Anal. Chem.

1997, 69, 4203-4209

Liquid Chromatographic Separation of Sediment, Rock, and Coal Extracts and Crude Oil into Compound Classes
Helmut Willsch, Heather Clegg, Brian Horsfield, Matthias Radke, and Heinz Wilkes*

Institut fu r Erdo l und Organische Geochemie, Forschungszentrum Ju lich GmbH, 52425 Ju lich, Germany

A method is described for the rapid fractionation of sediment, rock, and coal extracts and crude oils into compound classes. The method is based on combined polarity/affinity chromatography of soluble organic matter. Five heterocompound fractions are obtained in addition to the conventional saturated and aromatic hydrocarbon fractions. Model compound studies show that those fractions are chemically well-defined. The applicability to a variety of geological materials is demonstrated by analyzing a North Sea crude oil and solvent extracts of Posidonia shale, coal, and recent lake sediment samples. Generally reproducibilities and linearities are satisfactory. The total recoveries vary between 76% and 57%, depending on the sample type. The method is shown to be wellsuited for the bulk compositional characterization of soluble organic matter. Separations can be performed on both the analytical and the semipreparative scales. Isolated fractions are amenable directly or after derivatization to qualitative and quantitative analysis by gas chromatography and gas chromatography/mass spectrometry.
Sediment, rock, and coal extracts, as well as crude oils, are composed of extremely complex mixtures of compounds belonging to a variety of compound classes. Beside the saturated and aromatic hydrocarbons, heterofunctionalized organic compounds containing nitrogen, oxygen, and sulfur (NSO compounds) are important constituents of soluble organic matter (SOM) from geological materials. A separation of SOM into compound classes is generally required prior to characterization at the molecular level. A procedure commonly used in fossil fuel research is the separation into saturates, aromatics, resins, and asphaltenes,1 and high throughput has been made possible by automated mediumpressure liquid chromatography.2 Saturates and aromatics have been analyzed in much detail, especially by gas chromatography and gas chromatography/mass spectrometry. The aromatic hydrocarbon fractions generally include a range of very low polar heterocompounds such as benzothiophenes, dibenzothiophenes, and dibenzofurans.3-5 Individual NSO compound groups occurring in the resin and asphaltene fractions such as porphyrins,6
(1) Snyder, L. R.; Buell, B. E.; Howard, H. E. Anal. Chem. 1968, 40, 13031317. (2) Radke, M.; Willsch, H.; Welte, D. H. Anal. Chem. 1980, 52, 406-411. (3) Galpern, G. D. Chem. Heterocycl. Compds. 1985, 44, 325-351. (4) Arpino, P. J.; Ignatiadis, I.; de Rycke, G. J. Chromatogr. 1987, 390, 329348. (5) Orr, W.; Sinninghe Damste , J. S. In Geochemistry of Sulfur in Fossil Fuels; Orr, W., White, C. M., Eds.; American Chemical Society: Washington, DC, 1990; pp 2-29. S0003-2700(97)00381-8 CCC: $14.00 1997 American Chemical Society

azaarenes,7 and carboxylic acids8 have been investigated at the molecular level. However, a substantial proportion has not been characterized to a comparable extent due to their extreme complexity. Prior work has been concentrated mostly on the isolation of distinct NSO compound classes from SOM. Often, selective extraction methods are applied as described for the isolation of, e.g., phenols,9,10 fatty acids,11 and basic nitrogen compounds such as benzoquinolines12 from different matrices. A nonaqueous ion exchange method has been used for the separation of liquid fossil fuels into acid, base, and neutral concentrates.13 The acid concentrate obtained in this way can be separated into subfractions using in situ tetraalkylammonium hydroxide-modified silica high performance liquid chromatography (HPLC).14 Li et al. describe a method for the isolation of basic and nonbasic azaarenes from crude oil based on the use of aminocyano-bonded silica HPLC columns.15 An overview concerning recent developments in the use of liquid chromatographic techniques in petroleum geochemistry is provided by Rowland and Revill.16 However, in most cases, the described methods are of limited applicability with respect to the analyzable materials and/or the isolable compound classes. The present paper describes a liquid chromatographic method for the fractionation of crude oils and bitumens into compound classes that substantially extends the capabilities of previous methods by Schmitter et al.17,18 It was our main objective to develop a method which allows both the determination of the bulk composition of SOM and the isolation of chemically defined fractions for further investigations. Moreover, the method should be applicable to a wide variety of different materials, provide good overall recoveries, reproducibilities, and linearities with varying
(6) Baker, E. W.; Palmer, S. E. In The Porphyrins, Vol. 1; Dolphin, D., Ed.; Academic Press: London, 1978; pp 486-552. (7) Schmitter, J. M.; Arpino, P. J. Mass Spectrom. Rev. 1985, 4, 87-121. (8) Seifert, K. Fortschr. Chem. Org. Naturst. 1975, 32, 1-49. (9) Ioppolo, M.; Alexander, R.; Kagi, R. I. Org. Geochem. 1992, 18, 603-609. (10) Bennett, B.; Bowler, B. F. J.; Larter, S. Anal. Chem. 1996, 68, 3697-3702. (11) Rezanka, T. J. Chromatogr. 1992, 627, 241-245. (12) Yamamoto, M.; Taguchi, K.; Sasaki, K. Chem. Geol. 1991, 93, 193-206. (13) Green, J. B.; Hoff, R. J.; Woodward, P. W.; Stevens, L. L. Fuel 1984, 63, 1290-1301. (14) Green, J. B. J. Chromatogr. 1986, 358, 53-75. (15) Li, M.; Larter, S. R.; Stoddart, D.; Bjory, M. Anal. Chem. 1992, 64, 13371344. (16) Rowland, S. J.; Revill, A. T. In Chromatography in the Petroleum Industry; Adlard, E. R., Ed.; Journal of Chromatography Library 56; Elsevier: Amsterdam, 1995; pp 127-141. (17) Schmitter, J. M.; Arpino, P. J.; Guiochon, G. J. Chromatogr. 1978, 167, 149158. (18) Schmitter, J. M.; Vajta, Z.; Arpino, P. J. In Advances in Organic Geochemistry 1979; Douglas, A. G., Maxwell, J. R., Eds.; Pergamon Press: Oxford, 1980; pp 67-76.

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amounts of organic material used for separation, and be suitable for automation. The procedure described here separates SOM into five fractions based on polarity and/or affinity to different sorbents. The fraction of lowest polarity is subsequently separated into three subfractions by medium-pressure liquid chromatography as described previously.2 EXPERIMENTAL SECTION Apparatus. The pump was a Knauer (Berlin, Germany) HPLC pump, Model C64, equipped with a preparative pump head. The injector was a Valco (Houston, TX) six-way rotary valve. The columns were standard 10 mm i.d. glass columns (Omnifit, Cambridge, U.K.). The three precolumns were 100 mm, and the main column was 250 mm in length. The tubing was 1/16 in. o.d. 0.8 mm i.d. PTFE tubing equipped with Omnifit gripper fittings. For the elution of the bases, acids, and high polarity compounds from the precolumns, pressurized solvent bottles were used for solvent delivery. The pressure rating was 0.7 bar. The samples were concentrated in a Zymark (Hopkinton, MA) Turbovap 500 sample concentrator. The fraction weights were determined with a Mettler (Greifensee, Switzerland) balance, Type AE163, showing a resolution of 0.01 mg. For solvent removal from the modified silica types, a Bu chi (Flawil, Switzerland) rotary evaporator, Type RE 110, was used. Materials. Analytical reagent grade solvents, silica gel (Type 100), sodium chloride, potassium hydroxide, and sulfuric acid were obtained from E. Merck (Darmstadt, Germany). The solvents were distilled prior to use. The prepacked LiChrosorb Si60 main column was obtained from MKW (Ju lich, Germany). Standard compounds were obtained from Aldrich, Serva, Fluka, and E. Merck. Samples and Sample Preparation. The sediment sample from Lake Holzmaar, Germany (E44488), was obtained from the depth interval from 30.0 to 35.0 cm of a gravity core taken on November 7, 1995. The freeze-dried, homogenized sample contained 4.3 wt % organic carbon. A sample of Jurassic (Lias epsilon) Posidonia shale (E38) was obtained by blasting from a quarry near Dotternhausen, Germany. It contained 7.8 wt % organic carbon. The Westphalian bituminous coal (E2235) was a core sample from Seam 14 of the Sandbochum borehole drilled in the Ruhr Basin, Germany. It contained 51.2 wt % organic carbon. The oil sample (E36129) was derived from a Middle Jurassic North Sea reservoir. Finely ground sediment, rock, and coal samples were extracted using the flow-blending method19 with an azeotropic mixture of chloroform (47 wt %), methanol (23 wt %), and acetone (30 wt %). After centrifugation of the extraction slurry, the supernatant liquid was decanted over a folded filter, concentrated in a Turbovap 500, and transferred into preweighed 10 mL vials. The residual solvent was removed by keeping the vials at normal pressure and ambient temperature in the dark for several hours. For separation, the samples were taken up in known amounts of dichloromethane with 1% methanol. For evaluation of evaporation losses, one aliquot was injected into a preweighed vial. After being dried, the vial was weighed again. Preparation of Silica Gel. For the separation, three different types of silica were used. Deactivated silica, Type 100, was prepared as described elsewhere.2 Acidic silica was prepared by the following procedure. Hydrogen chloride gas, produced by treating sodium chloride with concentrated sulfuric acid, was fed
(19) Radke, M.; Sittardt, H. G.; Welte, D. H. Anal. Chem. 1978, 50, 663-665.

into cold methanol. Type 100 silica was dispensed in this solution for 1 h. Subsequently, the solvent was removed using a rotary evaporator. The silica was stored at -20 C. Basic silica was prepared by treating 100 g of Type 100 silica with 100 mL of a solution of 5% potassium hydroxide in methanol. The solvent was removed using a rotary evaporator. Column Preparation. The three precolumns were filled with the appropriate silica using the tap-fill procedure. Precolumn 1 was filled with Type 100 silica, precolumn 2 with acidic silica, and precolumn 3 with basic silica. All column end-fittings were equipped with 5 m PTFE frits, except precolumn 1, whose inlet had no frit to prevent the system from plugging. All columns were mounted in series. Air was completely removed from the packings by washing with solvent (dichloromethane with 1% methanol). The three precolumns were repacked after each run. The main column was replaced after coloration became obvious. Procedure. Initial experiments were conducted manually. After injection of the sample, the low-polarity compounds and the hydrocarbons were collected for 5.5 min at a flow rate of 8 mL/ min, corresponding to an elution volume of 44 mL. The mediumpolarity fraction was eluted for an additional 7.5 min. The total elution volume was 104 mL. After this step, the precolumns were disconnected from the system. Each precolumn was connected to a pressurized PFA solvent bottle via a short PTFE tubing. The high-polarity fraction was washed from precolumn 1 with methanol. The elution volume was 50 mL. Precolumn 2, containing the basic compounds in the form of their corresponding hydrochlorides, was connected to a column filled with basic silica. The hydrochlorides were washed from precolumn 2 with methanol. The free bases were released from their salts on the column filled with basic silica. The elution volume was 50 mL. The acids were washed from precolumn 3 with dichloromethane containing 1% formic acid. The elution volume was 50 mL. All fractions were evaporated to dryness. The residue was taken up in a small volume of dichloromethane and transferred into preweighed vials. To examine the recoveries obtainable with solvents of higher polarity, all four columns were subsequently eluted with 50 mL of the azeotropic mixture used for extraction as described above. Yields from this elution step were determined gravimetically after removal of the solvent. For later separations, an MKW heterocompound class separator system was used. Once the sample is loaded to the sample loop, all further processing is done fully automatically. The total time required for the separation of one sample using this instrument is 20 min. Medium-Pressure Liquid Chromatography (MPLC). For the fractionation of low-polarity NSO compounds and hydrocarbons, an MKW MPLC system was used. Hexane solutions were injected into the system and automatically separated into three fractions, containing the saturated hydrocarbons, the aromatic hydrocarbons, and the low-polarity fraction, as described previously.2 Derivatization. Acid fractions were methylated using a solution of diazomethane in diethyl ether prior to gas chromatographic and gas chromatographic/mass spectrometric analysis. For derivatization of the medium-polarity NSO compounds, Silyl991 (Macherey & Nagel, Du ren, Germany) was used. Gas Chromatography (GC). For GC analysis of the saturated hydrocarbons, a Hewlett Packard 5890B instrument was used, equipped with an on-column injector with EPC and a standard

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flame ionization detector (FID). Hydrogen (1.6 bar at starting temperature) was used as the carrier gas. The gas chromatograph was equipped with an Ultra No. 1 fused silica capillary column (Hewlett Packard) of 50 m length, 0.2 mm i.d., and 0.33 m film thickness. The oven temperature was programmed from 90 C (hold time, 4 min) to 310 C (hold time, 50 min) at 3 C/min. The aromatic hydrocarbons were analyzed using a Hewlett Packard 5890B instrument which was equipped with an on-column injector with EPC, FID, and a sulfur-selective Hall electrolytic conductivity detector (HECD). Helium (4.4 bar at starting temperature) was used as the carrier gas. The gas chromatograph was equipped with a BPX5 fused silica capillary column (SGE) of 50 m length, 0.15 mm i.d., and 0.25 m film thickness. The oven temperature was programmed from 90 C (hold time, 4 min) to 120 C (hold time, 1 min) at 50 C/min and then to 310 C (hold time, 50 min) at 3 C/min. The polar fractions were analyzed using a Hewlett Packard 5890B instrument which was equipped with a temperature-programmable injection system (Gerstel KAS 3), FID, and a nitrogen-specific HECD. Helium (3.5 bar) was used as the carrier gas. The gas chromatograph was equipped with a BPX5 fused silica capillary column (SGE) of 50 m length, 0.15 mm i.d., and 0.25 m film thickness. The oven temperature was programmed from 120 C (hold time, 2 min) to 310 C (hold time, 50 min) at 3 C/min. The GC data were stored and processed using a Multichrom 2 on-line data system (VG Data Systems, Fisons Instruments). Gas Chromatography/Mass Spectrometry (GC/MS). For GC/MS analysis, a Finnigan MAT 95SQ mass spectrometer was used that was coupled to a Hewlett Packard 5890B gas chromatograph. The mass spectrometer was operated in EI mode at an ionization energy of 70 eV and a source temperature of 260 C. Full-scan mass spectra were recorded over the mass range of 35800 Da at a scan cycle time of 1.4 s. The gas chromatograph was equipped with a temperature-programmable injection system (Gerstel KAS 3) and a BPX5 fused silica capillary column (SGE) of 50 m length, 0.22 mm i.d., and 0.33 m film thickness. Helium was used as the carrier gas. The oven temperature was programmed from 60 to 340 C (final hold time, 8 min) at 3 C/min. RESULTS AND DISCUSSION General Description of the Method. A schematic outline of the method is given in Figure 1. The system consists of three precolumns and one main column from which five fractions are obtained. High-, medium-, and low-polarity compounds including aromatics and saturates are separated according to their polarity. Acids and bases are retained according to their affinity to modified basic and acidic silica, respectively. In the present procedure, conventional MPLC2 is used to subdivide the low-polarity compounds into saturates, aromatics, and low-polarity NSO compounds. Notably, at least a portion of the asphaltenes will be lost because, for MPLC, n-hexane solutions of SOM are used. Thus, NSO fractions obtained by MPLC do not necessarily represent the total NSO compounds of SOM. The present method circumvents this problem by starting the separation procedure with solutions of SOM in dichloromethane containing 1% methanol. For none of the materials investigated in this study was a loss of bitumen due to insolubility in the starting solvent observed. The yield and composition of SOM from sediments and coals strongly depend on the solvent used for extraction. The azeotropic mixture of chloroform, methanol, and acetone used in the present study has two main advantages. First, the solvent has proved

Figure 1. Schematic representation of the liquid chromatographic separation procedure.

suitable for the extraction of structurally diverse organic compounds from geological samples.20 Second, due to the azeotropic nature of the solvent, its composition does not change when solvent is being evaporated during extraction. Other solvent systems may also be used; however, it cannot be excluded that, in such cases, a different behavior of SOM during compound class separation will be observed, especially when using solvents of higher polarity for extraction. It should be noted that, according to our observations, high amounts of salts are coextracted when using the azeotropic mixture for young marine sediments. Model Compound Study. The elution behavior of the 86 compounds of potential geochemical significance investigated is demonstrated by Table 1, where compounds eluting in each fraction are listed in the order of increasing retention volume. Among the low-polarity NSO compounds, 2-nonadecanone was eluted later than all other ketones, aldehydes, carboxylic acid esters, and nonbasic nitrogen compounds examined. Generally, ethers also occurred in this fraction. However, heterocyclic aromatic ethers such as dibenzofuran and dibenzothiophene were eluted already, along with the aromatic hydrocarbons. Finally, phenols bearing alkyl substituents in the ortho position, with the exception of 2-methylphenol, were also constituents of the lowpolarity NSO fraction. All tested alcohols and alkylphenols, with the exception of o-alkylphenols, showed higher retention volumes than 2-methylphenol and were contained in the medium-polarity NSO fraction. Considering these results, we conclude that, with the present method, a chemically well-defined compound class separation for medium- and low-polarity NSO compounds is achieved. For bases and acids, the retention volumes were determined for their elution from precolumns 1 and 2, respectively.
(20) Given, P. H. In Coal Science, Vol. 3; Gorbaty, M. L., Larsen, J. W., Wender, I., Eds.; Academic Press: London, 1984; pp 63-252.

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Table 1. Retention Volumes of Standard Compounds compound squalane n-hexadecane coronene benzo[ghi]perylene 1-phenylhexane chrysene perylene 1-methylfluorene dibenzofuran molecular formula retention volume (mL) compound molecular formula C17H36 C16H32 C12H8S C13H10 C14H10 C8H6S C7H8 C10H8 retention volume (mL) 27.2 27.6 27.8 27.9 28.0 28.1 28.2 28.3

Low-Polarity FractionsSaturates C30H62 26.9 2-methylhexadecane C16H34 27.1 1-hexadecene Low-Polarity FractionsAromatics C24H12 27.2 dibenzothiophene C22H12 27.3 fluorene C12H18 27.4 phenanthrene C18H12 27.7 benzothiophene C20H12 27.7 toluene C14H12 27.7 naphthalene C12H8O 27.8

Low-Polarity FractionsLow-Polarity NSO Compounds 5,10,15,20-tetraphenyl-21H,23H-porphinenickel(II) C44H28N4Ni 26.2 2,4-dimethylbenzaldehyde hexadecyl docosanoate C38H76O2 26.6 dibutyl ether trimyristine C45H86O6 27.1 dimethyl decanedioate 5R-cholestan-3-yl stearate C45H82O2 27.1 4-methylacetophenone diphenyl ether C12H10O 27.2 4-methyl-1-tetralone 2-methoxybiphenyl C13H12O 28.0 5-methoxy-1-tetralone 1,7-dimethoxynaphthalene C12H12O2 28.2 4-decanolide 10-nonadecanone C19H38O 28.9 2,6-dimethylphenol tetradecanal C14H28O 29.5 5-decanolide carbazole C12H9N 29.6 2-hexadecanone 9-fluorenone C13H8O 30.2 2-nonadecanone methyl benzoate C8H8O2 30.5 2-propylphenol indole C8H7N 30.8 2-(methylethyl)phenol 4-ethylbenzaldehyde C9H10O 31.1 2-ethylphenol 2-methylphenol 1-naphthol 1,12-dodecanediol 4-ethylbenzyl alcohol 1,2-dipalmitoyl glycerol 11-pentacosanol 1-monopalmitoyl rac-glycerol 4-methylbenzyl alcohol 9,10-diaminophenanthrene 3-aminofluoranthene 1-aminoanthracene 1-amino-7-nitrofluorene benzo[c]cinnoline 2-phenylpyridine cis-octadec-9-enoic acid 3,4-dimethylbenzoic acid trans-octadec-9-enoic acid 2-hydroxybenzoic acid decanedioic acid hexadecanedioic acid hexadecanoic acid Medium-Polarity Fraction C7H8O 48.3 2-pentacosanol C10H8O 51.6 phenol C12H26O2 54.2 2-hydroxydibenzofuran C9H12O 57.4 3-methylphenol C35H68O5 57.6 1-pentadecanol C25H52O 58.3 4-methylphenol C19H38O4 58.4 2-undecanol C8H10O 59.0 C14H12N2 C16H11N C14H11N C13H10N2O2 C12H8N2 C11H9N C18H34O2 C9H10O2 C18H34O2 C7H6O3 C10H18O4 C16H30O4 C16H32O2 Bases 5.6 6.1 6.5 13.0 19.2 22.6 Acids 17.2 19.2 19.5 19.5 19.9 20.0 20.1 acridine 3-phenylpyridine quinoline isoquinoline 4-phenylpyridine pyridine octadecanoic acid 2,5-dihydroxybenzoic acid 4-methylphenylacetic acid 16-hydroxyhexadecanoic acid 2-naphthoic acid phenylacetic acid 1-naphthoic acid

C9H10O C8H18O C12H22O4 C9H10O C11H12O C11H12O2 C10H18O2 C8H10O C10H18O2 C16H32O C19H38O C9H12O C9H12O C8H10O C25H52O C6H6O C12H8O2 C7H8O C15H32O C7H8O C11H24O

31.3 32.0 33.2 33.5 33.5 33.7 34.2 34.7 37.1 39.3 39.9 40.8 41.0 43.6 59.6 62.2 62.4 62.9 63.6 64.9 65.6

C13H9N C11H9N C9H7N C9H7N C11H9N C5H5N C18H36O2 C7H6O4 C9H10O2 C16H32O3 C11H8O2 C8H8O2 C11H8O2

22.6 27.9 28.6 31.5 31.8 33.4 20.5 20.6 20.7 22.0 28.4 29.0 30.9

The bases showed increasing retention volumes with increasing basicity. Low retention volumes were observed for aromatic amino compounds such as 1-aminoanthracene, while pyridine showed the largest retention volume. Most of the acids eluted within a relatively narrow retention volume range. A number of aromatic acids (e.g., benzoic acid, 1- and 2-naphthalenecarboxylic acid), however, showed an unexpected chromatographic behavior when injected as pure compounds; i.e., they were not at all or only very slowly eluted from precolumn 1 and/or precolumn 2. This effect was not observed when those compounds were injected together with small amounts of aliphatic acids (e.g., acetic acid) or when complex natural mixtures of SOM were applied. None of the model compounds was retained on precolumn 1. As discussed below, the high-polarity NSO compounds are more or less quantitatively important constituents of SOM, depending on
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sample origin. Considering the results of the present model compound study, no insight is obtained into the structural composition of this fraction. It can, however, be assumed that it consists of relatively complex polyfunctionalized compounds. Application to Geological Samples. SOM from four samples of different origin was analyzed in detail (Table 2). The North Sea crude oil, the Posidonia shale extract, and the coal extract are typical representatives of ancient organic matter for which different bulk compositions of SOM could be anticipated. The Lake Holzmaar sample was selected as an example of a subrecent sediment. Recovery. The highest recovery (76.3%) was obtained for the Posidonia shale extract, while lower recoveries were obtained for the North Sea crude oil (65.5%), the coal extract (65.8%), and the Lake Holzmaar sediment extract (56.6%) (Table 2). Evaporation

Table 2. Results of the Liquid Chromatographic Separation of Soluble Organic Matter of Different Geological Origins sample type (sample no.) North Sea crude oil (E36129) yield (%) acids bases high-polarity compds medium-polarity compds low-polarity compds aromatics saturates total I acids IIa bases IIa high-polarity compds IIa medium-polarity compds IIa total II total I + II
a

Posidonia shale extract (E38) SD 0.47 0.09 0.70 0.41 0.37 0.20 0.18 1.11 0.21 0.01 0.20 0.39 0.38

Westphalian coal extract (E2235) SD 0.88 0.24 1.90 0.44 1.01 0.10 0.11 2.9 0.39 0.14 2.18 0.42 2.26 RSD (%) 5.9 16.6 10.3 4.9 7.2 1.7 6.1 4.4 20.7 65.0 60.7 10.7 23.5

Lake Holzmaar sediment extract (E44488) yield (%) 6.5 1.0 32.6 5.8 4.2 0.5 7.0 56.6 1.1 0.2 2.5 3.1 6.8 63.4 SD 0.38 0.16 3.52 0.45 0.44 0.05 0.56 3.62 0.50 0.13 0.55 0.10 0.81 RSD (%) 5.9 16.5 10.8 7.9 10.4 9.6 8.0 6.4 49.8 56.8 22.3 3.2 11.9

SD 0.04 0.17 0.36 0.21 0.27 0.42 0.64 0.73 0.12 0.08 0.18 0.36 0.41

RSD (%) yield (%) 7.2 5.5 11.3 6.7 1.5 2.4 3.3 1.1 36.5 4.6 6.5 10.4 4.9 3.5 1.6 10.1 7.8 19.0 13.8 20.6 76.3 0.4 <0.1 3.8 4.4 8.5 84.8

RSD (%) yield (%) 13.6 5.8 6.9 5.2 1.9 1.5 0.9 1.5 55.4 173.2 5.4 9.0 4.5 15.0 1.4 18.5 9.0 14.0 6.1 1.8 65.8 1.9 0.2 3.6 3.9 9.6 75.4

0.6 3.1 3.2 3.2 18.1 17.8 19.7 65.5 0.3 1.8 2.9 3.4 8.5 74.0

Second elution with an azeotropic mixture of chloroform, methanol, and acetone.

losses are unlikely to contribute largely to the total losses, since the sample amounts used for separation were corrected for evaporation losses. It cannot be ruled out, however, that evaporation may be enhanced with isolated fractions as compared to total SOM. To investigate the influence of the solvent on recovery, all four columns were subsequently eluted with the azeotropic mixture used for extraction. The summed amounts of additionally recovered material vary between 6.8% and 9.6% (Table 2). For all four samples, the major portion of this material is obtained from precolumn 1 and from the main column. A significant amount of material is recovered from precolumn 2 only for the oil sample and from precolumn 3 only for the coal extract and the Lake Holzmaar sample. For these samples, bases or acids, respectively, show elevated yields in the main eluates. Until now, the composition of the material obtained by the second elution step has not been examined. It cannot be ruled out that it consists at least partially of silica. In summary, it appears that at least 15% of SOM is lost due to irreversible adsorption on the stationary phases. Reproducibility. Five replicate analyses for the North Sea crude oil, the coal extract, and the Lake Holzmaar sediment extract and four replicate analyses for the Posidonia shale extract have been performed. The injected amounts of SOM varied between 27.1 and 45.3 mg. Generally, the relative standard deviations (RSDs) are below 11% (Table 2). The only exceptions are the yields of bases in the extract of the Lake Holzmaar sediment and the coal extract, where RSDs are distinctly higher (16%-17%). In both cases, the yields of bases are low (<2% of SOM), which might affect the accuracy of the gravimetic determination of the fraction yield. Considering the results shown in Table 2, we conclude that the compound class separation presented here is well-suited for the determination of the bulk composition of SOM. Linearity. Figure 2 shows the fraction yields obtained upon separation of different amounts of Posidonia shale extract. The data indicate satisfactory linearity for all seven fractions. The separation method presented here is, therefore, appropriate for fractionation of SOM on both the analytical and the semipreparative scales.

Figure 2. Results of a linearity test performed for different amounts of a Posidonia shale extract (E38).

Bulk Compositional Characteristics of SOM of Different Geological Origin. The compound class distributions of the four samples analyzed in this study are depicted as bar charts in Figure 3. In the North Sea crude oil saturates, aromatics and the lowpolarity NSO compounds, present in similar relative amounts, constitute more than 55% of SOM (Figure 3a). Particularly for the oil sample, evaporation loss of volatile compounds, especially from the saturates and aromatics, might result in an underestimation of the relative contribution of these fractions to total SOM. The amount of low-polarity NSO compounds is of the same order as the summed amount of all other heterocompound fractions. The oil is poorest in acids and richest in bases as compared to the other samples. In the Posidonia shale extract saturates, aromatics and low-polarity NSO compounds are the most abundant fractions (Figure 3b). In contrast to the North Sea crude oil, however, other heterocompound fractions, and particularly the
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Figure 3. Bulk composition of soluble organic matter of different origin as determined by liquid chromatographic fractionation: (a) North Sea crude oil (E36129), (b) Posidonia shale extract (E38), (c) Westphalian coal extract (E2235), and (d) Lake Holzmaar sediment extract (E44488). 1, acids; 2, bases; 3, high-polarity NSO compounds; 4, medium-polarity NSO compounds; 5, low-polarity NSO compounds; 6, aromatics; 7, saturates.

high- and medium-polarity compounds, play a more important role. The present sample of the Posidonia shale is of relatively low thermal maturity. Application of the compound class separation procedure to a maturity sequence of Posidonia shales revealed that the bulk composition of source rock extracts depends on maturity, i.e., decreasing relative amounts of high-polarity compounds, acids, and bases with increasing maturity, while the opposite is true for the saturates.21 Completely different patterns are observed for the coal extract and the Lake Holzmaar sediment extract. In the coal sample, all heterocompound classes with the exception of the bases are relatively abundant (Figure 3c). On the other hand, aromatics and saturates constitute only a minor portion of the coal extract. As one would expect for young sediments where diagenesis has not altered the organic matter to a very high extent, the high-polarity compounds are the quantatively most important fraction in the Lake Holzmaar sediment extract (Figure 3d). In this particular sample, the amount of high-polarity compounds is even higher than the summed amounts of all other isolated fractions. It should also be noted that the relative amount of saturates in this sample is overestimated, since relatively high amounts of elemental sulfur are present in this fraction. Molecular Compositional Characteristics of Selected Heterocompound Fractions. According to GC and GC/MS investigations, the molecular composition of saturates and aromatics obtained by the present separation method does not differ from those obtained by conventional MPLC as described by Radke et al.2 The high-polarity compounds have not yet been further
(21) Wilkes, H.; Clegg, H.; Disko, U.; Willsch, H.; Horsfield, B. Submitted to Fuel.

Figure 4. (a) Partial extracted ion chromatogram (m/z ) 167 + 181 + 195 + 217) for the low-polarity NSO compounds of a North Sea crude oil (E36129). Peak identification: 1, carbazole; 2, 1-methylcarbazole; 3, 3-methylcarbazole; 4, 2-methylcarbazole; 5, 1,8dimethylcarbazole; 6, 4-methylcarbazole; 7, 1-ethylcarbazole. 8, benzo[a]carbazole; 9, benzo[c]carbazole; b, C2-carbazoles. (b) Partial total ion chromatogram for the medium-polarity NSO compounds of a Lake Holzmaar sediment extract (E44488). All assigned alcohols are present as trimethysilyl ethers; numbers indicate chain length of n-alkanols. Other peaks: A, phytol; B, cholesterol; C, cholestanol; D, campesterol; E, 24-methylcholestanol; F, stigmasterol; G, -sitosterol; H, stigmastanol. (c) Partial total ion chromatogram for the acid fraction of a coal extract (E2235). All assigned acids are present as methyl esters; numbers indicate chain length of n-fatty acids. Other peaks: b, n-fatty acids; O, hopanecarboxylic acids; A, benzoic acid; B, 2-methylbenzoic acid; C, 3-methylbenzoic acid; D, 4-methylbenzoic acid; E, 3,7,11-trimethyldodecanoic acid; F, 2-naphthalenecarboxylic acid; G, phytanoic acid. (d) Partial extracted ion chromatogram (m/z ) 143 + 157 + 171 + 185 + 199 + 213) for the bases of a Posidonia shale extract (E38), representing the C1-C6 quinolines and/or isoquinolines. Peaks: b, C1; O, C2; 9, C3; 0, C4; 2, C5; 4, C6.

characterized, since the major portion of their constituents is expected not to be amenable to GC. Evaluation of GC and GC/ MS data of all other heterocompound fractions is consistent with the model compound study discussed above, hence suggesting

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that the retention behavior of structurally defined compounds in SOM generally is predictable. A complete characterization at the molecular level of all heterocompound fractions of the different geological materials investigated here is out of the scope of this paper. The chromatograms shown in Figure 4 give an impression of the structural diversity of compounds in selected heterocompound fractions, as revealed by GC/MS. Figure 4a shows an extracted ion chromatogram of the low-polarity NSO compounds of a North Sea crude oil representing selected carbazoles and benzocarbazoles. These compounds, which are currently attracting much interest as a tool for the assessment of migration distances,22 were also detected as constituents of the low-polarity NSO fraction of the Posidonia shale extract and the coal extract, where they, however, exhibited different distribution patterns. The carbazoles are easily quantified in low-polarity NSO fractions by GC/MS using an internal standard.23 Besides the carbazoles and benzocarbazoles, also their dibenzo and/or naphtho analogues are detected as constituents of the low-polarity NSO fraction.21 Other types of compounds identified in the low-polarity NSO fraction of ancient materials are, e.g., aromatic aldehydes and ketones, particularly fluoren-9-ones.21 In the low-polarity NSO fractions from young sediments, mainly oxidation products of biogenic lipids, e.g., steroidal ketones and 2-alkanones, have been identified. The precursors of these carbonyl compounds are mainly alcohols and sterols, which occur in the medium-polarity NSO compound fraction and fatty acids. The distribution of n-alkanols and sterols in the Lake Holzmaar sediment extract is shown as an example in Figure 4b. Among the samples investigated in this study, the
(22) Larter, S. R.; Bowler, B. F. J.; Li, M.; Chen, M.; Brincat, D.; Bennett, B.; Noke, K.; Donohoe, P.; Simmons, D.; Kohnen, M.; Allan, J.; Telnaes, N.; Horstad, I. Nature 1996, 383, 593-597. (23) Clegg, H.; Wilkes, H.; Horsfield, B. Geochim. Cosmochim. Acta, in press.

coal extract was particularly rich in carboxylic acids (Figure 3c). Figure 4c shows that the distribution of carboxylic acids in this sample is extremely complex. Among the acids identified as their methyl esters after derivatization are straight-chain and branched fatty acids, aromatic carboxylic acids, and hopanecarboxylic acids. The basic fractions have not been characterized in much detail up to now. As an example, the extracted ion chromatogram in Figure 4d shows the distribution of C1-C6 quinolines and/or isoquinolines in the Posidonia shale sample. Alkylated quinolines, isoquinolines, and benzoquinolines were also detected in the North Sea crude oil and the coal extract. CONCLUSIONS The liquid chromatographic procedure for compound class separation of SOM from geological samples presented in this paper is applicable to a broad range of different geological sample types. This is a major advantage over other methods, which are mostly restricted to certain sample materials and/or compound classes. Importantly, the method provides information on the bulk chemical composition of SOM while producing compound class fractions which are amenable to further characterization by, e.g., GC and GC/MS. ACKNOWLEDGMENT The authors thank U. Disko, F. J. Keller, W. Laumer, and A. Ropertz for technical assistance, D. Erdmann for a gift of 2-pentacosanol and 11-pentacosanol, and the Ruhrkohle AG, Essen, for providing the coal sample. Received for review April 10, 1997. Accepted July 18, 1997.X
AC9703818
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Abstract published in Advance ACS Abstracts, September 1, 1997.

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