Resolution and Identification of Elemental Compositions for More than 3000 Crude Acids in Heavy Petroleum by Negative-Ion Microelectrospray High-Field Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Kuangnan Qian* and Winston K. Robbins ExxonMobil Research and Engineering, 1545 Route 22 East, Annandale, New Jersey 08801 Christine A. Hughey,�� Helen J. Cooper, Ryan P. Rodgers, and Alan G. Marshall*,�� Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310 Received May 17, 2001. Revised Manuscript Received July 13, 2001 Although crude acids are minor constituents in petroleum, they have significant implications for crude oil geochemistry, corrosion, and commerce. We have previously demonstrated that a single positive-ion electrospray ionization (ESI) high-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) experiment can resolve and identify 3000 chemically different elemental compositions of bases (basic nitrogen compounds) in a crude oil. Here, we show that negative-ion ESI high-field FT-ICR MS can selectively ionize and identify naphthenic acids without interference from the hydrocarbon background. When combined with prechro- matographic separation, ESI FT-ICR MS reveals an even more detailed acid composition. An average mass resolving power, m/��m50% g 80 000 (��m50% is mass spectral peak full width at half-maximum peak height) across a wide mass range (200 m/z 1000), distinguishes as many as 15 distinct chemical formulas within a 0.26 Da mass window. Collectively, more than 3000 chemically different elemental compositions containing O2, O3, O4, and O2S, O3S, and O4S were determined in a South American heavy crude. Our data indicates that the crude acids consist of a mixture of structures ranging from C15-C55 with cyclic (1-6 rings) and aromatic (1-3 ring) structures. The acid composition appears to be simpler than that of the corresponding hydrocarbon analogues. Introduction Crude acids are minor constituents in petroleum with special significance in geochemistry, corrosion, and commerce.1 Crude oils are considered acidic if their total acid number (TAN) exceeds 0.5 mg KOH/g by nonaque- ous titration. Petroleum acids are found predominantly in immature, biodegraded, heavy crudes.2,3 The rela- tionships between these acids and their hydrocarbon counterparts in crudes have been studied in petroleum formation and migration.4,5 In refineries, they distill into the gas oil and vacuum gas oil fractions and cause liquid-phase corrosion at process temperatures of 250- 400 ��C.6-8 Commercial naphthenic acids extracted from gas oil find applications as specialty chemicals.1 Because naphthenic acids are surface active and marginally water-soluble, their release to wastewaters is closely monitored.9 Historically, crude oil acids have had to be isolated from the hydrocarbon matrix before they can be posi- tively characterized by spectroscopic techniques. The acids isolated by amine-silica gel chromatography10 have been examined by FTIR11 and 13C NMR.12 Most char- acterization of acids, however, has come from a variety of mass spectrometric (MS) techniques. For example, exhaustive extraction and selective reduction to parent * To whom correspondence may be addressed. �� Department of Chemistry, Florida State University. (1) Brient, J. Wessner, P. J. Doyle, M. N. Naphthenic Acids, 4th ed. Brient, J., Wessner, P. J., Doyle, M. N. Ed. 1995 Vol. 16, pp 1017-1029. (2) Ahsan, A. Karlsen, D. A. Patience, R. L. Mar. Pet. Geol. 1997, 14, 55-64. (3) Jaffe, R. Gardinali, P. Wolff, G. A. Org. Geochem. 1992, 18, 195- 201. (4) Jaffe, R. Albrecht, P. Oudin, J. L. Geochim. Cosmochim. Acta 1988, 52, 2599-2607. (5) Koike, L. Reboucas, L. M. C. Reis, F. D. M. Marsaioli, A. J. Richnow, H. H. Michaelis, W. Org. Geochem. 1992, 18, 851-860. (6) Gutzeit, J. Mater. Perform. 1977, 16, 24-35. (7) Piehl, R. NACE Conf. 1987, paper no. 196. (8) Babaian-Kibala, E. al., e. Mater. Perform. 1993, 50-55. (9) Lai, J. W. S. Pinto, L. J. Kiehlmann, E. BendellYoung, L. I. Moore, M. M. Environ. Toxicol. Chem. 1996, 15, 1482-1491. (10) Morrison, B. DeAngelis, D. Bonnette, L. Wood, S. Presented at PittCon, New Orleans, 1992. (11) Li, K. Zhang, J. Zhao, X. Luo, Y. Xu, C. Acta Petrolei Sinica 1995, 6, 100-108. (12) Shinn, J. Robinson, R. Rechsteiner, C. Tomczyk, N. Winans, R. Presented at the World Petroleum Congress, Beijing, 1995 Forum 15, Poster 5. 1505 Energy & Fuels 2001, 15, 1505-1511 10.1021/ef010111z CCC: $20.00 �� 2001 American Chemical Society Published on Web 08/28/2001

hydrocarbons have been combined with high-resolution MS to identify 3-ring and 4-ring steroid carboxylic acids in a California crude.13,14 A wider range of linear and acyclic isoprenoid acids has been esterified and identi- fied by GC/MS.15,16 Isolated acid fractions have also been characterized by negative-ion mass spectrometry tech- niques to generate ring type and carbon number dis- tributions. A range of acid molecules with 0-6 naph- thenic rings and carbon numbers 10-35 has been reported by fluoride negative ion chemical ionization (NICI) MS of California crudes.17 Negative-ion fast atom bombardment (FAB) MS18 has been used to analyze acids isolated by ion exchange from California, Mon- tana, and Louisiana crudes, showing a wider carbon range of naphthenic acids (C10-C50) with 0-6 rings. Aliphatic and naphthenic acids have been characterized by GC/MS following nonaqueous solid-phase extraction and methylation.19 Overall, MS data suggest that the naphthenic acids are closely related to the distribution of naphthenic hydrocarbons in their source oils. In addition, MS analyses reveal the presence of aromatic and hetero-aromatic acids (N, S, NOx, and SOx) at levels close to those for the ���true��� naphthenic acids in the California and Venezuelan crudes.14,20-22 Direct analysis of oxidized hydrocarbons (alcohol, ketone and acids) in lube oil has been accomplished by on-line liquid chro- matography negative ion isobutane chemical ionization mass spectrometry.23 Finally, direct characterization of crude acids by negative ion atmospheric pressure chemi- cal ionization (APCI) has been reported 24 however, the acid composition was not determined due to limited mass resolving power. In our previous efforts, we have explored the use of Electrospray Ionization (ESI) high field Fourier trans- form ion cyclotron resonance (FT-ICR) MS to character- ize nitrogen-containing aromatics25 and petroporphy- rins26 in crude oils. With high field positive-ion ESI FT- ICR MS, we were able to identify elemental compositions for more than 3000 basic nitrogen molecules in a South American heavy petroleum crude.25 In this work, we explore negative-ion ESI high field FT-ICR MS for characterizing the heavy petroleums. Acidic hydrocar- bons in the petroleum crude are directly speciated in extremely high detail. Isolated acid fractions from the crude were also analyzed by the same technique for comparison. Experimental Methods Samples. The South American heavy crude oil analyzed in this work has previously been characterized by positive-ion ESI FT-ICR mass spectrometry.25 It contains ���50% of 566 ��C boiling point hydrocarbons, and contains 4.02% sulfur and 0.65% nitrogen. It has a TAN number of 3.2. Acid fractions were isolated from the crude by solid-phase extraction (see below). Isolation of Acid Fractions. One hundred grams of the total crude oil was diluted with 700 mL (70:30) of toluene and methanol, and loaded onto 50 g of amino-propel silica (APS, Baker). After standing overnight, the bulk oil solution was removed by filtration and solvent wash. The acid-loaded APS was Soxhlet extracted with 30% acetic acid in toluene. The extract was water washed to remove residual acetic acid and then rotovapped to remove solvent. The residue was re- extracted with hexane. The hexane-soluble fraction is hereafter designated as the ���acid fraction��� (2.34 g), and the hexane- insoluble fraction as ���acidic asphaltene��� (0.37 g). ESI Sample Preparation. Crude oil samples were pre- pared by dissolving ���10-20 mg in 3 mL toluene, and then diluting with 17 mL MeOH. Fifty to 100 ��L of ammonium hydroxide solution (30%) was added to facilitate deprotonation of the acids and neutral nitrogen compounds to yield [M-H]- ions. Less ammonium hydroxide (a few microliters) was added to the acidic asphaltene fraction to reduce the amount of precipitate that formed upon addition of the base. Electrospray Ionization High-Field FT-ICR Mass Spec- trometry. The extra heavy oil was analyzed at the National High Magnetic Field Laboratory (NHMFL) with a home-built 9.4 T FT-ICR mass spectrometer.27 Ions were generated externally by a microelectrospray source28 and samples deliv- ered by a syringe pump at a rate of 300 nL/min. 2.2 kV was applied between the capillary needle and ion entrance. The externally generated ions were accumulated in a short (45 cm) rf-only octopole for 5-10 s and then transferred via a 200 cm rf-only octopole ion guide to a Penning trap. Ions were excited by frequency-sweep (100-725 kHz at 150 Hz/��s at an ampli- tude of 200 Vp-p across a 10 cm diameter open cylindrical cell). The time-domain ICR signal was sampled at 1.28 Msample/s for 1.63 s to yield 2 Mword time-domain data. Two hundred data sets were co-added, zero-filled once, Hanning apodized, and fast Fourier transformed with magnitude computation. A continuous-wave 40 W CO2 laser (Synrad E48-2-115, Bothell, WA) was used to dissociate noncovalent ion complexes. Mass Calibration. The high field FT-ICR mass spectra were frequency-to-m/z calibrated internally, with respect to a #G2421A electrospray ���tuning mix��� from Agilent (high mass) and stearic acid (low mass). The full range mass spectrum was then converted to the Kendrick mass scale (see below) with high accuracy by use of identified sample peak(s): C19H33O2 (Kmass ) 292.9211) and C39H73O2 (Kmass ) 572.9211). Data Analysis and Interpretation It is helpful to convert the mass spectral data from the IUPAC mass scale (based on the 12C atomic mass as exactly 12 Da) to the Kendrick mass (KMass) scale to facilitate the identification of petroleum homologues. (13) Seifert, W. Teeter, R. Howells, W. Cantow, M. Anal. Chem. 1969, 41, 1639-1646. (14) Seifert, W. Fortzchr. Chem. Org. Naturst. 1975, 32, 1-49. (15) Green, J. B. Stierwalt, B. K. Thomson, J. S. Treese, C. A. Anal. Chem. 1985, 57, 2207-2211. (16) Green, J. B. Yu, S. K. T. Vrana, R. P. HRC, J. High Resolut. Chromatogr. 1994, 17, 427-438. (17) Dzidic, I. Somerville, A. C. Raia, J. C. Hart, H. V. Anal. Chem. 1988, 60, 1318-1323. (18) Fan, T. P. Energy Fuels 1991, 5, 371-375. (19) Jones, D. M. Watson, J. S. Meredith, W. Chen, M. B., B. Anal. Chem. 2001, 73, 703-707. (20) Seifert, W. Teeter, R. Analytical Chemistry 1970, 42, 750-758. (21) Green, J. B. Analysis of Heavy Oils: Method Development and Application to Cerro Negro Heavy Petroleum. U.S. D. O. E. Report No. DE0000200 Green, J. B., Ed. U.S. Department of Energy: Washington, DC, 1989 Vol. NIPER Publication-452. (22) Tomczyk, N. Winans, R. Shinn, J. Identification of Acidic Constituents in a California Heavy Crude. Tomczyk, N., Winans, R., Shinn, J., Ed. ACS Division of Fuel Preprints, American Chemical Society: Washington, DC, 1997 pp 339-343. (23) Qian, K. Hsu, C. Robbins, W. Rose, K. In Proceedings of the 40th ASMS Conference on Mass Spectrometry and Allied Topics, Washington, DC, 1992 pp 758-759. (24) Hsu, C. S. Dechert, G. J. Robbins, W. K. Fukuda, E. K. Energy Fuels 2000, 14, 217-223. (25) Qian, K. Rodgers, R. P. Hendrickson, C. L. Emmett, M. R. Marshall, A. G. Energy Fuels 2001, 15, 492-498. (26) Rodgers, R. P. Hendrickson, C. L. Emmett, M. R. Marshall, A. G. Greaney, M. Qian, K. Can. J. Chem. 2001, 79, 546-551. (27) Senko, M. W. Hendrickson, C. L. Pasa-Tolic, L. Marto, J. A. White, F. M. Guan, S. Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1824-1828. (28) Senko, M. W. Hendrickson, C. L. Emmett, M. R. Shi, S. D.- H. Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. 1506 Energy & Fuels, Vol. 15, No. 6, 2001 Qian et al.