Factors Affecting the Chromatographic Analysis of Asphaltic Petroleum and of Recent Marine Sediment Organic Matter PAULETTE HAMWAY, MICHAEL CEFOLA, and BARTHOLOMEW NAGY Fordham University, New York 58, N. Y.
b This investigation was undertaken to define better some of the factors which influence the elution chromatography of rock and sediment organic matter containing high molecular weight components. The separation efficiencies of a normal paraffin hydrocarbon, n-octadecane-1 -C4, of an aromatic hydrocarbon, phenanthrene9-C14, and of an aliphatic acid, octadecanoic-1 -C14 acid, in the presence of asphaltic petroleum or of Recent marine sediment organic matter have been determined on alumina columns. The presence of high molecular weight material on the columns caused the retention of some of the radioactive eluates beyond their normal points of effluence. Experimental evidence suggests association of asphaltic material with a t least some of the C'*-labeled substances. The relative inefficiency of chromatographic separations of the three tracer compounds was noted even in the absence of bituminous material.
c
One the primary tools in the analysis of petroleum and of Recent marine sediment organic matter. The chromatographic analysis of petroleum distillate fractions is now routine; separation efficiencies in distillate fractions have been studied by Fink, Lewis, and Weiss (8), among others. I n certain types of investigations, however, such as in geochemical studies, it is desirable to work with samples in their original state rather than with distillate fractions. This poses some difficulty because, as Witherspoon (IO)has shown, crude petroleum has colloidal components. Winniford (9) reported that the molecular weights of the asphaltene colloids may go as high as 300,000. These high molecular weight particles seem to interfere with the chromatographic separations of petroleum constituents. Kleinschmidt (S),therefore, suggested the removal of the asphaltenes prior to chromatography. Schwarta and Brasseaux (7) worked out a method whereby the asphaltenes were separated from the analytical sample within the HROMSTOGRAPHY iS
chromatographic column. The separation efficiency of a saturated hydrocarbon from crude oil has been studied (5) with asphaltenes present on the column. The relative inefficiency of chromatographic separations was noted in this study. It was, therefore, considered desirable to evaluate the effect of high molecular weight material on separation efficiencies in alumina columns. REAGENTS AND SAMPLES
n-O~tadecane-l-C'~, New England Nuclear Corp.; specific activity 2.04 me. per mmole. Phenanthrene-9-CI4, Nuclear Chicago Corp.; specific activity 1.93 mc. per mmole. Octadecanoic-l-CI4 acid (stearic acid), Xew England Nuclear C o w : specific activity 8.42 me. per mmole. Alumina, neutral grade Woelm, Alupharm Chemicals, Inc., used as received; and Fisher Scientific Co., Catalog No. A-540 (described as alkaline but not so specified in the Chemical Index No. 123), used as received. Silica gel, Davison Chemical Co., grade 923, 100- to 200-mesh, used as received. Eluents, used always in the following order: n-heptane, technical grade; carbon disulfide, carbon tetrachloride, benzene, methylene chloride, chloroform, anhydrous ether, pyridine, ethyl acetate, methanol, all c. P. grade; absolute ethyl alcohol, U.S.P. grade. For the elution of stearic acid, glacial acetic acid, c. P. grade, and 90% formic acid, technical grade, followed the others. This list of eluents was modified from a series of solvents recommended by the manufacturers of Woelm alumina for the chromatography of unknown mixtures. All eluents were used as received. The petroleum was obtained from the Athabasca deposit, Abasand Quarry, near McMurray, Alberta, Canada. It was extracted with methylene chloride a t room temperature, the mineral matter was separated by centrifugation a t 1725 r.p.m. for several minutes, and the solvent was evaporated a t room temperature. It is an asphaltic petroleum, rich in aromatic compounds and in sulfur. Analysis has indicated the presence of naphthenes, normal and isoparafis, alkyl benzenes, cycloalkyl _
benzenes, naphthalenes, and phenanthrenes (4). The asphaltene content is 22.3% as determined by coagulation with pentane. The gasoline boiling range distillate fraction of a Perry, Okla., crude oil was purified by extraction with cold, concentrated sulfuric acid, followed by elution on alumina and silica gel columns. The Recent marine sediment organic matter came from Long Island Sound, New York. The sample was collected a t a depth of 30 feet of water, one and a quarter miles off-shore, near Lloyd's Point. Organic matter was separated from clay minerals by Soxhlet extraction with a mixture of 70% benzene, 15% methanol, and 15% acetone a t temperatures ranging up to 79" C., according to the method of Smith (8). The solvent was evaporated from the sample over a steam bath.
I
EXPERIMENTAL
The octadecane-1-GI4 was dissolved in heptane, the phenanthrene-9-C14 in methylene chloride, and the octadecanoic-l-C14 acid in diethyl ether. The quantities of radioactive compounds used in any of the chromatographic experiments were always calculated by taking aliquot portions from these solutions of known concentrations. All elution experiments were performed in glass chromatographic columns fitted with fritted-glass disks sealed in a t the bottom. Two columns were used: 10 X 300 mm. for small samples, 20 X 400 mm. for large ones. Adsorbent-sample ratios n-ere always at least 300 to 1 (w./w.) and usually much higher for the purification steps. The alumina was prewet with 10 ml. of heptane. Once the elution was started, solvent was added in consecutive portions, so that its level was always well above that of the adsorbent. Furthermore, a calcium chloride drying tube was placed a t the top of the column to prevent the entrance of moisture. Tenmilliliter fractions of the eluent material were collected, evaporated, redissolved in methylene chloride, and transferred to planchets. The samples were counted on thin, end-window Geiger counters. Samples giving counts per minute in excess of 400 were determined with an accuracy of a t least 20/, a t the 68.3'-70 confidence level; those in excess of 1000 c.p.m., with 1% accuracy a t the 68.3y0 conVOL 34, NO. 1, JANUARY 1962
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fidence level. The accuracy of some of the counting was reduced by geometric effects, by creepage from the planchets, and by absorption of beta radiation by the bitumen or by Recent marine sediment organic matter. The recovery of the radioactive eluate fractions is reported in percentages of the total counts per minute to facilitate comparison of experiments. The purity of the C14-labeled compounds was determined by elution chromatography. Small portions of their solutions were evaporated, the radioactive compounds were redissolved in heptane, and an elution pattern on alumina was obtained for each. It wa8 found that impurities were present and it was necessary to remove them before mixing with petroleum or Recent marine sediment organic matter. The purification of octadecane-1-C14 was carried out by three different procedures. The first method was the obtaining of the heptane eluate fraction of the labeled material from a Woelm alumina column. The second method involved rechromatographing the heptane eluate fraction from the alumina column with heptane on a silica gel column. Finally, a purification method was used in which the octadecane material was first extracted with cold concentrated sulfuric acid; this was followed by chromatography with heptane on an alumina column and finally on a silica gel column. The elution patterns of the purified samples were determined with the same series of solvents as used for the bitumen and the results are listed in Table I. Although the third method-acid extraction-alumina-silica gel chromatography-gave the best results, the elution curves of all of the purified octadecane-l-C14samples still contained some radioactive eluates coming off with a number of solvents regardless of the method of purification. All elution experiments were performed within three days of the purification. It is unlikely that any condition existed which would have led to the decomposition of octadecane after purification. Furthermore, since the product of carbon-14 decay is stable nitrogen-14, any new compounds resulting from the decay or any recoil fragments from bond rupture would not account for the radioactive post-heptane eluates. Following purification, the asphaltic petroleum- and the Recent marine sediment organic matter-octadecane-lC14 mixtures were studied, and the results of some of these elution experiments are given in Table 11. Known portions of the labeled compounds were added to samples of asphaltic petroleum or of organic matter from the Recent marine sediments and stirred in methylene chloride. The solvent was evaporated from the mixtures, which were then slurried in heptane and quantitatively transferred onto the chromatographic columns.
To determine the effect of high molecular weight substances on the elution pattern, control columns were run with samples of the Athabasca petroleum and of the Perry, Okla., crude oil which had been vacuum-distilled under nitrogen. The gasoline boiling range fractions obtained were purified, mixed with ~ c t a d e c a n e - l - C ~and ~ , chromatographed in the usual manner. Table I11 shows the results obtained. Experiments were performed to determine the composition of the radioactive post-heptane eluates of purified octadecane-l-C14. -4n ultraviolet spectrum of the methylene chloride fraction of the octadecane chromatographed alone m s obtained. The absorption pattern was characteristic of saturated hydrocarbons such as those reported by Potts (6). I n another experiment, one of the pyridine eluates of a petroleum-octadecane-l-C14 mixture was rerhromatographed with the usual series of solvents on alumina. Counts were obtained chiefly in the chloroform fraction, but also in the methylene chloride, diethyl ether, pyridine, ethyl acetate, and methanol fractions. The original pyridine fraction was not soluble in heptane; however, it did dissolve in methylene chloride. Il'o radioactivity was recorded before the methylene chloride fraction mas reached. Prior to the purification of phenanthrene-9-C14, an experiment was carried out using unlabeled phenanthrene only. A sample having a melting point range of 99' to 100" C. was dissolved in heptane and eluted on an alumina column with the usual series of solvents. The great majority of the sample came off with carbon disulfide, but some came off with benzene. The ultraviolet spectrum of the carbon disulfide fraction was characteristic of phenanthrene, whereas the spectrum of the benzene fraction was caused by impurities. Consequently, the CI4-labeled phenanthrene was purified by collecting the second eluate fraction-that is, the carbon disulfide fraction-from alumina columns. High counts were recorded in the heptane fraction; an ultraviolet spectrum of this fraction showed a weak phenanthrene pattern. The octadecanoic-l-C14 acid was eluted with the same series of solvents, followed by glacial acetic acid and 90% formic acid. Radioactive material was not recovered in quantity until the last two eluents were reached. An experiment with unlabeled octadecanoic acid having a melting range of 69.0" to 72.0' C. was performed to identify a white solid which resulted from the evaporation of the acetic acid eluate. This precipitate was tested for aluminum ion by the aluminon reagent test and for acetate ion by the ester test. These analyses, together with a partial darkening of the precipitate
U
VOL. 34, NO. 1, JANUARY 1962
45
upon heating, indicated the presence of aluminum acetate as well as of some alumina. Therefore, the C14-Iabeled octadecanoic acid was purified by obtaining the heptane-soluble portion of the acetic acid eluate. Purified phenanthrene - 9 - C14 was mixed with the Athabasca bitumen and the octadecanoic-l-C14 acid was mixed with both the bitumen and the Recent marine sediment organic matter; elution patterns &-ere obtained in the usual manner. Finally, because some of the solvents discolored the alumina columns, it was necessary to determine whether any interaction had taken place between them and the adsorbent. Consequently, an elution experiment was performed in which only the pure solvents in their usual sequence were passed through a column. Some of the same color effects were observed in this experiment as in those containing petroleum or Recent marine sediment organic matter -4yellow ring developed a t the top of the column during heptane elution; a stronger yellow color was noted throughout the entire column during elution with carbon disulfide. Subsequently, this color disappeared but it developed again during elution with pyridine. Reaction products were not revealed by ultraviolet spectra. FACTORS AFFECTING CHROMATOGRAPHIC SEPARATIONS
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2 46
ANALYTICAL CHEMISTRY
Tno of the prime factors influencing any elution pattern are the chemical composition of the sample and the nature of the solvents employed. Each of the eluents in the specified series brought down a characteristic fraction from petroleum and from the Recent marine sediment organic matter. The C14tracers were included to define better the type of compounds which could be expected in the various eluate fractions. Under the experimental conditions described, the majority of the octadecane-l-C14 was eluted with heptane and octadecanoic-l-C14 acid with acetic acid from both the petroleum and Recent marine sediment organic matter mixtures. Phenanthrene-g-Cl4, which F a s mixed only with petroleum, came off with benzene. Some of the results are shown in Figures 1 and 2; the former was plotted on an expanded vertical scale to demonstrate the postheptane eluates of octadecane-l-CI4. Another important factor in chromatographic separations is the effect of high molecular weight substances in the column. Mixtures of bitumen with the radioactive hydrocarbon compounds resulted in the holding back of some of the normal points of eluate effluence. The post-heptane eluate fraction of octadecane-l-CI4 came off mainly nTith pyridine after the sample was mixed
* M I X T U R E OF n-OCTADECANE.I-C14 A N D ATHABASCA B I T U M E N ; A D S O R B E N T TO S A M P L E RATIO 0
Figure 1.
Elution patterns
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of octadecane-1 -C14 mixed with a crude oil and with a gasoline boiling range distillate fraction
Octadecane-1 -C14 and distillate fraction were purified b y extraction with cold, concentrated sulfuric acid followed b y chromatography on both alumina and silica gel columns
with the Xthabasca petroleum. The organic matter from Recent marine sediments also caused a retainment of the post-heptane eluate fractions of octadecane-l-C14. However, as Table 111 demonstrates, in the absence of the high molecular weight substances, when the gasoline boiling range distillate fractions were mixed with the octadecane tracer and eluted, the post-heptane eluates came off not with pyridine mainly, but with benzene, methylene chloride, chloroform, or ether. Similarly, phenanthrene was not eluted until benzene when it mixed with petroleum, as shown in Figure 2. The reason for these phenomena may be some type of association with or dissolution of the hydrocarbons and/or impurities in the high molecular weight si1bs t ances .
A fourth factor affecting the separations is the quantity of solvent employed. If a sufficient amount of eluent was not used, the eluate was not fully recovered until certain subsequent fractions. The eluted material usually appeared in high concentration during the first few 10-ml. portions collected from an eluent. Then the concentration gradually decreased and usually disappeared during the last portions. When it did not disappear, the remainder came down with later eluents, but not necessarily with the one immediately following. Evans, Kenny, Meinschein, and Bray reported similar phenomena with a mixture of alkanes eluted on alumina ( I ) . This effect is illustrated with unlabeled phenanthrene in Figure 3. The ssmple was not fully recovered with
100 ml. of carbon disulfide. This is demonstrated by the fact that the phenanthrene concentration did not reach zero in the final portions of the carbon disulfide elution. The next solvent, carbon tetrachloride, eluted only insignificant quantities. Benzene, however, brought down a signscant amount of material. A more efficient recovery was obtained by 300 ml. of carbon disulfide, although even this was not sufficient to eliminate the benzene eluate. The use of 500 ml. of carbon disulfide resulted in an almost complete recovery of the sample with that solvent. However, ultraviolet spectra showed that the eluate recovered with benzene during the 300-ml. run contained impurities and that the carbon disulfide eluate was composed of pure phenanthrene. It was concluded
PHENANTHRENE.9-CI4, PURIFIED B Y ELUTION ON ALUMINA, MIXED WiTH ATHABASCA BITUMEN, A D S O R B E N T TO S A M P L E R A T I O ' 3 1 9 / 1
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that under the experimental conditions described, the use of 300 ml. of carbon disulfide led to the most efficient separation of impurities from phenanthrene. A. similar effect was observed with octadecane-l-C14. Experiment 1 in Table I involved the chromatography of C14-labeled octadecane alone, using 100 ml. of heptane as the first eluent. I n experiment 2, 500 ml. of heptane were used. None of the purification processes resulted in the elimination of the radioactive post-heptane eluates; however, their concentrations were lower when 500 ml. of heptane were used. These results point out that it is difficult to predict optimum conditions for chromatography because the use of both insufficient as well as too great amounts of eluent may hinder sharp separations under a n otherwise identical experimental setup. At the same time, one may speculate that the postheptane eluates still contain some octadecane due to a Molecular Sieve type of action. This view is supported by the spectral evidence referred to previously. Whatever the cause, the presence of these eluates points out that chromatography, under the conditions described, does not effect complete separations. However, the relative quantities of these eluates are usually low. The combined postheptane eluates account for less than 1% of the total counts in experiment 4 of Table I. Other factors are the adsorbentsample ratios, the nature of the adsorbents, the types and the sequence of eluents, the flow rates, etc. Most of these have been discussed in the literaANALYTICAL CHEMISTRY
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Influence of eluent volumes o n recovery of p h e n a n t h r e n e ture. Two brief comments will suffice here. First, it was confirmed that adsorbent-sample ratios much lower than 300 to 1 do not permit effective separations of asphaltic mixtures. Second, it was reaffirmed that the same type of adsorbent prepared by different manufacturers may have substantially different properties. The Fisher alumina gave lower post-heptane counts with octadecane-l-C14 than the Koelm alumina. The complexity of the various factors precluded a quantitative account of the tracer compounds. CONCLUSIONS
The following made :
Observations
were
1. The majority of octadecane-l-C14 was eluted with heptane, phenanthrene9-CI4 with benzene, and octadecanoicl-Cl4 acid with acetic acid from alumina columns. 2. The presence of high molecular weight substances in the columns retarded some of the points of eluate effluence. This may have been caused by a n association between these substances and the tracer compounds. 3. Separation efficiencies were dependent on the volumes of eluents employed. 4. During the chromatography of octadecane-l-C14 alone, it was not possible t o restrict the appearance of some radioactive eluates beyond the heptane fraction even when very rigorous purification procedures were employed. This may be due to a Molecular Sieve effect. 5 . The complexity of factors makes sharp separations difficult to obtain
during the chromatography of asphaltic petroleum and of organic matter from Recent marine sediments. The applicability of observations 1 to 5 to systems other than the ones described was not examined. ACKNOWLEDGMENl
The authors acknowledge the assistance of Sister Mary Carol Bitz and George C. Gagnon, Fordham Cniversity, and thank Maxine Brenneman, Esso Research and Engineering Co., for her valuable suggestions and critical reading of the manuscript. LITERATURE CITED
(1) Evans, E. D., Kenny, G. S., Meinschein, W. G., Bray, E. E., - 4 5 ~ ~ . CHEM.29, 1858 (1957). (2) Fink, D. F., Lewis, R. JT., Keiss, F. T., Ibid., 22,858 (1950). (3) Kleinschmidt, L. R., J . Research Satl. Bur. Standards 54, 163-6 (1955). (4) Nagy, B., Gagnon, G. C., Geochim. et Cosmochim. Acta 23, 155-85 (1961). (5) Kagy, B., Hamway, P., Gagnon, G. C., Cefola, M., Ibid., 21, 151-5 (1960). (6) Potts, W. J., Jr., J . Chem. Phys. 20, 810 (1952). (7) Schwartz, R. D., Brasseam, D. J., ANAL.CHEM.30, 1999-2002 (1958). (8) Smith, P. V., Jr., Bull. Am. Assoc. Petrol. Geologists 38, 380 (1954). (9) Rinniford, R. S., Division of Petroleum chemistry, 138th Meeting, ACS, New York, ?;. Y., September 1960, Preprint 5 , 4A-11 (1960). (10) Witherspoon, P. rl., Ill. State Geol. Surv., Rept. Invest. 206 (1958).
RECEIVEDfor review June 28, 1961. Accepted October 23, 1961. Work sponsored by the National Science Foundation.
