Preparative HPLC with ultrastable-Y zeolite for
compound-speci®c carbon isotopic analyses
Fabien Kenig
a,b,*, Brian N. Popp
a, Roger E. Summons
caDepartment of Geology and Geophysics, University of Hawaii, 2525 Correa Road, Honolulu, HI 96822 USA bDepartment of Earth and Environmental Sciences (M/C 186), University of Illinois at Chicago, 845 W. Taylor Street, Chicago,
IL 60607-7059, USA
cAustralian Geological Survey Organisation, PO Box 378, Canberra, 2601 Australia
Received 11 April 2000; accepted 24 August 2000 (returned to author for revision 15 June 2000)
Abstract
Preparative high pressure liquid chromatography on US-Y zeolite shape-selective molecular sieve was studied for carbon isotopic fractionation eects. We tested a standard mixture [17b, 21b(H)-hopane, 5a-cholestane] and complex natural hydrocarbon mixtures dominated by tetra- and pentacyclic triterpenoids extracted from Oxford Clay shales. We con®rmed that steroids and hopanoids were separated on the basis of stereochemical con®guration while isotopic analysis of eluents indicated that shape-selective chromatography did not result in isotopic fractionation. US-Y zeolite chromatography can be used to simplify hydrocarbon mixtures and prepare well resolved mixtures of molecular fossils for compound-speci®c isotopic analyses (CSIA).#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Liquid chromatography; Zeolite; Compound speci®c isotope analysis; Biomarkers; Isotope chromatographic eect; Oxford Clay
1. Introduction
Extractable organic matter in contemporary oceanic environments and in preserved sediments comprises complex mixtures of organic compounds. This com-plexity results from the diversity of source inputs and is compounded by the transformation of biomolecules during transport and diagenesis. Chemical and stable isotopic characterization of organic compounds can provide insight into origins and fates of organic matter (e.g. Freeman et al., 1990; Kohnen et al., 1992; Kenig et al., 1994a, 1995). However, to be useful for quantitative and qualitative analysis, these complex mixtures are best separated into subfractions of well resolved compounds. The recent development of compound-speci®c iso-topic analysis (CSIA, e.g. Matthews and Hayes, 1978;
Hayes et al., 1990) has enabled a much clearer de®nition of geospheric organic matter origins through measure-ment of carbon isotopic composition. In turn, this has led to signi®cant revision of environmental and paleoenvironmental understanding (e.g. Hayes et al., 1987, 1989, 1990; Engel et al., 1990; Freeman et al., 1990, 1994; Jasper and Hayes, 1990; Freeman and Wakeham, 1992; Wakeham et al., 1993; Kenig et al., 1994a, b, 1995; Jasper et al., 1994; Laws et al., 1995; Bidigare et al., 1997). Although isotope ratio monitoring gas chromatography-mass spectrometry (irm-GC±MS) instruments can provide accurate carbon isotopic com-positions of individual compounds, the performance of CSIA is commonly limited by chromatographic resolution of individual compounds (see Hayes et al., 1990; Fig. 4).
Following early reports of isotopic chromatographic eects (Liberti et al., 1965; Hook, 1969; Gunter and Gleason, 1971), Hayes et al. (1990) observed that capil-lary gas chromatographic columns tend to slightly separate organic molecules enriched in13C from those
more depleted in 13C. 13C-enriched molecules elute at
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* Corresponding author. Tel.: 312-996-3020; fax: +1-312-413-2279.
the front of the chromatographic peak while13C
deple-ted counterparts elute toward the tail (Hayes et al., 1990). Accordingly, an accurate carbon isotopic com-position for a single compound using irm-GC±MS can only be obtained when the entire peak, free from co-eluting compounds, is integrated.
High pressure liquid chromatography (HPLC) is often used for preparative chromatographic separation of organic compounds. However, Bidigare et al. (1991) found random but signi®cant stable carbon isotopic variation in fractions collected across a peak of chlor-ophyll-a obtained by C18 reversed phase HPLC. An
isotopic chromatographic eect equivalent to that observed on capillary columns, with13C enriched peak
front and13C depleted peak tail, was observed by Martin
Schoell (1996, personal communication) during reverse phase HPLC preparative separation of steranes, with a maximum13C isotopic fractionation of 18%across a peak.
Thus, the use of preparative HPLC for CSIA is conditional on the quantitative recovery of the compounds analyzed (Bidigare et al., 1991) and this compromises its widespread application.
5 AÊ and silicalite molecular sieves have proven very useful for compound class separations preceding CSIA (Hoering and Freeman, 1984; Kenig et al., 1994a; Dowling et al., 1995). Recently, Armanios et al. (1992, 1994) demonstrated that excellent chromatographic separation of petroleum hopanoids could be achieved utilizing the molecular sieve properties of ultrastable-Y (US-Y) zeolite. These authors concluded that the observed separation with US-Y zeolite is based on molecular cross-sectional dierences with the preferential retention of larger compounds on the phase. This approach does not involve the same type of column phase/eluent interactions (sorption/desorption) that cause isotopic chromatographic eects during reverse phase HPLC or gas-chromatographic separations (Liberti et al., 1965).
The goals of this investigation were to determine if shape-selective chromatography could usefully separate compounds other than pentacyclic triterpanes and to determine the scale of isotopic chromatographic eects, if any. Speci®cally, we wished to determine if co-eluting acyclic isoprenoid, tetra- and pentacyclic hydrocarbon biomarkers in immature sediment extracts could be resolved suciently for CSIA using the shape-selective properties of ultrastable-Y zeolite.
2. Sample materials
2.1. Zeolite
Ultrastable Zeolite (US-Y, PQ Corporation, KS, USA) was obtained from R. Alexander (Curtin Uni-versity) and activated at 350C overnight, followed by storage at 120C.
2.2. Standards
Pure 5a-cholestane and 17b,21b(H)-hopane from the Australian Geological Survey Organisation (AGSO) standards library were mixed in a 60:40 (wt.%) sterane: hopane ratio.
2.3. Samples
Two samples of the Peterborough Member of the Oxford Clay Formation (Callovian) were collected in Central England in the Dogsthorpe (sample P89-4) and Bletchley (sample B89-8) brick pits. Location and stra-tigraphy of the brick pits are described in Kenig et al. (1994a). P89-4 is a Gryphea shell bed with 4.2 wt.% total organic carbon (TOC) and ad13C
TOCvs. PDB of
ÿ27.4 %. B89-8 is a deposit feeder bituminous shale
with 4.2 wt.% TOC and ad13C
TOCvs. PDB ofÿ26.4%
(Kenig et al., 1994a). The Peterborough Member is an organic-rich mudrock with a TOC content ranging from 3 to 16.5 wt.% (Kenig et al., 1994a). The average hydrogen index (533 mg HC/gTOC), values of Tmax
(419C), high contents of unsaturated hydrocarbons and high abundance of biological stereoisomers (e.g., bb hopanes and aaa steranes) indicate that the organic matter in these samples is immature with respect to pet-roleum generation (Kenig et al., 1994a). Isotopic com-positions of TOC and of individual compounds (pristane, phytane and n-alkanes) indicated that the organic material was predominantly of marine origin (Kenig et al., 1994a). The saturated hydrocarbon fraction of these samples is characterized by a complex mixture of steranes and hopanes not readily amenable to CSIA even after adduction ofn-alkanes with silicalite.
3. Methods
3.1. Preparation of Oxford Clay sediment samples
Total extractable material was obtained by Soxhlet extraction of ®nely ground sediment (120 g) with di-chloromethane and methanol (1:1) for 48 h. Approxi-mately 10 g of ®nely powdered, solvent-extracted, hydrochloric acid-activated copper was added to each extraction ¯ask to remove elemental sulfur. All solvents were distilled in glass.
mesh) impregnated with 10% AgNO3in petroleum ether.
Saturated hydrocarbons were eluted with 50 ml of petro-leum ether and the unsaturated hydrocarbons were eluted with 50 ml of petroleum ether:dichloromethane (1:1). Finally, then-alkanes and branched/cyclic fractions of the saturated hydrocarbons were further separated with a silica molecular sieve (Silicalite, PQ Corporation, KS, USA) using the method of Hoering and Freeman (1984).
3.2. Molecular sieve chromatography with US-Y zeolite
Molecular sieve chromatography was performed using a modi®cation of the method of Armanios et al. (1992). Approximately 4 g of US-Y was dry packed into a 30010 mm i.d. or2 g packed into a 2504.6 mm i.d. stainless steel HPLC column. The small diameter column was used to separate the sterane/hopane stan-dard mixture and the large diameter column was used for the samples of the Oxford Clay. Columns were repacked between each run and were dried at 70C for 1 h prior to use. Each column was washed with approxi-mately three bed volumes ofn-pentane before injection of sample. Elution of sample was monitored using a refractive index (RI) detector. The RI detector revealed only the start and end of the elution of material through the column and did not identify discrete peaks. Approximately 1.4 mg of the hopane/sterane mixture was injected onto the 250 4.6 mm i.d. diameter col-umn at a constant ¯ow rate of 0.5 ml/min. Six con-secutive fractions were collected in 2 minute intervals beginning at 10 min. Approximately 3 mg of the Silica-lite non-adducted Oxford Clay material was dissolved in 60mln-pentane and 50ml injected onto the 30010 mm i.d. column at a ¯ow rate of 1.0 ml/min. After 1 min, ¯ow rate was slowed to 0.05 ml/min for 5.75 min, then increased slowly to the original ¯ow rate. Collection of seven or eight consecutive fractions commenced after the RI detector revealed elution of material (typically within 16 min of injection for the 30010 mm i.d. col-umn). For Oxford Clay sample P89-4, 1 ml samples were collected for fractions 1±6 and 4 ml samples for fraction 7, whereas for sample B89-8, 1.5 ml samples were collected for fractions 1 and 2, 1 ml samples for fractions 3±6 and 4.5 ml samples for fractions 7 and 8. Fractions were dried under N2and the yield determined
by weight of the fraction when possible.
3.3. GC, GC±MS and irm-GC±MS analyses
Relative concentrations in the sterane/hopane com-pound mixtures were determined at AGSO by gas chromatography using a Hewlett-Packard 5890 Series II GC equipped with ¯ame ionization detector and using hydrogen as the carrier gas. The GC was ®tted with a 25 m 0.2 mm i.d. HP Ultra-1 column and programmed from 60 to 300C at 6C/min. The samples were injected
using an Hewlett-Packard cold on-column injector. Compounds in the extracts of the Oxford Clay were identi®ed by gas chromatography±mass spectrometry at AGSO using a Finnigan INCOS 50 GCMS. The GCMS was ®tted with a 30 m0.25 mm i.d. J&W DB-5 column and was programmed from 60 to 300C at 6C/min with helium (30 psi) as carrier. Samples were injected with a Varian SPI Injector at 60C and temperature pro-grammed from 50 to 300C at 100C/min. The MS source was operated at 250C and 70 eV.
Compound-speci®c isotopic analyses were performed by irm-GC±MS at the University of Hawaii using a Finnigan Delta-S with a Hewlett-Packard 5890 GC. The GC was equipped with a 50 m0.32 mm i.d. Ultra-1 column (Hewlett-Packard) with a ®lm thickness of 0.52 mm and used He as the carrier gas. The column was temperature programmed from 50 to 150C at 10C/ min, from 150 to 320C at 3C/min and then held at 320C for 30 min. Samples were injected using a Hew-lett-Packard cold on-column injector. All compound-speci®c isotopic results reported in this study were col-lected using techniques described by Hayes et al. (1990), Merritt and Hayes (1994), and Merritt et al. (1995). Carbon isotopic compositions are reported in standard d-notation where all values ofdrefer tod13C relative to
the Pee Dee belemnite (PDB) standard.
4. Results and discussion
4.1. Standard
Shape selective chromatography of US-Y zeolite was tested for possible isotopic chromatographic eect with a 60:40 wt.% mixture of 5a-cholestane and 17b, 21b-hopane of known isotopic composition (Fig. 1). 17b, 21b-Hopane was preferentially retained on the US-Y medium and the separation produced nearly pure 5a-cholestane in fraction 1 (Fig. 1b). Post separation isotopic analyses fall within the range of uncertainty of thed-values of the compounds in the original mixture (Fig. 1a). These results suggest that there is no systematic isotopic dis-crimination associated with separation of these com-pounds using US-Y zeolite. Concentrations of 17b, 21b -hopane in fraction 1 and concentration of 5a-cholestane in fraction 6 were below the detection limit of the irm-GC±MS used in this study. The response of the Refractive Index detector shown in Fig. 1c was typical for this procedure and indicates only the beginning and end of the elution of compounds.
4.2. Natural mixture of hydrocarbons
biomarkers in extracts of immature sediments can be resolved suciently for CSIA without inducing isotopic fractionation. The original silicalite non-adduct and the fractions separated by US-Y zeolite are shown in Fig. 2a (sample P89-4) and Fig. 3a (sample B89-8).
4.2.1. Separation of hydrocarbons
The trace of the silicalite non-adduct (SNA) hydro-carbon fractions and subfractions obtained by US-Y phase HPLC are shown in Figs. 2 (sample P89-4) and 3 (sample B89-8). For both samples, acyclic isoprenoids are concentrated in early eluting fractions. This is parti-cularly evident for sample B89-8 in which fraction 1 (Fig. 3b) exclusively contains the acyclic isoprenoids pristane, phytane (not shown in ®gure), squalane (1), lycopane (34) and an unidenti®ed acyclic isoprenoid (32). Pristane and phytane were the two major com-pounds of the SNA hydrocarbon fraction of sample B89-8 with squalane and lycopane undetected. In both samples, pristane and phytane were the most abundant compounds of fraction 1 and 2, very minor compounds in fraction 3 and not detectable at all in the later eluting fractions.
Separation of steroidal hydrocarbons by US-Y zeolite followed stereochemical con®guration. For sample P89-4, early eluting fractions preferentially contained 5a -steranes whereas later eluting fractions exclusively held the 5bstereoisomers (Fig. 2, Table 1). For example, the most abundant steranes in fractions 1±3 are 5a -choles-tane (peak3, Fig. 2, Table 1) and 5a-24-ethylcholestane (16). These compounds were virtually absent from frac-tions 4±7. On the other hand, 5b-cholestane (2), and 5b -24-ethylcholestane (12) were absent from fractions 1 but the dominant peaks in fractions 3±7 (Fig. 2). The 5b,14a,17a-steroid hydrocarbons were the only com-pounds eluting in fractions 5±7. For sample B89-8 (Fig.3), all the 5b,14a,17a;- steroid hydrocarbons (2,5,
12,19), and 4-methyl-5b,14a,17a-steroid hydrocarbons (4, 11) were concentrated in fraction 8 and perfectly resolved from the co-eluting compounds that were evident in the intact SNA fraction. 5a-Steroid hydrocarbons were concentrated in fractions 2±4 (Fig. 3c±e). It is also important to note that 4b-23,24-trimethylcholestane (dinosterane, 25) was a component of an unresolved mixture in the SNA trace (Fig. 3a) and coeluting with 4b-methyl-24-ethylcholestane (26) in fraction 3 (Fig. 3d). Fig. 1. Results of shape-selective sorption chromatographic
separation of 17b,21bH)-hopane and 5a-cholestane. (a) Plot of carbon isotopic composition of compounds in the original mixture and as a function of retention time. Error bars are one standard deviation of the average of two or more isotopic ana-lyses (n). (b) Plot of relative concentration of 17b,21b(H)-hopane and 5a-cholestane in the original mixture and as a function of retention time. (c) Plot showing the response of the refractive index detector.
In fractions 4 and 5 (Fig. 3e±f), however, dinosterane was completely free from co-eluting hydrocarbons.
Dierences in the distribution of compounds in the subfractions of the two samples (compare Figs. 2 and 3) resulted from changes in the collection times of the fractions, from dierences in the activation of the US-Y zeolite and probably from density dierences of the zeolite into the column.
Armanios et al. (1992, 1994) showed that liquid chromatography using US-Y zeolite as a stationary phase provided a means of separating hopanoid classes on the basis of their shapes and size. These authors did not report on separation of other compounds (i.e. acyc-lic isoprenoids, tetracycacyc-lic triterpanes). We have shown here that acyclic isoprenoids are least retained by US-Y zeolite, as expected from its shape selective properties. Experimental separations using the SNA fraction from the immature Oxford Clay also revealed that US-Y zeolite separates steroidal hydrocarbons on the basis of their stereochemistry at C5 and also gives a partial resolution of steranes from hopanes.
4.2.2. Isotopic analysis
Tables 2 and 3 summarize the results of compound speci®c carbon isotope analysis of SNA hydrocarbon subfractions of Oxford Clay samples obtained by US-Y phase-HPLC. As with the hopane-sterane standard mixture, US-Y phase chromatography of the SNA hydrocarbons does not produce an observable isotope eect (Tables 2 and 3). For example, isotopic values of pure 5b-cholestane (Table 2) are within the analytical uncertainty even though this compound was analyzed in six separate fractions. Similarly, 24-ethyl-5b-cholestane (Table 2) and 17b,21b(H) homohopane (Table 3) gave well correlatedd13C values when analyzed in ®ve
sepa-rate fractions. Pristane and phytane were only present in one or two subfractions and their d13C values were
indistinguishable (Tables 2 and 3). The isotopic compo-sitions of squalane (1) and lycopane (34), compounds which were not resolved from the background in the Fig. 3. Partial total ion current traces of (a) silicalite
non-adduct fraction and (b)±(i) US-Y separated subfractions (F1± F8) of the silicalite non-adduct hydrocarbon fraction of Oxford Clay sample B89-8. The numbered peaks are identi®ed on Table 1. Numbers in parentheses refer to the collection time (min) of the subfractions.
Table 1
Compounds identi®ed in the silicalite non-adduct hydrocarbon fraction and US-Y separated hydrocarbon fractions of sample P89-4 (Fig. 2) and B89-8 (Fig. 3)
Peak Structure
1 2,6,10,15,19,23-Hexamethyltetracosane (squalane) 2 5b-Cholestane
3 5a-Cholestane
4 5b(H)-4a-Methylcholestane 5 24-Methyl-5b-cholestane 6 17aH)-Trisnorhopane 7 5aH)-4a-Methylcholestane 8 5aH)-24-Methylcholestane 9 17b(H)-Trisnorhopane 10 5aH)-4b-Methylcholestane 11 5b(H)-4a-24-Dimethylcholestane 12 5b-24-Ethylcholestane
13 17a,21bH)-Bisnorhopane 14 5a-4a,24-Dimethylcholestane
15 23,24-Dimethylcholestane (4-desmethyldinosterane) 16 5a-24-Ethylcholestane
17 17b,21a(H)-Bisnorhopane 18 17a,21bH)-Norhopane 19 5b-24-Propylcholestane 20 5a(H)-4b,24-Dimethylcholestane 21 17b,21aH)-Norhopane
22 4a,23,23-Trimethylcholestane (dinosterane)
23 4a-Methyl-24-ethyl-cholestane+5a(H)-24-propylcholestane 24 17a,21b(H)-Hopane
25 4b,23,24-Trimethylcholestane (dinosterane) 26 4b-Methyl-24-ethyl-cholestane
27 17b,21aH)-Hopane 28 17a,21bH)-Homohopane 29 17b,21bH)-Hopane 30 17b,21aH)-Homohopane 31 17a,21bH)-Bishomohopane 32 Unidenti®ed acyclic isoprenoid 33 17b,21bH)-Homohopane
SNA fraction (Fig. 3a), were measurable for fraction 1 (Fig. 3b, Table 3).
Isotopic variability beyond the analytical uncertainty was observed for some compounds (e.g., 24-methyl-5a -cholestane (8), 24-ethyl-5a-cholestane (16); Table 2).
However, this is almost certainly due to the presence, in some fractions, of co-eluting components rather than an isotope eect associated with chromatography. 24-Methyl-5a-cholestane (8) coelutes with 17b (H)-trisnor-hopane (9) in the SNA fraction and in fractions 1 to 4
Table 2
Results of compound speci®c carbon isotopic analyses of US-Y separated subfractions of Oxford Clay sample P89-4 with standard deviations (n=2). Numbers in parentheses refer to peak numbering in Fig. 2 and Table 1
Compound d13C (%vs. PDB)
Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7
Pristane ÿ32.00.1
Phytane ÿ32.20.1 ÿ32.00.1
5b-Cholestane (2) ÿ31.00.8a
ÿ31.80.5 ÿ31.40.3 ÿ31.70.5 ÿ31.80.6 ÿ31.60.1 5a-Cholestane (3) ÿ32.10.3 ÿ32.11.1 ÿ30.60.2b
4a-Methyl-5b-cholestane (4) ÿ32.20.4
24-Methyl-5b-cholestane (5) ÿ31.00.1b
ÿ31.40.6 ÿ31.70.1 4a-Methyl-5a-cholestane (7) ÿ31.10.3 ÿ30.70.9 ÿ31.70.3b
24-Methyl-5a-cholestane (8) ÿ31.20.1 ÿ31.41.0 ÿ30.70.5 ÿ29.60.8b 4b-Methyl-5a-cholestane (10) ÿ32.60.1 ÿ30.90.5
24-Ethyl-5b-cholestane (12) ÿ30.60.3 ÿ30.80.7 ÿ31.00.1 ÿ30.90.9 ÿ30.80.1 4a,24-Dimethyl-5a-cholestane (14) ÿ33.00.4
24-Ethyl-5a-cholestane (16) ÿ30.60.2b
ÿ29.80.2b
ÿ30.40.4b
ÿ29.40.3c 4b,24-Dimethyl-5a-cholestane (20) ÿ32.40.6 ÿ32.50.1 ÿ31.80.1 ÿ31.70.3 17a,21b(H)-Hopane (28) ÿ29.10.6
17b,21b(H)-Homohopane (33) ÿ28.70.3 ÿ28.41.0
a Values obtained on low intensity peaks.
bValues obtained on compounds coeluting with others.
Table 3
Results of compound speci®c carbon isotopic analyses of US-Y separated subfractions of Oxford Clay sample B89-8 with standard deviations (n=2). Numbers in parentheses refer to peak numbering in Fig. 3 and Table 1
Compound d13C (%vs. PDB)
Fraction 1 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8
Pristane ÿ31.70.1
Phytane ÿ31.30.1 ÿ31.10.1
Squalane (1) ÿ31.30.5
Lycopane (34) ÿ29.40.1
5b-Cholestane (2) ÿ31.90.1
5a-Cholestane (3) ÿ31.70.4 ÿ32.10.5
4a-Methyl-5b-cholestane (4) ÿ30.60.7a
24-Methyl-5b-cholestane (5) ÿ32.50.1
24-Methyl-5a-cholestane (8) ÿ29.90.4b
4a-24-Dimethyl-5b-cholestane (11) ÿ30.81.3a
24-Ethyl-5b-cholestane (12) ÿ31.20.4
24-Ethyl-5a-cholestane (16) ÿ29.60.7b
24-Propyl-5b-cholestane (19) ÿ31.40.1a
4b-23,24-trimethyl-5a-cholestane (25) ÿ28.61.0
17a(H)-Trisnohopane (6) ÿ28.00.7
17a,21bH)-Hopane (24) ÿ28.30.1 ÿ28.20.3 ÿ28.10.6 ÿ27.30.5 ÿ28.00.7
17b,21aH)-Hopane (27) ÿ27.60.6
17a,21b(H)-homohopane (28) ÿ27.90.3 ÿ28.50.5 ÿ28.60.1 ÿ27.90.1 ÿ28.40.8 17b,21b(H)-Homohopane (33) ÿ27.70.3 ÿ28.21.0 ÿ27.80.1 ÿ28.00.6 ÿ28.20.2 17b,21bH)-Bishomohopane (35) ÿ27.90.1 ÿ28.30.4 ÿ28.1n=1 ÿ27.90.1
a Values obtained on low intensity peaks.
(Fig. 2). Similarly, 24-ethyl-5a-cholestane (16) coelutes partly with 23,24-dimethylcholestane (15) and 17b,21a(H)-bisnorhopane (17) in the SNA fraction and in fractions 1±4 (Fig. 2). Isotopic compositions of 24-methyl-5b-cholestane of sample P89-4 was measured in fractions 3±5 (Table 2). In fraction 3, 24-methyl-5b -cholestane is not completely resolved from 4a -methyl-5a-cholestane (7; Fig. 2d) but is well resolved in frac-tions 4 and 5 (Fig. 2e and f). This explains the dierent isotopic composition of 24-methyl-5b-cholestane in fractions 3, 4 and 5 (Table 2). Compounds which were well isolated (e.g. 5b-cholestane, 24-ethyl-5b-cholestane) showed less variability than compounds which were in a complex portion of the chromatogram. Although sig-ni®cantly more work is involved, accuracy and con-®dence in measurements of the isotopic compositions of complex mixtures of acyclic and polycyclic hydro-carbons can be enhanced with the aid of separations based on shape-selective US-Y zeolite.
5. Conclusions
Shape-selective chromatography on US-Y zeolite can be used to improve the separation of complex mixtures of tetra- and pentacyclic compounds by yielding discrete fractions containing simple hydro-carbon mixtures. US-Y zeolite preferentially retains tetra- and pentacyclic triterpenoids versus acyclic iso-prenoids. Steroid hydrocarbons are separated on the basis of their stereochemical con®guration, with 5b(H) isomers preferentially retained over their 5a(H) coun-terparts. Pentacyclic triterpenoids are also separated from steroids and are separated on the basis of their stereochemical con®guration similarly to the observations by Armanios et al. (1992, 1994). These results indicate that US-Y zeolite HPLC is sensitive to both compound type and stereochemical con®guration and is in agree-ment with the cross-sectional dimension selectivity of US-Y zeolite suggested by Armanios et al. (1992, 1994).
No isotopic fractionation was observed during pre-parative US-Y phase-HPLC of an arti®cial mixture of standard compounds and of natural hydrocarbon frac-tions. The shape selective properties of US-Y zeolite do not involve the phase/eluent interaction (sorption/des-orption) that underlies the origin of chromatographic eect in reverse phase HPLC and gas chromatography. Separation of silicalite non-adduct hydrocarbon frac-tions of Oxford Clay shales (Callovian, UK) with US-Y zeolite allowed compound speci®c isotopic analyses of biomarkers that usually are not resolved by other means of preparative separation. Therefore, US-Y zeolite HPLC can be used to produce subfractions amenable to compound speci®c isotope analysis from complex mix-tures of hydrocarbons.
Acknowledgements
We wish to thank Janet Hope (AGSO) and Terri Rust (UH) for their patience and assistance with this research. This work was partially supported by NSF-EAR 9304401 grant to BNP and NSF-NSF-EAR 9614769 grant to F.K. We would like to thank Joe Werne and Bob Alexander for their reviews. This is SOEST con-tribution 5249.
Associate EditorÐS. George
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