Anal. Chem. 2001, 73, 703-707
Determination of Naphthenic Acids in Crude Oils Using Nonaqueous Ion Exchange Solid-Phase Extraction D. M. Jones,* J. S. Watson, W. Meredith, M. Chen, and B. Bennett
Fossil Fuels and Environmental Geochemistry (Postgraduate Institute), NRG, The University, Drummond Building, Newcastle upon Tyne, NE1 7RU, U.K.
A method is presented for the routine, rapid, and quantitative analysis of aliphatic and naphthenic acids in crude oils, based on their isolation using nonaqueous ion exchange solid-phase extraction cartridges. The isolated acid fractions are methylated and analyzed by gas chromatography and gas chromatography/mass spectrometry. The method is effective on both light and heavy oils and is capable of providing mechanistic information of geochemical significance on the origin of the acids in the oils. Analysis of oils that were solvent extracted from laboratory and field mesocosm marine sediment oil degradation studies indicate that this new method of analyzing the products of hydrocarbon biodegradation may be a useful tool for monitoring the progress of bioremediation of oil spills in the environment. Carboxylic acids in crude oils are important because of the corrosion problems they cause during refining,1,2 their surfactant properties,3,4 their geochemical significance,5,6 and their commercial uses as wood preservatives, paint additives, etc.7 The presence of carboxylic acids in petroleum was discovered more than 100 years ago, when they were termed “naphthenic acids”.8 Currently, although alicyclic (naphthenic) acids appear to predominate in crude oils, it is generally recognized that other types of carboxylic acids are also present.9 A large number of carboxylic acid classes were identified in a heavy, immature Californian crude oil containing ∼2.5 wt % carboxylic acids, and the most abundant species were reported to be naphthenic and naphthenoaromatic acids.3,10,11 The acid content of crude oils has been reported to vary from nondetectable levels to as high as 3 wt %.8 * Corresponding author: (e-mail)
[email protected]; (fax) + (0)191 2225431. (1) Derungs, W. A. Corrosion 1956, 12, 41-46. (2) Babaian-Kibala, E.; Petersen, P. R.; Humphries, M. J. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 106-110. (3) Seifert, W. K.; Howells, W. G. Anal. Chem. 1969, 41, 554-568. (4) Acevedo, S.; Escobar, G.; Ranaudo, M. A.; Khazen, J.; Borges, B.; Pereira, J. C.; Mendez, B. Energy Fuels 1999, 13, 333-335. (5) Mackenzie, A. S.; Wolff, G. A.; Maxwell, J. R. In Advances in Organic Geochemistry 1981; Bjoroy M., et al., Eds.; Wiley: Chichester, 1983; pp 637649. (6) Jaffe, R.; Gallardo, M. T. Org. Geochem. 1993, 20, 973-984. (7) Brient, J. A. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 131-133. (8) Lochte, H. L.; Littman, E. R. The Petroleum Acids and Bases; Chemical Publishing Co. Inc.: New York, 1955. 10.1021/ac000621a CCC: $20.00 Published on Web 12/29/2000
© 2001 American Chemical Society
The determination of total acidity in oils is routinely carried out by standard methods in the oil industry using ASTM D 974 (IP 139/86) and ASTM D 664 (IP 177/96). Both of these methods only provide information on the total acidity (total acid number (TAN) or neutralization number) and they do not provide any significant compositional information on the organic acids in the oils. Some of the methods reported for obtaining carboxylic acid fractions from crude oils use relatively simple base extraction or saponification steps,12-14 while others5,15-17 use modified versions of a KOH impregnated silica gel method.18 Polar fractions containing naphthenic acids have also been obtained using a technique where the polars are backflushed from a cyano-modified silica column after elution of nonpolar hydrocarbons.19 Nonaqueous ion exchange-based methods have been used to isolate acidic fractions from petroleums for many years,20,3,21-23 but they often involved complex multistep separations using relatively large scale ion exchange chromatographic columns, sometimes in combination with base extraction stages and large volumes of solvents. Although conventional nonaqueous ion exchange resin methods for the separation of liquid fossil fuels into fractions have a number of advantages over other methods, they are still laborious, require careful preparation of the ion exchange resins prior to use, require (9) Speight, J. G. The Chemistry and Technology of Petroleum, 2nd ed.; Marcel Dekker: New York, 1991. (10) Seifert, W. K.; Teeter, R. M. Anal. Chem. 1969, 41, 786-795. (11) Seifert, W. K.; Teeter, R. M. Anal. Chem. 1970, 42, 180-189. (12) Behar, F. H.; Albrecht, P. Org. Geochem. 1984, 6, 597-604. (13) Dzidic, I.; Somerville, A. C.; Raia, J. C.; Hart, H. V. Anal. Chem. 1988, 60, 1318-1323. (14) Barth, T.; Moen, L. K.; Dyrkorn, C. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 134-136. (15) Douglas, A. G.; Powell, T. G. J. Chromatogr. 1969, 43, 241-246. (16) Ramljak, Z.; Solc, A.; Arpino, P.; Schmitter, J.-M.; Guiochon, G. Anal. Chem. 1977, 49, 1222-1225. (17) Jaffe, R.; Albrecht, P.; Oudin, J. L. Geochim. Cosmochim. Acta 1988, 52, 2599-2607. (18) McCarthy, R.; Duthie, A. J. Lipid Res. 1962, 3, 117-119. (19) Yepez, O.; Lorenzo, R.; Callarotti, R.; Vera, J. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 114-122. (20) Webster, P. V.; Wilson, J. N.; Franks, M. C. Anal. Chim. Acta 1972, 38, 193-200. (21) Jewell, D. M.; Weber, J. H.; Bunger, J. W.; Plancher, H.; Latham, D. R. Anal. Chem. 1972, 44, 1391-1395. (22) Green, J. B.; Hoff, R. J.; Woodward, P. W.; Stevens, L. L. Fuel 1984, 63, 1290-1299. (23) Tomczyk, N. A.; Winans, R. E.; Shinn, J. H.; Robinson, R. C. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 123-125.
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regeneration after use, and can also require the use of noxious reagents.22 Isolated carboxylic acid fractions have been analyzed by a number of methods including infrared spectroscopy,16,22,24 gas chromatography,5,14,17 mass spectrometry,11,13,23,25,26 and gas chromatography/mass spectrometry.6,11,14,27A variety of derivatization techniques have been used for the isolated acid fractions prior to analysis by GC and MS including methylation using BF3/ methanol23 or diazomethane5,6,14 and esterification with fluoro alcohols.27 However, few of the reports examined give absolute quantitations on the carboxylic acids analyzed and none appear to give quantitation and reproducibility validation, probably due to the extensive and time-consuming nature of each analysis. Nevertheless, these are essential measurements if a useful and mechanistic interpretation of the origins of acidity in petroleums is to be gained. One of the key aims of this present study was therefore to develop a practical, rapid, and quantitative method for analyzing carboxylic acids in oils. Experience in this laboratory has suggested that solidphase extraction (SPE) procedures couple the advantages of low solvent volume usage with the potential to effect separations of both petroleum hydrocarbons28 and non-hydrocarbons such as phenols and carbazoles.29,30 This together with phase uniformity prevalent in SPE approaches indicated that an SPE-based method to acid recovery was potentially a viable procedure. This we describe below. EXPERIMENTAL SECTION A SAX quaternary amine (10 g) SPE ion exchange column (International Sorbent Technology) was conditioned with 30-40 mL of n-hexane (Fisher, CertiFi HPLC grade). The oil sample (1-2 g) was spiked with 1-adamantanecarboxylic acid (Fluka), and 5β-cholanic acid (Sigma) as recovery (surrogate) standards (at approximate concentrations of 75 and 50 µg/g of oil, respectively) and then pipetted onto the column. After the sample had absorbed onto the column, interferences were removed by eluting with 80 mL of n-hexane followed by 80 mL of dichloromethane (Fisher Chemicals, Distol grade) and then the residual solvent was carefully removed by an air-flush. The acid fraction was eluted with 55 mL of diethyl ether (BDH, chromatography grade), containing 2% (v/v) formic acid (BDH, analytical grade). After removal of solvent by rotary evaporation, the acid fraction was redissolved in ∼1 mL of dichloromethane (DCM), ∼1 mL of 14% v/v BF3/methanol methylating agent (Fisher, GC grade) was added, and the vial placed in a water bath (50-60 °C) for 1 h.31 The derivatized sample was transferred to a flask containing distilled water (10 mL) and extracted three times with 3 mL of petroleum ether (Fisher Chemicals, Distol grade). The solvent in this extract was evaporated down to ∼100 µL with dry nitrogen. (24) Olsen, S. D. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 142-145. (25) Hsu, C. S.; Dechert, G. J.; Robbins, W. R. Prepr.-Am. Chem. Soc., Div. Pet. Chem. 1998, 3, 127-130. (26) Fan, T.-P. Energy U Fuels 1991, 5, 371-375. (27) Green, J. B.; Yu, S. K-T.; Vrana, R. P. J. High Resolut. Chromatogr. 1994, 17, 427-438. (28) Bennett, B.; Larter, S. R. Anal. Chem. 2000, 72, 1039-1044. (29) Bennett, B.; Bowler, B. F. J.; Larter, S. R. Anal. Chem. 1996, 68, 36973702. (30) Bowler, B. F. J.; Larter, S. R.; Clegg, H.; Wilkes, H.; Horsfield, B.; Li, M. Anal. Chem. 1997, 69, 3128-3129. (31) Metcalfe, L. D.; Schmitz, A. A. Anal. Chem. 1961, 33, 363-364.
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An alternative methylation procedure using diazomethane was also tested for a number of samples. The diazomethane was produced by the reaction of a solution of 43 g of N-methyl-N-nitrosotoluenep-sulfonamide (Sigma) in 250 mL of diethyl ether (Fisher) added dropwise to a solution of 10 g of potassium hydroxide in 15 mL of water and 50 mL of 96% ethanol (Aldrich) in a water bath-heated (65 °C) flask fitted with a condenser. This latter procedure was carried out using safety precautions appropriate to the reagent toxicities and tendency of explosive decomposition of diazomethane.32 The condensed ethereal diazomethane was collected in a cooled (ice/salt) flask, and aliquots ( ∼0.25 mL) of it were added to the oil acid fractions. Excess reagent was removed by evaporation with dry nitrogen. A silica (1 g) SPE column (IST Technologies) was conditioned with hexane (5-10 mL). The derivatized sample extract was loaded onto the column with the washings (using the minimum amount of hexane), interferences were eluted with hexane (4 mL), and the methyl esters were eluted with 10 mL of hexane/DCM (6:4 v/v). The carboxylic acid methyl esters were concentrated by rotary evaporation, ∼50 µg of the internal standard (methyl ester of 1-phenyl-1-cyclohexanecarboxylic acid) was added, and the mixture was analyzed by gas chromatography and gas chromatography/mass spectrometry. The methyl ester of 1-phenyl-1-cyclohexanecarboxylic acid was prepared by methylation (BF3/methanol) of 1-phenyl-1-cyclohexanecarboxylic acid (Acros Organics) and then purification (silica column chromatography) of the ester. Gas chromatographic analyses of the carboxylic acid methyl esters were performed on a Carlo Erba 5160 series gas chromatograph equipped with a cold on-column injector and a flame ionization detector (FID). Separation of the injected sample was performed on a Hewlett-Packard HP-5 fused-silica capillary column (30 m × 0.25 mm i.d.; film thickness, 0.25 µm). The oven temperature was held at 50 °C for 2 min and then programmed at 6 °C/min to 300 °C where it was held for 25 min. The carrier gas used was hydrogen. The GC data were collected and processed using a Fisons Multichrom or LabSystems XChrom data system. GC/MS analysis of carboxylic acid methyl esters was performed on a Hewlett-Packard 5890 II gas chromatograph linked to a HP 5972 MSD. Data acquisition, controlled by a HP PC Chemstation, was in full-scan mode. Samples were injected by a splitless injection technique using a HP 7673 autosampler. Separations were performed on a HP-5 column (30 m × 0.25 mm i.d.; film thickness, 0.25 µm). The GC oven temperature was programmed from 40 to 300 °C at 4 °C/min, where it was held for 20 min. The carrier gas used was helium. The n-alkanoic acids (C10-C30), and other GC resolved components were quantified as FID peak areas against the internal standard (methyl ester of 1-phenyl-1-cyclohexanecarboxylic acid) using an assumed response factor of 1 and taking into account the recoveries measured from the surrogate standard (e.g., a surrogate standard recovery of 90% meant that the analyte concentrations measured were corrected by multiplying them by 1.11). The total acid fractions were quantified by measurement of the total GC-FID chromatogram areas between 5 and 80 min (32) Vogel, A. I.; Furniss, B. S. Vogels’s Textbook of Practical Organic Chemistry, 5th ed.; Longman: London. 1989.
above the baseline of a blank (DCM) analysis against that of the internal standard peak, again assuming a response factor of 1 and taking into account the recoveries measured from the surrogate standard. Since individual response factors were not used the data should be regarded as semiquantitative, though it is unlikely that GC-FID response factors will vary significantly for the similar carboxylic acid esters quantified. The hopanoic acids present were identified by comparison of their mass spectra with those previously reported,33 together with their relative retention times in the m/z 235, 249, and 263 mass chromatograms.6,33 RESULTS AND DISCUSSION Development of SPE Method. The method was developed on the basis of experience gained in the use of SPE for the analysis of alkylphenols in crude oils and rock extracts.29 Initially, C18 reversed-phase SPE cartridges were tried for the separation of a synthetic combination of hydrocarbons and acids, and a light North Sea crude oil (M) spiked with acids, using various amounts of hexane to remove the hydrocarbons and various amounts of DCM to elute the polars. A further fractionation of the polars was then attempted using a KOH modified silicic acid minicolumn. These efforts were not successful, so the use of ion exchange SPE cartridges, specifically the 10-g SAX quaternary amine SPE cartridges (IST Technology), was investigated. A large number of combinations of different solvents (including hexane, DCM, methanol, and diethyl ether and with various amounts of formic acid modifiers), different solvent volumes, and different oil loadings were tried. Furthermore, it was discovered that the final acid fractions from this SAX column was also quite heavily colored and impure, especially that from a heavy, biodegraded oil which was also tested. A methylation step (BF3/methanol) and further cleanup of the methylated fraction on a normal-phase silica SPE cartridge using hexane as eluent was therefore introduced into the procedure. This provided usable and reproducible carboxylic acid fractions for GC and GC/MS analyses, which did not cause undue deterioration of the GC capillary columns used. Routine procedural blank analyses showed that nC16 and nC18 alkanoic acids were frequent low-level contaminants, which proved impossible to eliminate completely. They were thought to be mainly from the polypropylene housings from the SPE cartridges used. For oils with naturally very low levels of acids, these two acids were often the most prominent in the chromatograms, but the proportions of these contaminants could be accounted for during procedural blanks. Attempts to prewash the SPE cartridges with various solvents prior to addition of the oils resulted in much poorer recoveries and reproducibilities. However, it was subsequently discovered that the use of 4-g SAX SPE cartridges with glass housings (IST), together with a proportionate reduction in the solvent elution volumes used, compared with the 10-g SAX SPE cartridge method, produced comparable results but without any significant contamination. Routine procedural blanks using these cartridges showed that the C16 and C18 n-alkanoic acids were generally present in amounts lower than 1 µg and other individual acids were generally less than 0.05 µg/analysis. Procedural blanks carried out with each batch of samples were used to correct the sample results. However, less sample can be loaded onto the smaller glass columns, so for oil samples with a low concentration (33) Jaffe, R.; Albrecht, P.; Oudin, J. L. Org. Geochem. 1988, 13, 483-488.
Table 1. Reproducibilities of Oil Acid Fraction Measurementsa oil sample
TGCD acids (µg/g)
A (n ) 4) B (n ) 3) M (n ) 3)
4202 167 142
SD (µg/g)
RSD (%)
n-acids (µg/g)
SD (µg/g)
RSD (%)
314 23.5 25.5
7.5 14.1 18.0
nm 10.7 7.5
nm 1.5 1.1
nm 14.2 14.5
a TGCD refers to the total gas chromatography detectable acids (µg of acid/g of oil); SD and RSD are the standard deviations and standard deviations relative to the mean, respectively; nm is not measurable.
Table 2. Recoveries of Acid Spikes from Oil Ma spike acid compound benzoic 2-naphthoic nC10:0 nC14:0 nC22:0 nC30:0 1-adamantane acid 5β-cholanic a
recovery (%)
SD (n ) 4)
14 35 55 98 81 37 68 94
2.8 5.9 7.0 10.2 12.2 8.8 6.2 3.6
Quantitations used n-eicosane as internal standard.
of carboxylic acids the large polypropylene cartridges may still have to be used if similar-sized carboxylic acid fractions are required. Reproducibility and Recovery. The results of quantitative replicate analyses of the total GC detectable acids and n-alkanoic acids in light, unbiodegraded, low-acid North Sea oils B and M (with a TAN values of 0.05 and 0.25 mg of KOH/g, respectively) and a heavy, biodegraded, acidic North Sea oil A (with a TAN value of 1.85 mg of KOH/g) are shown in Table 1. The recoveries of a number of different acids added to an aliquot of oil M at concentrations of ∼10 µg/g of oil, varied from over 90% in the case of a four-ringed C28 alicylic acid (5β-cholanic acid) to below 15% for benzoic acid (see Table 2). Recoveries of straight-chain components tested varied from 98% for the nC14 acid to 37% for nC30. The reproducibilities of the recoveries ranged from 2.8 to 12.2% SD (Table 2). The apparently low recoveries of the aromatic acids were partly due to their poor derivatization efficiencies when BF3/methanol was used for methylation, which is an effect that has been noted previously.12 Further recovery tests with oils (n ) 4) spiked with aromatic acid standards showed that using diazomethane as the methylating agent increased the recoveries of naphthoic acid from 35 to 96% (SD 5.2%) and pyrene-1-carboxylic acid from 15 to 87% (SD 7.0%). The recovery of benzoic acid using diazomethane was lower than that using BF3/methanol at 7% (SD 4.8%), indicating that losses of the latter compound occurred elsewhere in the fractionation procedure. However, benzoic acid and its homologues with up to three alkyl carbons are present in very low concentrations in carboxylic acid fractions isolated from petroleum.27 To test the effects that the two different derivatization procedures had on the quantitation of crude oil total acid fractions, eight different oils containing varying concentrations of acids were analyzed and the results shown in Figure 1. These results show that for the six oils measured that had acid fraction concentrations greater than 250 µg/g, the average difference between the measurements made using the BF3/methanol and the diazoAnalytical Chemistry, Vol. 73, No. 3, February 1, 2001
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Figure 1. Comparison of the carboxylic acid fraction concentrations measured in eight different crude oils using BF3/methanol and diazomethane for methylation. Oil sample codes are the same as those given in Meredith et al.,36 where additional information on these oils is also given.
methane derivatization procedures was less than 5.1% (SD 4.4%). Gas chromatograms (not shown) of the acid fractions derivatized by the two methods were also almost identical. As another method test, a sample of a commercially available naphthenic acid mixture (Kodak Fine Chemicals, Rochester, NY) was split into two aliquots, one of which was analyzed by the full procedure detailed above and another aliquot was simply spiked with quantitation standards and methylated (BF3/methanol) before GC analysis. The resulting chromatograms were very similar in appearance, and quantitation of the total GC detectable acids in the aliquot that had been through the complete procedure showed a value of 65.5% of the starting weight, while the aliquot that had been derivatized only showed a value of 70.1%. These values were consistent with the recoveries of the individual carboxylic acids in the oil-spiking tests. The remaining 30% not measured in the derivatized-only aliquot is thought to be due to a combination of response factor effects, low derivatization efficiency of aromatic acids, and the presence of non-GC amenable components, though the relative importance of these factors is not known at present. Application 1. Gas chromatograms of the derivatized carboxylic acid fractions from the undegraded North Sea oil M and the biodegraded heavy North Sea oil A are shown in Figure 2a and b, respectively. As seen in Figure 2a, although the dominant peaks are the added quantitation standards and the nC16 and nC18 acids, resolved peaks due to nC12-nC30 n-alkanoic acids can be seen on a relatively flat baseline. However, the dominant feature of the degraded oil acid fraction chromatogram is a large unresolved complex mixture (UCM) or hump ranging from about C12 to >C30 and maximizing around C23, with the only significant resolved components being due to added standards. The quantitation data for the two oils clearly show that the undegraded oil contained low, but measurable amounts of n-carboxylic acids (Table 1). Also, although no resolved components are accurately quantifiable in the gas chromatogram of the degraded oil sample, the total GC detectable acids were very much higher, by a factor of ∼25 (Table 1). Comparison of the concentrations of the acid fractions in degraded and undegraded oils shows that although some increase in the relative abundance of the acid fraction may be due to preferential degradation of more easily biodegraded components, such as saturated hydrocarbons, this would not result 706 Analytical Chemistry, Vol. 73, No. 3, February 1, 2001
Figure 2. Gas chromatograms of methylated carboxylic acid fractions from (a) undegraded North Sea oil M and (b) biodegraded North Sea oil A. SS1 and SS2 are the surrogate standards 1-adamantanecarboxylic acid and 5β-cholanic acid, respectively. IS is the internal standard 1-phenyl-1-cyclohexanecarboxylic acid methyl ester; 16 and 18 are C16 and C18 n-alkanoic acids.
in the measured enrichment in acids and therefore the majority of the acids must be newly formed during the in-reservoir biodegradation process. The neoformed acids may be derived from the incomplete oxidation of the petroleum hydrocarbons34 or from the lipid biomass of the microorganisms responsible for the biodegradation. This interpretation is supported by recent work, which noted a 100-fold increase in the concentration of carboxylic acids in a crude oil biodegraded in the laboratory.35 It has also been shown that, with a few exceptions, there is a very good correlation between the carboxylic acid content of crude oils measured by this method, the neutralization or total acid number value of the oil, and the extent of biodegradation of the oils.36 The isolated carboxylic acid fractions can be analyzed by GC/ MS to show and quantify the distributions of biomarker compounds such as hopanoic acids (e.g., Figure 3), whose abundances and distributions have been used to provide indicators of both biodegradation6,36 and migration in crude oils.6 The composition of these isolated acid fractions can therefore provide information on a number of important petroleum geochemical processes. Application 2. The method was also used to separate carboxylic acid fractions from crude oils degraded in the labora(34) Atlas, R. M. Petroleum Microbiology; Collier Macmillen Publishers: London, 1984. (35) Watson, J. S.; Jones D. M.; Swannell, R. P. J. In In Situ Bioremediation of Petroleum Hydrocarbon and Other Organic Compounds; Alleman, B. C., Leeson, A., Eds.; Battelle Press: Columbus, OH, 1999; pp 251-255. (36) Meredith, W.; Kelland, S.-J.; Jones, D. M. Org. Geochem. 2000, 31, 10591073.
Table 3. Concentrations of Acids (mg/g of Dry Sediment) in Unoiled (1) and Oiled (2) Sediments from a Field Marine Mesocosm Experiment Ten Months after Oil Application
Figure 3. Mass chromatograms (m/z 235, 249, and 263) of a methylated carboxylic acid fraction from a moderately biodegraded crude oil, showing the distributions of C30, C31, and C32 hopanoic acids, respectively. The 17(H), 21(H), and 22S/R isomer peak identifications are as given in Jaffe and Gallardo:6 (1) RβS; (2) RβR; (3) βRS; (4) βRR; (5) ββR; (6) ββS.
tory using natural seawater and sand/seawater mixtures and also from oils degraded in field mesocosm-scale oil spill bioremediation experiments in a marine fine-grained sand environment.35 Preliminary results from these studies show that the method is able to measure the increase in the concentration of carboxylic acids with degradation time in oils that were recovered by solvent extraction from these matrixes. Like the in-reservoir and laboratory degradation studies, the oils degraded in the marine environment show significant increases in their carboxylic acid concentrations, though unlike the former studies, their acid distributions were dominated by C10-C30 n-alkanoic acids (see Table 3) rather than a complex mixture of branched and cyclic acids. The reasons for the difference in the acid compositions between the marine environment degraded oils and the laboratory and reservoir studies have not yet been fully elucidated and are currently being studied but probably relate to differences in the biodegradation mechanisms operating. However, since the amounts of carboxylic acids added to the sediments with the oil were negligible in comparison to the background concentrations of acids in original sediment, the increases in the amounts of n-acids and total acids in the oiled sediment after degradation probably represent an input of bacterial biomass-derived fatty acids as well as mostly nonresolved acidic petroleum biodegradation products. Nevertheless, the measurable differences in acid concentrations with degradation time in the oiled sediment are clearly another method for monitoring the progress of biodegradation, with potential for the discovery of acidic molecular markers of specific organisms or redox processes. CONCLUSIONS A routine, quantitative, and reproducible solid-phase extraction method has been developed for the analysis of aliphatic and
n-C14:0 n-C15:0 n-C16:0 n-C17:0 n-C18:0 n-C19:0 n-C20:0 n-C21:0 n-C22:0 n-C23:0 n-C24:0 n-C25:0 n-C26:0 n-C27:0 n-C28:0 summed n-acidsa TGCDa
1 (mg/g)
2 (mg/g)
1.31 1.71 7.69 0.58 5.35 0.07 0.18 0.03 0.19 0.07 0.47 0.09 0.27 0.68 0.28 6.02 22.67
4.39 1.77 34.24 1.69 52.02 0.20 0.92 nm 0.46 0.14 1.44 0.28 0.76 0.20 0.91 13.26 104.6
a Values exclude added standards and n-C16:0 and n-C18:0 acids; nm is not measurable.
naphthenic acids in crude oils using low solvent volumes. It has been demonstrated to be effective on light and heavy oils, and it enables the provision of fractions suitable for subsequent GC and GC/MS analysis, which in turn are capable of providing mechanistic information on the origin of the acids in the oils. The method is not efficient for the analysis of benzoic acid though the recovery of higher (two-four-ringed) aromatic acids is efficient if diazomethane rather than BF3/methanol is used for the derivatization procedure. The similarity of acid fractions isolated from crude oils and methylated using the two derivatization procedures indicates that two-four-ringed aromatic acids are not quantitatively important components of them. Analysis of the oils, which were solvent extracted from laboratory and field mesocosm marine sediment oil degradation studies, indicate that this new method of analyzing the products of hydrocarbon biodegradation may be a useful tool for monitoring the progress of bioremediation of oil spills in the environment. ACKNOWLEDGMENT We are very grateful to Norsk Hydro a.s. and Saga Petroleum (Norway) for funding much of this method development work and for their permission to publish it. We are also grateful to Enterprise Oil plc (U.K.) for funding refinement work on the method, for the supply of supply of oil samples and ancilliary data, and for permission to publish data on their samples. International Sorbent Technology (IST), U.K., are thanked for supplying various SPE cartridges and advice during the method work. We acknowledge valuable discussions with Steve Larter and technical support from K. Noke, P. Donohoe, I. Harrison, and E. Hart. Received for review May 31, 2000. Accepted November 21, 2000. AC000621A
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