Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue11.Nov2000:

Organic Geochemistry 31 (2000) 1087±1094
www.elsevier.nl/locate/orggeochem

Preparative HPLC with ultrastable-Y zeolite for
compound-speci®c carbon isotopic analyses
Fabien Kenig a,b,*, Brian N. Popp a, Roger E. Summons c
a
Department of Geology and Geophysics, University of Hawaii, 2525 Correa Road, Honolulu, HI 96822 USA
Department of Earth and Environmental Sciences (M/C 186), University of Illinois at Chicago, 845 W. Taylor Street, Chicago,
IL 60607-7059, USA
c
Australian Geological Survey Organisation, PO Box 378, Canberra, 2601 Australia

b

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 e€ects. 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 e€ect; Oxford
Clay

1. Introduction
Extractable organic matter in contemporary oceanic
environments and in preserved sediments comprises
complex mixtures of organic compounds. This complexity 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 isotopic analysis (CSIA, e.g. Matthews and Hayes, 1978;

* Corresponding author. Tel.: +1-312-996-3020; fax: +1312-413-2279.
E-mail address: fkenig@uic.edu (F. Kenig).

Hayes et al., 1990) has enabled a much clearer de®nition
of geospheric organic matter origins through measurement 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 compositions 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
e€ects (Liberti et al., 1965; Hook, 1969; Gunter and

Gleason, 1971), Hayes et al. (1990) observed that capillary gas chromatographic columns tend to slightly
separate organic molecules enriched in 13C from those
more depleted in 13C. 13C-enriched molecules elute at

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1088

F. Kenig et al. / Organic Geochemistry 31 (2000) 1087±1094

the front of the chromatographic peak while 13C depleted counterparts elute toward the tail (Hayes et al.,
1990). Accordingly, an accurate carbon isotopic composition for a single compound using irm-GC±MS can
only be obtained when the entire peak, free from coeluting 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 chlorophyll-a obtained by C18 reversed phase HPLC. An
isotopic chromatographic e€ect equivalent to that

observed on capillary columns, with 13C enriched peak
front and 13C depleted peak tail, was observed by Martin
Schoell (1996, personal communication) during reverse
phase HPLC preparative separation of steranes, with a
maximum 13C 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 crosssectional di€erences 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
e€ects 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 e€ects,
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 suciently 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 University) and activated at 350 C overnight, followed by
storage at 120 C.

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 stratigraphy 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 a d13CTOC vs. PDB of
ÿ27.4 %. B89-8 is a deposit feeder bituminous shale
with 4.2 wt.% TOC and a d13CTOC vs. PDB of ÿ26.4 %
(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
(419 C), 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 petroleum generation (Kenig et al., 1994a). Isotopic compositions 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 of n-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 dichloromethane and methanol (1:1) for 48 h. Approximately 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.
The extractable materials were separated using column chromatography on silica-gel (12 g, Merck 40, 70±
230 mesh). A hydrocarbon fraction was eluted with 40
ml of petroleum ether, an aromatic fraction was eluted
with 50 ml of petroleum ether and dichloromethane
(1:1) followed by elution of a polar fraction with 40 ml

of chloroform:methanol (1:1). The hydrocarbon fraction was further separated into saturated and unsaturated fractions using silica-gel (12 g, Merck 40, 70±230

F. Kenig et al. / Organic Geochemistry 31 (2000) 1087±1094

mesh) impregnated with 10% AgNO3 in petroleum ether.
Saturated hydrocarbons were eluted with 50 ml of petroleum ether and the unsaturated hydrocarbons were eluted
with 50 ml of petroleum ether:dichloromethane (1:1).
Finally, the n-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 300  10 mm i.d. or 2 g packed into a 250  4.6 mm
i.d. stainless steel HPLC column. The small diameter
column was used to separate the sterane/hopane standard 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 70 C for 1 h

prior to use. Each column was washed with approximately three bed volumes of n-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 column at a constant ¯ow rate of 0.5 ml/min. Six consecutive fractions were collected in 2 minute intervals
beginning at 10 min. Approximately 3 mg of the Silicalite non-adducted Oxford Clay material was dissolved in
60 ml n-pentane and 50 ml injected onto the 300  10 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 300  10 mm i.d. column). 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 N2 and 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 compound 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 300 C at 6 C/min. The samples were injected

1089

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 m  0.25 mm i.d. J&W DB-5 column
and was programmed from 60 to 300 C at 6 C/min with
helium (30 psi) as carrier. Samples were injected with a
Varian SPI Injector at 60 C and temperature programmed from 50 to 300 C at 100 C/min. The MS
source was operated at 250 C 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 m  0.32 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 150 C at 10 C/
min, from 150 to 320 C at 3 C/min and then held at
320 C for 30 min. Samples were injected using a Hewlett-Packard cold on-column injector. All compoundspeci®c isotopic results reported in this study were collected 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 of d refer to d13C 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 e€ect 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 the d-values of the
compounds in the original mixture (Fig. 1a). These
results suggest that there is no systematic isotopic discrimination associated with separation of these compounds using US-Y zeolite. Concentrations of 17b, 21bhopane in fraction 1 and concentration of 5a-cholestane
in fraction 6 were below the detection limit of the irmGC±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
The silicalite non-adduct fraction of two samples of
the Oxford Clay (Callovian, UK) were separated using
US-Y zeolite to determine if co-eluting hydrocarbon

1090

F. Kenig et al. / Organic Geochemistry 31 (2000) 1087±1094

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 analyses (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.

biomarkers in extracts of immature sediments can be
resolved suciently 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) hydrocarbon 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 particularly 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 compounds 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.

Fig. 2. Partial total ion current traces of (a) silicalite nonadduct fraction and (b)±(h) US-Y separated subfractions (F1±
F7) of the silicalite non-adduct hydrocarbon fraction of Oxford
Clay sample P89-4. The numbered peaks are identi®ed in Table
1. Numbers in parentheses refer to the collection time (min) of
the subfractions.

Separation of steroidal hydrocarbons by US-Y zeolite
followed stereochemical con®guration. For sample P894, early eluting fractions preferentially contained 5asteranes whereas later eluting fractions exclusively held
the 5b stereoisomers (Fig. 2, Table 1). For example, the
most abundant steranes in fractions 1±3 are 5a-cholestane (peak 3, Fig. 2, Table 1) and 5a-24-ethylcholestane
(16). These compounds were virtually absent from fractions 4±7. On the other hand, 5b-cholestane (2), and 5b24-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 compounds 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).

F. Kenig et al. / Organic Geochemistry 31 (2000) 1087±1094

1091

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

Fig. 3. Partial total ion current traces of (a) silicalite nonadduct 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.

In fractions 4 and 5 (Fig. 3e±f), however, dinosterane
was completely free from co-eluting hydrocarbons.
Di€erences 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 di€erences in the activation of the US-Y
zeolite and probably from density di€erences 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. acyclic isoprenoids, tetracyclic 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.

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

2,6,10,15,19,23-Hexamethyltetracosane (squalane)
5b-Cholestane
5a-Cholestane
5b(H)-4a-Methylcholestane
24-Methyl-5b-cholestane
17aH)-Trisnorhopane
5aH)-4a-Methylcholestane
5aH)-24-Methylcholestane
17b(H)-Trisnorhopane
5aH)-4b-Methylcholestane
5b(H)-4a-24-Dimethylcholestane
5b-24-Ethylcholestane
17a,21bH)-Bisnorhopane
5a-4a,24-Dimethylcholestane
23,24-Dimethylcholestane (4-desmethyldinosterane)
5a-24-Ethylcholestane
17b,21a(H)-Bisnorhopane
17a,21bH)-Norhopane
5b-24-Propylcholestane
5a(H)-4b,24-Dimethylcholestane
17b,21aH)-Norhopane
4a,23,23-Trimethylcholestane (dinosterane)
4a-Methyl-24-ethyl-cholestane+5a(H)-24-propylcholestane
17a,21b(H)-Hopane
4b,23,24-Trimethylcholestane (dinosterane)
4b-Methyl-24-ethyl-cholestane
17b,21aH)-Hopane
17a,21bH)-Homohopane
17b,21bH)-Hopane
17b,21aH)-Homohopane
17a,21bH)-Bishomohopane
Unidenti®ed acyclic isoprenoid
17b,21bH)-Homohopane
2,6,10,14,19,23,27,31-Octamethyldotriacontane (lycopane)
17b,21bH)-Bishomohopane

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
e€ect (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 correlated d13C values when analyzed in ®ve separate fractions. Pristane and phytane were only present in
one or two subfractions and their d13C values were
indistinguishable (Tables 2 and 3). The isotopic compositions of squalane (1) and lycopane (34), compounds
which were not resolved from the background in the

1092

F. Kenig et al. / Organic Geochemistry 31 (2000) 1087±1094

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-5acholestane (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 e€ect associated with chromatography. 24Methyl-5a-cholestane (8) coelutes with 17b(H)-trisnorhopane (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

Pristane
Phytane
5b-Cholestane (2)
5a-Cholestane (3)
4a-Methyl-5b-cholestane (4)
24-Methyl-5b-cholestane (5)
4a-Methyl-5a-cholestane (7)
24-Methyl-5a-cholestane (8)
4b-Methyl-5a-cholestane (10)
24-Ethyl-5b-cholestane (12)
4a,24-Dimethyl-5a-cholestane (14)
24-Ethyl-5a-cholestane (16)
4b,24-Dimethyl-5a-cholestane (20)
17a,21b(H)-Hopane (28)
17b,21b(H)-Homohopane (33)
a
b

ÿ32.00.1
ÿ32.20.1

Fraction 2

ÿ32.10.3

ÿ32.00.1
ÿ31.00.8a
ÿ32.11.1

ÿ31.10.3
ÿ31.20.1
ÿ32.60.1

ÿ30.70.9
ÿ31.41.0
ÿ30.90.5

ÿ33.00.4
ÿ30.60.2b
ÿ32.40.6
ÿ29.10.6
ÿ28.70.3

ÿ29.80.2b
ÿ32.50.1

Fraction 3

Fraction 4

Fraction 5

Fraction 6

Fraction 7

ÿ31.80.5
ÿ30.60.2b
ÿ32.20.4
ÿ31.00.1b
ÿ31.70.3b
ÿ30.70.5

ÿ31.40.3

ÿ31.70.5

ÿ31.80.6

ÿ31.60.1

ÿ31.40.6

ÿ31.70.1

ÿ29.60.8b

ÿ30.60.3

ÿ30.80.7

ÿ30.90.9

ÿ30.80.1

ÿ30.40.4b
ÿ31.80.1

ÿ29.40.3c
ÿ31.70.3

ÿ31.00.1

ÿ28.41.0

Values obtained on low intensity peaks.
Values 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

Pristane
Phytane
Squalane (1)
Lycopane (34)
5b-Cholestane (2)
5a-Cholestane (3)
4a-Methyl-5b-cholestane (4)
24-Methyl-5b-cholestane (5)
24-Methyl-5a-cholestane (8)
4a-24-Dimethyl-5b-cholestane (11)
24-Ethyl-5b-cholestane (12)
24-Ethyl-5a-cholestane (16)
24-Propyl-5b-cholestane (19)
4b-23,24-trimethyl-5a-cholestane (25)
17a(H)-Trisnohopane (6)
17a,21bH)-Hopane (24)
17b,21aH)-Hopane (27)
17a,21b(H)-homohopane (28)
17b,21b(H)-Homohopane (33)
17b,21bH)-Bishomohopane (35)
a
b

ÿ31.70.1
ÿ31.30.1
ÿ31.30.5
ÿ29.40.1

Fraction 3

Fraction 4

Fraction 5

Fraction 6

Fraction 7

Fraction 8

ÿ31.10.1

ÿ31.90.1
ÿ31.70.4

ÿ32.10.5
ÿ30.60.7a
ÿ32.50.1

ÿ29.90.4b
ÿ30.81.3a
ÿ31.20.4
ÿ29.60.7b
ÿ31.40.1a

ÿ28.30.1

ÿ28.61.0
ÿ28.00.7
ÿ28.20.3

ÿ27.90.3
ÿ27.70.3
ÿ27.90.1

ÿ28.50.5
ÿ28.21.0
ÿ28.30.4

Values obtained on low intensity peaks.
Values obtained on compounds coeluting with others.

ÿ28.10.6

ÿ27.30.5

ÿ28.60.1
ÿ27.80.1

ÿ27.90.1
ÿ28.00.6
ÿ28.1 n=1

ÿ28.00.7
ÿ27.60.6
ÿ28.40.8
ÿ28.20.2
ÿ27.90.1

F. Kenig et al. / Organic Geochemistry 31 (2000) 1087±1094

(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 24methyl-5b-cholestane of sample P89-4 was measured in
fractions 3±5 (Table 2). In fraction 3, 24-methyl-5bcholestane is not completely resolved from 4a-methyl5a-cholestane (7; Fig. 2d) but is well resolved in fractions 4 and 5 (Fig. 2e and f). This explains the di€erent
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 signi®cantly more work is involved, accuracy and con®dence in measurements of the isotopic compositions of
complex mixtures of acyclic and polycyclic hydrocarbons 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 hydrocarbon mixtures. US-Y zeolite preferentially retains
tetra- and pentacyclic triterpenoids versus acyclic isoprenoids. Steroid hydrocarbons are separated on the
basis of their stereochemical con®guration, with 5b(H)
isomers preferentially retained over their 5a(H) counterparts. 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 agreement with the cross-sectional dimension selectivity of
US-Y zeolite suggested by Armanios et al. (1992,
1994).
No isotopic fractionation was observed during preparative US-Y phase-HPLC of an arti®cial mixture of
standard compounds and of natural hydrocarbon fractions. The shape selective properties of US-Y zeolite do
not involve the phase/eluent interaction (sorption/desorption) that underlies the origin of chromatographic
e€ect in reverse phase HPLC and gas chromatography.
Separation of silicalite non-adduct hydrocarbon fractions 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 mixtures of hydrocarbons.

1093

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 NSFEAR 9304401 grant to BNP and NSF-EAR 9614769
grant to F.K. We would like to thank Joe Werne and
Bob Alexander for their reviews. This is SOEST contribution 5249.
Associate EditorÐS. George

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