmed at 40–300 C at 5 C min1, hold 10 min; injector 250 C; detector 280 C.
P.A. Sutton et al. / Organic Geochemistry 36 (2005) 963–970
ies followed by ion cyclotron resonance MS and recently Booth (2004) showed that some dialkyl tetralins were indeed as resistant to biotransformation as some components of an aromatic UCM. The same compounds were toxic to mussels (Smith et al., 2001), emphasising the environmental significance of these persistent bioaccumulative residues. The reported toxicity of aromatic UCMs to marine organisms (Smith et al., 2001) suggests that separation of individual components from aromatic UCMs warrants further study. The recent coupling of GC · GC to time of flight mass spectrometers (e.g. Dallu¨ge et al., 2002; Hamilton and Lewis, 2003) may provide more routine methods of analysis of UCMs, but there is still a need for complementary methods that provide information about individual UCM components. In the present study, we have therefore used a combination of preparative chromatographic techniques, including open column chromatography, normal phase high performance liquid chromatography (HPLC) and preparative capillary GC on two stationary phases to isolate individual and groups of aromatic hydrocarbons from the UCM recovered from a biodegraded crude oil. The components of the fractions become progressively more resolved with each chromatography step. Although these methods are probably too lengthy and time-consuming to be considered routine, their application to a few UCMs nonetheless has allowed the first identification of compounds which were insufficiently abundant and present in mixtures too complex to be resolved previously. Furthermore, the assumption that the amounts of compounds
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in the UCM are about equal allowed a very approximate estimate of the number of compounds present in a UCM to be made. We illustrate the approach by the isolation of some known triaromatic steroids and the identification by MS of a novel isomer, but the method is also applicable to identification of many other UCM components.
2. Experimental A flow diagram of the isolation procedures is shown in Fig. 1. 2.1. Solvents and chemicals Solvents were obtained from Rathburn Chemicals Ltd., Scotland and used as received after checking the purity of a concentrated aliquot (·100) with GC. The crude oil was a heavily biodegraded crude from Tia Juana Pesado, Venezuela. 2.2. Open column chromatography Crude oil (8 · ca. 0.7 g) in hexane was rotary evaporated to near dryness in the presence of Al2O3 (BDH, England; grade 1, neutral, 150 mesh) and was loaded on to a silica/alumina column [0.6 g cm3 SiO2 (Sigma–Aldrich Co. Ltd.; grade 645, 60–100 mesh), 5% H2O deactivated; Al2O3, 1.5% H2O deactivated; 50%, w/w, Al2O3:SiO2] and the column eluted with 170 ml
5 10DBE
7DBE 6DBE
4
Mass (mg)
5DBE
3
4 DBE
UCM UV absorbance (254 nm)
2
1
18 20 22 24 26
0
Retention time (min)
Fig. 3. HPLC UV chromatogram (254 nm) of Tia Juana Pesado, Venezuela, aromatic-1 fraction and gravimetric determination of HPLC 1-min cuts. Maximum retention times of the most condensed forms of a series of reference compounds (4 double bond equivalents, DBEs, benzene; 5 DBEs, tetralin; 6 DBEs, indene; 7 DBEs, naphthalene; 10 DBEs, anthracene) are indicated and the rerun of the 22–23 min cut shown inset. HPLC conditions: 3 · 25 cm · 1 cm · 8 lm HSAPS-2 columns in series; 2 ml min1 flow rate; 0– 40 min 100% hexane, 40–45 min gradient to 100% DCM, 65–70 min gradient to 100% hexane; UV detection at 254 nm.
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of hexane (HPLC grade), hexane/toluene (glass distilled grade; 3:1, v/v), hexane/toluene (1:1, v/v) and dichloromethane (DCM, HPLC grade), to yield non-aromatic, aromatic-1, aromatic-2 and polar fractions, respectively. The fractions were rotary evaporated to dryness, weighed and examined by GC–MS. The proportion of residue remaining on the column was determined by difference.
250 mm · 10 mm · 8 lm columns in series with a guard column; flow rate 2 ml min1; 0–40 min (100% hexane), 40–45 min (gradient to 100% DCM), 65–70 min (gradient to 100% hexane), with a Dionex AD20 UV absorbance detector at 254/280 nm]. Collected fractions were blown down to dryness (N2) and weighed before re-dissolving in hexane to a known concentration for analysis using GC–MS and preparative GC (PGC).
2.3. HPLC 2.4. Preparative GC The aromatic-1 fraction (10 · 800 ll, 20 mg ml1 in hexane) was further fractionated (1 min cuts from 10 to 74 min) using normal phase HPLC [Dionex GP40 gradient pump (Dionex Corp., USA) with 3 · Hypersil Hyperprep HSAPS-2 (ThermoHypersil-Keystone)
(a)
21-22 min HPLC fraction
PGC analysis was carried out using an Agilent 6890 Series gas chromatograph (Agilent Technologies, Inc., USA) fitted with an Agilent 7683 Series injector and autosampler, and a Gerstel Preparative Fraction Collector (Gerstel GmbH & Co. KG, Germany). FID response was recorded using Turbochrom Navigator V6.1 software (Perkin–Elmer Corp., USA). Initial PGC was performed using a BP-5 column (15 m · 0.53 mm · 1.0 lm) and secondary PGC using a SolGel-Wax column (30 m · 0.25 mm · 0.25 lm), both from SGE Europe Ltd., UK. PGC conditions: injector 260 C; FID 300 C; transfer line/switching device 280 C; GC programme 40–280 C at 10 C min1, 280–300 C at 5 C min1 (BP-5) or 40–300 C at 10 C min1 (SolGel WaxTM), hold 10 min; traps at ambient. Fraction masses were estimated by relating integrated cut areas to mass injected.
38-39 min HPLC fraction
2.5. GC–MS
98 % UCM
RIC
(b)
GC–MS was performed using a HP5890 Series II gas chromatograph (Hewlett–Packard, USA) fitted with a
92 % UCM 2
3 A
(c)
Table 1 Retention times (RT)a and double bond equivalents (DBEs) for a range of reference compounds and model aromatic UCM hydrocarbons
m /z 217
CD B
4
m /z 231 m /z 245
1 54-55 min HPLC fraction
81 % UCM 5
10
15
20
25
30
35
retention time (min)
Fig. 4. Typical total ion chromatograms of 1-min HPLC cuts from Tia Juana Pesado, Venezuela, aromatic-1 fraction (a) 21–22, (b) 38–39, (c) 54–55 min (1 = C0–C2 fluorenes; 2 = methyl-dibenzothiophenes; 3 = dimethylnaphthothiophenes; 4 = trimethylnaphthothiophenes); compound A, m/z 217, tentatively identified as C17 demethylated C26 triaromatic steroid (TAS); compounds B–D, m/z 231 = methyl C26–C28 TAS; m/z 245 = dimethyl TAS. GC–MS conditions: Agilent Ultra-1 column (12.5 m · 0.20 mm i.d. · 0.33 lm) programmed from 40 to 300 C at 10 C min1, hold 10 min; injector 250 C; detector 280 C.
Compound
DBEs
RT (min)
1-Phenyldecane 1-(3 0 -Methylbutyl)-7-cyclohexyltetralin Benzene 1-n-Nonylcyclohexyltetralin 1-Methyl-7-cyclohexyltetralin Tetralin 6-Cyclohexyltetralin Indene 1-n-Nonylcyclohexylnaphthalene 4-Pentylbiphenyl Biphenyl Naphthalene Phenanthrene Anthracene
4 6 4 6 6 5 6 6 8 8 8 7 10 10
26.05 27.07 27.96 28.23 29.51 29.86 30.02 36.73 37.26 39.39 41.72 42.50 64.15 67.33
a HPLC conditions: 3 · 25 cm · 1 cm · 8 lm HSAPS-2 columns in series; 2 ml min1 flow rate; isocratic 100% hexane; UV detection at 254 nm.
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3. Results and discussion Gas chromatography of a DCM solution of Tia Juana Pesado crude oil (Venezuela) revealed few resolved components and a correspondingly large UCM (Fig. 2a, 97% unresolved). Open column chromatography on silica and elution with hexane, hexane/toluene (3:1), hexane/toluene (1:1) and DCM produced fractions designated non-aromatics (39.1% ± 1.8, mean ± r, n = 8), aromatics-1 (21.0% ± 1.6), aromatics-2 (6.6% ± 0.3) and polars (11.6% ± 2.9), respectively. Again, these were predominantly unresolved (resolved components, 3%, 8% and 19% for non-aromatic, aromatic-1 and aromatic-2, respectively) when examined by GC (Fig. 2b– d). HPLC examination of the aromatic-1 fraction with UV detection (254 nm) also revealed a prominent UCM (Fig. 3). Collection of fractions every 1 min through this HPLC separation (96% recovery by weight, n = 10 injections) and re-chromatography of selected fractions using HPLC, revealed reasonably Gaussian peaks (inset Fig. 3). Not surprisingly, gravimetry of the fractions (Fig. 3) demonstrated that UV detection did not quantitatively measure aromatic UCM components, most probably due to the presence of non-UV absorbing highly alkylated substituents on the aromatic rings. GC–MS examination of each HPLC fraction (e.g. Fig. 4) and mass chromatography of selected key ions revealed approximately the typical number of double bond equivalents (DBEs) in the components of each fraction (Fig. 3; Killops and Readman, 1985). For example, m/z 142, 156, 170 mass chromatograms revealed the presence of C1–3 alkylnaphthalenes (7 DBEs) in the 38– 39-min fraction (Fig. 4b), whereas the 54–55 and 55–56min fractions contained compounds ranging from 8 DBEs such as fluorene and dibenzothiophenes (Fig. 4c) to 11 DBEs such as the well-known triaromatic steroids (m/z 231). HPLC analysis of reference compounds (Table 1) confirmed the DBE assignments and showed that shorter retention times were associated with increasing degree of alkyl substitution and molecular geometry, in a similar manner to that previously reported (Akhlaq, 1993). Alkyl substitution, including the presence of the alicyclic D ring, of the phenanthrene moiety and the geometry of the phenanthrene moiety of the 11 DBE
triaromatic steroids likely explains why these compounds eluted earlier during HPLC analysis than less substituted 10 DBE triaromatics (e.g. C1–C3 phenanthrenes). For the purposes of illustration of the successive preparative GC methods, we chose to concentrate on the later HPLC fractions for this initial study. The 54–55 and 55–56-min fractions were combined and subjected to preparative GC on apolar BP-5 stationary phase. A fraction eluting between 28.3 and 33.5 min (so-called Primary Cut: Fig. 5, top) was collected and re-cut by preparative GC on polar SolGel-Wax stationary phase (Fig. 5, F1–F4). Successive fractions from this chromatographed sample (F1–F4) were collected in individual traps and examined using GC–MS on apolar
Primary Cut
A
F1
B
F2
RIC
HP5970 Series Mass Selective Detector and HP7673 autosampler. General conditions were: 1 ll splitless injection; injector temperature 250 C; column Agilent Ultra-1 (12.5 m · 0.20 mm · 0.33 lm) GC programme, 40–300 C at 5 C min1, hold 10 min; head pressure 40 kPa; electron multiplier, 1600 V; detector temperature 280 C; source 70 eV operated in full scan mode (50–500 Da). Compound masses were estimated by relating integrated peak/UCM areas to estimated mass injected.
967
C
F3
D
22.5
23.5
24.5
F4
25.5
retention time (min) Fig. 5. Total ion chromatograms of primary preparative GC cut using an apolar phase column (top) and secondary preparative GC cuts using a polar phase column (F1–F4). GC–MS conditions: Agilent Ultra-1 column (12.5 m · 0.20 mm · 0.33 lm) programmed at 40–300 C at 10 C min1, hold 10 min; injector 250 C; detector 280 C. Primary preparative GC conditions: SGE BP-5 column (15 m · 0.53 mm i.d. · 1.0 lm) programmed from 40 to 280 C at 10 C min1, 280–300 C at 5 C min1, hold 10 min; injector 260 C; FID 300 C; transfer line and switching device 260 C. Secondary preparative GC conditions: SolGel Wax column (30 m · 0.25 mm · 0.25 lm) programmed from 40 to 300 C at 10 C min1, hold 10 min; injector 260 C; FID 300 C; transfer line and switching device 280 C.
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Ultra-1 phase. The resulting chromatograms revealed varying degrees of resolution. Thus, fraction F1 was least resolved (8%) but significantly contained components which were not previously resolved (cf. Fig. 4c), whereas fractions F2–F4 were better resolved (28–50%; Fig. 5). Compounds A–D were identified from their electron impact mass spectra as a series of triaromatic steroids. The spectra of B–D (C26–C28 triaromatic steroids) were comparable to those of well known synthetic and petroleum-derived compounds (Mackenzie et al., 1981; Peters and Moldowan, 1993). Whilst such compounds are routinely assigned in petroleum fractions using m/z 231 mass chromatography (Peters and Moldowan, 1993), they have rarely, if ever, been isolated from crude oil in sufficient quantities to be detected as single components in total ion chromatograms (e.g. Fig. 5). Although this is not of particular significance in itself since the compounds are well known to organic geochemists, it nonetheless illustrates the successful isolation of resolved compounds (Fig. 5) from a previously unresolved complex mixture (Fig. 1). More importantly, the mass spectrum of compound A now resolved in F1 (Fig. 5) was consistent with its tentative assignment as a novel C17 demethylated C26 triaromatic steroid (Fig. 6). Thus, the molecular ion (m/z 344) and base peak ion (m/z 217) dominate the spectrum and are consistent with a C26 compound and alkyl side chain loss, as also observed in the spectrum of DielsÕ hydrocarbon (Rowland et al., 1986) and proposed previously (Mackenzie et al., 1981). However, no molecular ion was observed previously, even when chemical ionisation GC–MS was used (Mackenzie et al., 1981), suggesting that it was either below the limit of detection or was not present in the Lower Toarcian shales studied. In a similar manner in the present study, extracted ion chromatography and selected ion monitoring of m/z 344 of the aromatic-1 fraction was not conclusively diagnostic (data
100
not shown). Mass chromatography of selected ions at m/z 217, 330 (C25) and 358 (C27) from the GC–MS analysis of HPLC cuts and prep GC cuts indicated the presence of other demethylated homologues, but good quality mass spectra could not be obtained owing to their low concentrations and co-elution with other compounds. Whilst the m/z 217 base peak ion is also characteristic of ring D fragmentation in steranes (To¨ke´s and Djerassi, 1969), these compounds were present only in the hexane eluate fraction following open column chromatography (Fig. 1b). The presence of A is also consistent with the biodegraded status of TJP crude since demethylation is a known feature of metabolism of cyclic terpenoids in crude oils (Peters and Moldowan, 1993). Indeed, identification of such compounds in the UCMs of biodegraded oils may allow an extension of current biodegradation indices. The purification of a partially resolved fraction containing A using a combination of preparative open column chromatography, HPLC and GC and GC–MS identification via molecular and base peak ions is therefore a demonstration of the isolation of resolved components from a UCM and of the utility of the multi-stage preparative chromatography approach. On the basis of the isolated fractions (Fig. 5), the concentration of compound A was estimated as 4 lg g1 whole oil (B–D estimated as 5–7 lg g1 whole oil). The C28 (20R isomer) steroid (e.g. D) has been previously estimated as <10–300 lg g1 C15+ fraction in a series of non-biodegraded Monterey oils and up to 6000 lg g1 C15+ fraction in extensively biodegraded Lousiana oils (Requejo, 1992) but such steroids may be depleted by more extensive maturation (Requejo, 1992) or biodegradation (Volkman et al., 1984; Wardroper et al., 1984). If the concentration of A is taken as the very approximate average concentration of compounds in the UCM, then the total number of
217
relative intensity
75
50
m/z = 217
25
0 50
344
202
100
150
200
250
300
350
400
450
500
550
m/z
Fig. 6. Electron impact ionisation mass spectra of compound A, a novel C26 17-desmethyl triaromatic steroid (tentative identification).
P.A. Sutton et al. / Organic Geochemistry 36 (2005) 963–970
compounds in the whole oil UCM (Fig. 1a) would be about 250,000. Even if this estimate exaggerates the total number of UCM components by a factor of two, the complexity of such an unresolved mixture would (and certainly does) represent a major challenge for the future, even with the deployment of multidimensional GC methods. Nonetheless, this is an important goal, not the least because of the known toxicity of some aromatic UCMs (e.g. Donkin et al., 2003).
4. Conclusions Hydrocarbon fractions in which some individual hydrocarbons are now resolved were isolated from a previously unresolved (92%) complex mixture (UCM). As a preliminary example, one of these resolved compounds was tentatively identified using GC–MS as a novel C26 17-desmethyl triaromatic steroid. The proportion of UCM represented by such compounds is, however, still very small (ca. 0.0004%) and a very approximate estimation suggests that the UCM studied contains maybe 250,000 compounds. This indicates that oil UCMs are amongst the most complex mixtures of organic compounds on Earth. The results thus confirm the extreme complexity of crude oil UCMs and the difficulty of UCM identification. Nevertheless, they also indicate that the above approach can lead to the identification of novel compounds, which may have considerable geochemical, petrochemical and environmental significance.
Acknowledgements This work was supported by Natural Environment Research Council Grant No. GR3/13184. We thank Dr. P. Schaeffer and two anonymous reviewers for their useful comments on this manuscript. Associate Editor—Philippe Schaeffer
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ity, and aqueous solubility of monoaromatic compounds. Environmental Toxicology and Chemistry 20, 2428–2432. Thomas, K.V., 1995. Characterisation and environmental effects of unresolved complex mixtures of hydrocarbons. Ph.D. Thesis, University of Plymouth, UK. To¨ke´s, L., Djerassi, C., 1969. Mass spectrometry in structural and stereochemical problems. CLXXVI. Course of the electron impact induced fragmentation of androstane. Journal of the American Chemical Society 91, 5017– 5023. van Deursen, M., Beens, J., Reijenga, J., Lipman, P., Cramers, J., Blomberg, J., 2000. Group-type identification of oil samples using comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometer (GC · GC-TOF). Journal of High Resolution Chromatography 23, 507–510. Volkman, J.K., Alexander, R., Kagi, R.I., Rowland, S.J., Sheppard, P.N., 1984. Biodegradation of aromatic hydrocarbons in crude oils from the Barrow Sub-basin of Western Australia. In: Schenck, P.A., de Leeuw, G.W.M., Lijmbach, G.W.M. (Eds.), Advances in Organic Geochemistry 1983. Pergamon Press, Oxford, pp. 619–632. Wardroper, A.M.K., Hoffmann, C.F., Maxwell, J.R., Barwise, N.S., Goodwin, N.S., Park, P.J.D., 1984. Crude oil biodegradation under simulated and natural conditions – II. Aromatic steroid hydrocarbons. In: Schenck, P.A., de Leeuw, J.W., Lijmbach, G.W.M. (Eds.), Advances in Organic Geochemistry 1983. Pergamon Press, Oxford, pp. 605–617. Warton, B., 1999. Studies of the saturate and aromatic hydrocarbon unresolved complex mixtures in petroleum. Ph.D. Thesis, Curtin University of Technology, Perth, Australia. Warton, B., Alexander, R., Kagi, R.I., 1997. Identification of some single branched alkanes in crude oils. Organic Geochemistry 27, 465–476. Warton, B., Alexander, R., Kagi, R.I., 1999. Characterisation of the ruthenium tetroxide oxidation products from the aromatic unresolved complex mixture of a biodegraded crude oil. Organic Geochemistry 30, 1255–1272.
Isolation of individual hydrocarbons from the unresolved complex hydrocarbon mixture of a biodegraded crude oil using preparative capillary gas chromatography P.A. Sutton, C.A. Lewis *, S.J. Rowland Petroleum and Environmental Geochemistry Group, School of Earth, Ocean and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK Received 28 June 2004; accepted 22 November 2004 (returned to author for revision 27 September 2004) Available online 31 March 2005
Abstract We describe the isolation, using preparative capillary gas chromatography, of hydrocarbon fractions from an unresolved complex mixture (UCM) of hydrocarbons isolated from a biodegraded crude oil. Some of the individual hydrocarbons in these fractions were then resolved by gas chromatography (GC) and identified using GC–mass spectrometry (GC–MS). The mass spectra contained distinct molecular ions. As a preliminary example, after preparative open column chromatography, preparative HPLC (three columns in series) and preparative GC on two stationary phases, one of the compounds from the UCM was tentatively identified using electron impact ionisation GC–MS as a novel C26 17desmethyl triaromatic steroid. If the concentration of this compound (ca. 4 lg g1 whole oil) is typical of other individual compounds in the UCM, the proportion of UCM represented by each compound is obviously very small (<0.0004%) and suggests that the whole oil UCM contains in the order of 250,000 mostly unidentified compounds. This suggests that oil UCMs are amongst the most complex mixtures of organic compounds on Earth and explains the difficulty of UCM component identification. Nevertheless, it also indicates that multi-step chromatography can lead to the identification of individual compounds, some of which have geochemical, petrochemical and environmental significance. Acquisition of such knowledge is fundamental if we are to improve our understanding of petroleum. 2005 Elsevier Ltd. All rights reserved.
1. Introduction Despite the reliance of the economies and lifestyles of the industrialised world on crude oil, the chemical
* Corresponding author. Tel.: +44 1752 232988; fax: +44 1752 233035. E-mail address: [email protected] (C.A. Lewis).
composition of quite a large proportion of liquid petroleum remains a mystery (Altgelt and Boduszynski, 1993; Rowland and Revill, 1995). Although many of the hydrocarbons can be made volatile enough for gas chromatography (GC) analysis, relatively few are separable into individual, identifiable compounds using routine one-dimensional gas chromatography. Rather, most compounds appear as unresolved complex mixtures (UCMs) or GC ÔhumpsÕ (e.g. Gough and
0146-6380/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.orggeochem.2004.11.007
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Rowland, 1990). These UCMs are particularly obvious when biodegraded oils are examined using GC. A lack of detailed knowledge of UCM composition is a hindrance to the development of theories of the origin of petroleum. It is also a restriction on the prediction of the effects of refinery processing of crude oils and may be an important limitation to the assessment of the effects of unresolved oil residues in the environment (e.g. Rowland et al., 2001; Reddy et al., 2002; Donkin et al., 2003). GC–MS techniques have been routinely used to distinguish the presence of particular compounds within complex petroleum mixtures but use of GC–MS is usually confined to detection of those compounds which are resolved and have characteristic mass spectra (e.g. reviewed by Peters and Moldowan, 1993). Such methods leave most of the UCM hydrocarbons unidentified. However, a variety of techniques has been applied to try to identify some of the unresolved components within oils, including, for example, studies of oxidised UCMs (Gough and Rowland, 1990; Killops and Al-Juboori, 1990; Revill, 1992; Warton, 1999; Warton et al., 1999), isolation of individual or groups of compounds following treatment of oil UCMs with molecular seive (e.g.
Armanios et al., 1994; Ellis et al., 1994; Fazeelat et al., 1994), statistical deconvolution methods (Pool et al., 1997; Dagan, 2000; Demir et al., 2000), and multidimensional GC (Blomberg et al., 1997; Phillips and Beens, 1999; van Deursen et al., 2000; Reddy et al., 2002; Frysinger et al., 2003). These have led to proposed structures for some of the non-aromatic (Gough and Rowland, 1990; Warton et al., 1997) and aromatic (Gough and Rowland, 1990; Warton et al., 1999) hydrocarbons. Synthetic compounds, which exhibit the properties of some UCM components, have also been made. Thus, Gough et al. (1992) showed that simple ÔTÕ branched alkanes were relatively resistant to biodegradation and were oxidised to compounds similar to those obtained from oxidation of some UCMs. Later, Warton et al. (1997) showed that 3% of the alkanes from a crude oil were ÔTÕ branched. Similarly, Thomas (1995) suggested that some aromatic UCM components were substituted alkyl tetralins on the basis of oxidation stud-
(a)
97 % UCM (whole oil)
Whole oil
(b)
Open column chromatography (non-aromatic, aromatic-1, aromatic-2, polar, residual)
RIC
97 % UCM (non-aromatic)
Normal phase (HPLC) (cut each 1 min)
(c) 92 % UCM (aromatic-1)
Preparative (GC) (apolar phase) (ca 10 s cuts)
81 % UCM (aromatic-2)
(d)
Preparative (GC) (polar phase) (cut peaks)
5
15
25
35
45
55
retention time (min)
GC – MS Fig. 1. Flow diagram of sequential preparative chromatographic approach to resolving components from the UCM of a biodegraded oil.
Fig. 2. Total ion chromatograms of Tia Juana Pesado, Venezuela, oil (a) crude oil (b) non-aromatic fraction (c) aromatic-1 fraction (d) aromatic-2 fraction. GC–MS conditions: Agilent Ultra-1 column (12.5 m · 0.20 mm id · 0.33 lm) programmed at 40–300 C at 5 C min1, hold 10 min; injector 250 C; detector 280 C.
P.A. Sutton et al. / Organic Geochemistry 36 (2005) 963–970
ies followed by ion cyclotron resonance MS and recently Booth (2004) showed that some dialkyl tetralins were indeed as resistant to biotransformation as some components of an aromatic UCM. The same compounds were toxic to mussels (Smith et al., 2001), emphasising the environmental significance of these persistent bioaccumulative residues. The reported toxicity of aromatic UCMs to marine organisms (Smith et al., 2001) suggests that separation of individual components from aromatic UCMs warrants further study. The recent coupling of GC · GC to time of flight mass spectrometers (e.g. Dallu¨ge et al., 2002; Hamilton and Lewis, 2003) may provide more routine methods of analysis of UCMs, but there is still a need for complementary methods that provide information about individual UCM components. In the present study, we have therefore used a combination of preparative chromatographic techniques, including open column chromatography, normal phase high performance liquid chromatography (HPLC) and preparative capillary GC on two stationary phases to isolate individual and groups of aromatic hydrocarbons from the UCM recovered from a biodegraded crude oil. The components of the fractions become progressively more resolved with each chromatography step. Although these methods are probably too lengthy and time-consuming to be considered routine, their application to a few UCMs nonetheless has allowed the first identification of compounds which were insufficiently abundant and present in mixtures too complex to be resolved previously. Furthermore, the assumption that the amounts of compounds
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in the UCM are about equal allowed a very approximate estimate of the number of compounds present in a UCM to be made. We illustrate the approach by the isolation of some known triaromatic steroids and the identification by MS of a novel isomer, but the method is also applicable to identification of many other UCM components.
2. Experimental A flow diagram of the isolation procedures is shown in Fig. 1. 2.1. Solvents and chemicals Solvents were obtained from Rathburn Chemicals Ltd., Scotland and used as received after checking the purity of a concentrated aliquot (·100) with GC. The crude oil was a heavily biodegraded crude from Tia Juana Pesado, Venezuela. 2.2. Open column chromatography Crude oil (8 · ca. 0.7 g) in hexane was rotary evaporated to near dryness in the presence of Al2O3 (BDH, England; grade 1, neutral, 150 mesh) and was loaded on to a silica/alumina column [0.6 g cm3 SiO2 (Sigma–Aldrich Co. Ltd.; grade 645, 60–100 mesh), 5% H2O deactivated; Al2O3, 1.5% H2O deactivated; 50%, w/w, Al2O3:SiO2] and the column eluted with 170 ml
5 10DBE
7DBE 6DBE
4
Mass (mg)
5DBE
3
4 DBE
UCM UV absorbance (254 nm)
2
1
18 20 22 24 26
0
Retention time (min)
Fig. 3. HPLC UV chromatogram (254 nm) of Tia Juana Pesado, Venezuela, aromatic-1 fraction and gravimetric determination of HPLC 1-min cuts. Maximum retention times of the most condensed forms of a series of reference compounds (4 double bond equivalents, DBEs, benzene; 5 DBEs, tetralin; 6 DBEs, indene; 7 DBEs, naphthalene; 10 DBEs, anthracene) are indicated and the rerun of the 22–23 min cut shown inset. HPLC conditions: 3 · 25 cm · 1 cm · 8 lm HSAPS-2 columns in series; 2 ml min1 flow rate; 0– 40 min 100% hexane, 40–45 min gradient to 100% DCM, 65–70 min gradient to 100% hexane; UV detection at 254 nm.
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of hexane (HPLC grade), hexane/toluene (glass distilled grade; 3:1, v/v), hexane/toluene (1:1, v/v) and dichloromethane (DCM, HPLC grade), to yield non-aromatic, aromatic-1, aromatic-2 and polar fractions, respectively. The fractions were rotary evaporated to dryness, weighed and examined by GC–MS. The proportion of residue remaining on the column was determined by difference.
250 mm · 10 mm · 8 lm columns in series with a guard column; flow rate 2 ml min1; 0–40 min (100% hexane), 40–45 min (gradient to 100% DCM), 65–70 min (gradient to 100% hexane), with a Dionex AD20 UV absorbance detector at 254/280 nm]. Collected fractions were blown down to dryness (N2) and weighed before re-dissolving in hexane to a known concentration for analysis using GC–MS and preparative GC (PGC).
2.3. HPLC 2.4. Preparative GC The aromatic-1 fraction (10 · 800 ll, 20 mg ml1 in hexane) was further fractionated (1 min cuts from 10 to 74 min) using normal phase HPLC [Dionex GP40 gradient pump (Dionex Corp., USA) with 3 · Hypersil Hyperprep HSAPS-2 (ThermoHypersil-Keystone)
(a)
21-22 min HPLC fraction
PGC analysis was carried out using an Agilent 6890 Series gas chromatograph (Agilent Technologies, Inc., USA) fitted with an Agilent 7683 Series injector and autosampler, and a Gerstel Preparative Fraction Collector (Gerstel GmbH & Co. KG, Germany). FID response was recorded using Turbochrom Navigator V6.1 software (Perkin–Elmer Corp., USA). Initial PGC was performed using a BP-5 column (15 m · 0.53 mm · 1.0 lm) and secondary PGC using a SolGel-Wax column (30 m · 0.25 mm · 0.25 lm), both from SGE Europe Ltd., UK. PGC conditions: injector 260 C; FID 300 C; transfer line/switching device 280 C; GC programme 40–280 C at 10 C min1, 280–300 C at 5 C min1 (BP-5) or 40–300 C at 10 C min1 (SolGel WaxTM), hold 10 min; traps at ambient. Fraction masses were estimated by relating integrated cut areas to mass injected.
38-39 min HPLC fraction
2.5. GC–MS
98 % UCM
RIC
(b)
GC–MS was performed using a HP5890 Series II gas chromatograph (Hewlett–Packard, USA) fitted with a
92 % UCM 2
3 A
(c)
Table 1 Retention times (RT)a and double bond equivalents (DBEs) for a range of reference compounds and model aromatic UCM hydrocarbons
m /z 217
CD B
4
m /z 231 m /z 245
1 54-55 min HPLC fraction
81 % UCM 5
10
15
20
25
30
35
retention time (min)
Fig. 4. Typical total ion chromatograms of 1-min HPLC cuts from Tia Juana Pesado, Venezuela, aromatic-1 fraction (a) 21–22, (b) 38–39, (c) 54–55 min (1 = C0–C2 fluorenes; 2 = methyl-dibenzothiophenes; 3 = dimethylnaphthothiophenes; 4 = trimethylnaphthothiophenes); compound A, m/z 217, tentatively identified as C17 demethylated C26 triaromatic steroid (TAS); compounds B–D, m/z 231 = methyl C26–C28 TAS; m/z 245 = dimethyl TAS. GC–MS conditions: Agilent Ultra-1 column (12.5 m · 0.20 mm i.d. · 0.33 lm) programmed from 40 to 300 C at 10 C min1, hold 10 min; injector 250 C; detector 280 C.
Compound
DBEs
RT (min)
1-Phenyldecane 1-(3 0 -Methylbutyl)-7-cyclohexyltetralin Benzene 1-n-Nonylcyclohexyltetralin 1-Methyl-7-cyclohexyltetralin Tetralin 6-Cyclohexyltetralin Indene 1-n-Nonylcyclohexylnaphthalene 4-Pentylbiphenyl Biphenyl Naphthalene Phenanthrene Anthracene
4 6 4 6 6 5 6 6 8 8 8 7 10 10
26.05 27.07 27.96 28.23 29.51 29.86 30.02 36.73 37.26 39.39 41.72 42.50 64.15 67.33
a HPLC conditions: 3 · 25 cm · 1 cm · 8 lm HSAPS-2 columns in series; 2 ml min1 flow rate; isocratic 100% hexane; UV detection at 254 nm.
P.A. Sutton et al. / Organic Geochemistry 36 (2005) 963–970
3. Results and discussion Gas chromatography of a DCM solution of Tia Juana Pesado crude oil (Venezuela) revealed few resolved components and a correspondingly large UCM (Fig. 2a, 97% unresolved). Open column chromatography on silica and elution with hexane, hexane/toluene (3:1), hexane/toluene (1:1) and DCM produced fractions designated non-aromatics (39.1% ± 1.8, mean ± r, n = 8), aromatics-1 (21.0% ± 1.6), aromatics-2 (6.6% ± 0.3) and polars (11.6% ± 2.9), respectively. Again, these were predominantly unresolved (resolved components, 3%, 8% and 19% for non-aromatic, aromatic-1 and aromatic-2, respectively) when examined by GC (Fig. 2b– d). HPLC examination of the aromatic-1 fraction with UV detection (254 nm) also revealed a prominent UCM (Fig. 3). Collection of fractions every 1 min through this HPLC separation (96% recovery by weight, n = 10 injections) and re-chromatography of selected fractions using HPLC, revealed reasonably Gaussian peaks (inset Fig. 3). Not surprisingly, gravimetry of the fractions (Fig. 3) demonstrated that UV detection did not quantitatively measure aromatic UCM components, most probably due to the presence of non-UV absorbing highly alkylated substituents on the aromatic rings. GC–MS examination of each HPLC fraction (e.g. Fig. 4) and mass chromatography of selected key ions revealed approximately the typical number of double bond equivalents (DBEs) in the components of each fraction (Fig. 3; Killops and Readman, 1985). For example, m/z 142, 156, 170 mass chromatograms revealed the presence of C1–3 alkylnaphthalenes (7 DBEs) in the 38– 39-min fraction (Fig. 4b), whereas the 54–55 and 55–56min fractions contained compounds ranging from 8 DBEs such as fluorene and dibenzothiophenes (Fig. 4c) to 11 DBEs such as the well-known triaromatic steroids (m/z 231). HPLC analysis of reference compounds (Table 1) confirmed the DBE assignments and showed that shorter retention times were associated with increasing degree of alkyl substitution and molecular geometry, in a similar manner to that previously reported (Akhlaq, 1993). Alkyl substitution, including the presence of the alicyclic D ring, of the phenanthrene moiety and the geometry of the phenanthrene moiety of the 11 DBE
triaromatic steroids likely explains why these compounds eluted earlier during HPLC analysis than less substituted 10 DBE triaromatics (e.g. C1–C3 phenanthrenes). For the purposes of illustration of the successive preparative GC methods, we chose to concentrate on the later HPLC fractions for this initial study. The 54–55 and 55–56-min fractions were combined and subjected to preparative GC on apolar BP-5 stationary phase. A fraction eluting between 28.3 and 33.5 min (so-called Primary Cut: Fig. 5, top) was collected and re-cut by preparative GC on polar SolGel-Wax stationary phase (Fig. 5, F1–F4). Successive fractions from this chromatographed sample (F1–F4) were collected in individual traps and examined using GC–MS on apolar
Primary Cut
A
F1
B
F2
RIC
HP5970 Series Mass Selective Detector and HP7673 autosampler. General conditions were: 1 ll splitless injection; injector temperature 250 C; column Agilent Ultra-1 (12.5 m · 0.20 mm · 0.33 lm) GC programme, 40–300 C at 5 C min1, hold 10 min; head pressure 40 kPa; electron multiplier, 1600 V; detector temperature 280 C; source 70 eV operated in full scan mode (50–500 Da). Compound masses were estimated by relating integrated peak/UCM areas to estimated mass injected.
967
C
F3
D
22.5
23.5
24.5
F4
25.5
retention time (min) Fig. 5. Total ion chromatograms of primary preparative GC cut using an apolar phase column (top) and secondary preparative GC cuts using a polar phase column (F1–F4). GC–MS conditions: Agilent Ultra-1 column (12.5 m · 0.20 mm · 0.33 lm) programmed at 40–300 C at 10 C min1, hold 10 min; injector 250 C; detector 280 C. Primary preparative GC conditions: SGE BP-5 column (15 m · 0.53 mm i.d. · 1.0 lm) programmed from 40 to 280 C at 10 C min1, 280–300 C at 5 C min1, hold 10 min; injector 260 C; FID 300 C; transfer line and switching device 260 C. Secondary preparative GC conditions: SolGel Wax column (30 m · 0.25 mm · 0.25 lm) programmed from 40 to 300 C at 10 C min1, hold 10 min; injector 260 C; FID 300 C; transfer line and switching device 280 C.
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Ultra-1 phase. The resulting chromatograms revealed varying degrees of resolution. Thus, fraction F1 was least resolved (8%) but significantly contained components which were not previously resolved (cf. Fig. 4c), whereas fractions F2–F4 were better resolved (28–50%; Fig. 5). Compounds A–D were identified from their electron impact mass spectra as a series of triaromatic steroids. The spectra of B–D (C26–C28 triaromatic steroids) were comparable to those of well known synthetic and petroleum-derived compounds (Mackenzie et al., 1981; Peters and Moldowan, 1993). Whilst such compounds are routinely assigned in petroleum fractions using m/z 231 mass chromatography (Peters and Moldowan, 1993), they have rarely, if ever, been isolated from crude oil in sufficient quantities to be detected as single components in total ion chromatograms (e.g. Fig. 5). Although this is not of particular significance in itself since the compounds are well known to organic geochemists, it nonetheless illustrates the successful isolation of resolved compounds (Fig. 5) from a previously unresolved complex mixture (Fig. 1). More importantly, the mass spectrum of compound A now resolved in F1 (Fig. 5) was consistent with its tentative assignment as a novel C17 demethylated C26 triaromatic steroid (Fig. 6). Thus, the molecular ion (m/z 344) and base peak ion (m/z 217) dominate the spectrum and are consistent with a C26 compound and alkyl side chain loss, as also observed in the spectrum of DielsÕ hydrocarbon (Rowland et al., 1986) and proposed previously (Mackenzie et al., 1981). However, no molecular ion was observed previously, even when chemical ionisation GC–MS was used (Mackenzie et al., 1981), suggesting that it was either below the limit of detection or was not present in the Lower Toarcian shales studied. In a similar manner in the present study, extracted ion chromatography and selected ion monitoring of m/z 344 of the aromatic-1 fraction was not conclusively diagnostic (data
100
not shown). Mass chromatography of selected ions at m/z 217, 330 (C25) and 358 (C27) from the GC–MS analysis of HPLC cuts and prep GC cuts indicated the presence of other demethylated homologues, but good quality mass spectra could not be obtained owing to their low concentrations and co-elution with other compounds. Whilst the m/z 217 base peak ion is also characteristic of ring D fragmentation in steranes (To¨ke´s and Djerassi, 1969), these compounds were present only in the hexane eluate fraction following open column chromatography (Fig. 1b). The presence of A is also consistent with the biodegraded status of TJP crude since demethylation is a known feature of metabolism of cyclic terpenoids in crude oils (Peters and Moldowan, 1993). Indeed, identification of such compounds in the UCMs of biodegraded oils may allow an extension of current biodegradation indices. The purification of a partially resolved fraction containing A using a combination of preparative open column chromatography, HPLC and GC and GC–MS identification via molecular and base peak ions is therefore a demonstration of the isolation of resolved components from a UCM and of the utility of the multi-stage preparative chromatography approach. On the basis of the isolated fractions (Fig. 5), the concentration of compound A was estimated as 4 lg g1 whole oil (B–D estimated as 5–7 lg g1 whole oil). The C28 (20R isomer) steroid (e.g. D) has been previously estimated as <10–300 lg g1 C15+ fraction in a series of non-biodegraded Monterey oils and up to 6000 lg g1 C15+ fraction in extensively biodegraded Lousiana oils (Requejo, 1992) but such steroids may be depleted by more extensive maturation (Requejo, 1992) or biodegradation (Volkman et al., 1984; Wardroper et al., 1984). If the concentration of A is taken as the very approximate average concentration of compounds in the UCM, then the total number of
217
relative intensity
75
50
m/z = 217
25
0 50
344
202
100
150
200
250
300
350
400
450
500
550
m/z
Fig. 6. Electron impact ionisation mass spectra of compound A, a novel C26 17-desmethyl triaromatic steroid (tentative identification).
P.A. Sutton et al. / Organic Geochemistry 36 (2005) 963–970
compounds in the whole oil UCM (Fig. 1a) would be about 250,000. Even if this estimate exaggerates the total number of UCM components by a factor of two, the complexity of such an unresolved mixture would (and certainly does) represent a major challenge for the future, even with the deployment of multidimensional GC methods. Nonetheless, this is an important goal, not the least because of the known toxicity of some aromatic UCMs (e.g. Donkin et al., 2003).
4. Conclusions Hydrocarbon fractions in which some individual hydrocarbons are now resolved were isolated from a previously unresolved (92%) complex mixture (UCM). As a preliminary example, one of these resolved compounds was tentatively identified using GC–MS as a novel C26 17-desmethyl triaromatic steroid. The proportion of UCM represented by such compounds is, however, still very small (ca. 0.0004%) and a very approximate estimation suggests that the UCM studied contains maybe 250,000 compounds. This indicates that oil UCMs are amongst the most complex mixtures of organic compounds on Earth. The results thus confirm the extreme complexity of crude oil UCMs and the difficulty of UCM identification. Nevertheless, they also indicate that the above approach can lead to the identification of novel compounds, which may have considerable geochemical, petrochemical and environmental significance.
Acknowledgements This work was supported by Natural Environment Research Council Grant No. GR3/13184. We thank Dr. P. Schaeffer and two anonymous reviewers for their useful comments on this manuscript. Associate Editor—Philippe Schaeffer
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