Detection of Polyaromatic Sulfur Heterocycles in Crude Oil Using

Our protocol utilizes distillation, alumina, and ligand exchange chromatography and HPLC to partially fractionate the crude oil. Postcolumn addition o...
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Detection of Polyaromatic Sulfur Heterocycles in Crude Oil Using Postcolumn Addition of Tropylium and Tandem Mass Spectrometry Walter E. Rudzinski* and Vidya Rai Department of Chemistry and Institute for Environmental and Industrial Science, Texas State University, San Marcos, Texas 78666-4616 Received July 28, 2004. Revised Manuscript Received April 28, 2005

To complement the gas chromatography/mass spectrometric separation and detection of 3- and 4-ring organosulfur compounds, established methods for the separation of polyaromatic sulfur heterocycles (PASH) have been coupled to high-performance liquid chromatography/atmospheric pressure chemical ionization/tandem mass spectrometry (HPLC/APCI/MS/MS). The latter technique is capable of detecting both thermally unstable and nonvolatile PASH compounds in a crude oil. Our protocol utilizes distillation, alumina, and ligand exchange chromatography and HPLC to partially fractionate the crude oil. Postcolumn addition of tropylium cations prior to APCI/MS leads to an increase in the sensitivity of the detector toward 3- and 4-ring organosulfur compounds. Nonalkylated species primarily generate radical cations in the APCI source, whereas alkylated species exhibit both radical cation and product ions resulting from addition and/or loss of hydrogen and loss of alkyl groups. MS2 and MS3 fragmentation patterns confirm the presence of dibenzothiophene and benzonaphthothiophene derivatives in an Arabian crude oil.

Introduction Interest in the isolation and characterization of polyaromatic sulfur heterocycles (PASH) has increased for a number of reasons. PASH have potential mutagenic/ carcinogenic properties,1 they are difficult to desulfurize when attempting to produce low sulfur fuels, they photoreact in the aqueous phase after oil spills, they affect microbial metabolism, and they function as possible indicators for the maturity of crude oils and source rocks.2 Arguably, the most important reason for the interest in PASH is its combustion to form SO2, which contributes to acid rain.3 New legislation in Japan and Europe will limit the sulfur content in light oil to 50 parts per million (ppm) by 2005.4 The United States will limit the sulfur content to 15 ppm by the middle of 2006.5 Although a variety of techniques have been used to isolate and remove the sulfur, further developments are necessary to meet the new regulatory requirements.6 Since the properties of polyaromatic sulfur heterocycles (PASH) are very similar to those of polyaromatic hydrocarbons (PAH), finding methods for the isolation, chromatographic separation, and identification of PASH is difficult. For the fractionation of crude oil, hydrocarbon type analysis (HTA) by open-column chromatography is * To whom correspondence should be addressed. Phone: (512) 2453120; fax: (512) 245-2374; e-mail: [email protected]. (1) Delgado, M. A.; Tena, R. C.; Montelango, F. I Chromatogr. Italia 1999, 50, 235-238. (2) Andersson, J. T.; Schmid, B. J. Chromatogr. A 1995, 693, 325338. (3) Monticello, D. J. CHEMTECH 1998, 28, 38-45. (4) Off. J. Eur. Commun. December 28, 1998, L350, 58. (5) EPA Federal Register 2001, 66, 5001. (6) Whitehurst, D. D.; Isoda, T.; Mochida, I. Adv. Catal. 1998, 42, 345-471.

employed initially. Here, the goal of chromatographic separation is to prepare chemically meaningful and operationally well-defined compound class fractions for further molecular characterization.7 A classic example of this type of analysis is the saturate-aromatic-resinasphaltene (SARA) method, which is based on adsorption chromatography using solvent mixtures of increasing eluotropic strength.8 For the separation of PASH, further fractionation is needed.9 Nishioka developed a ligand exchange chromatography (LEC) approach using PdCl2 and CuCl2 on silica gel columns.10-14 CuCl2 can be used to isolate thiophenols and sufides,13 while PdCl2 can be used to separate PASH compounds.11 Unfortunately, the selectivity of PdCl2 varies with the organosulfur ring size. Milenkovic et al.15 and Rudzinski and co-workers16 found that the selectivity decreased in the order of 3-ring > 2-ring > 1-ring PASH compounds. After the initial open column chromatographic separation of compound classes, GC or GC/MS is often used for the separation of PASH compounds. The Nishioka (7) Boduszynski, M. M. Energy Fuels 1988, 2, 597-613. (8) Ali, M. F.; Bukhari, A.; Misbah-ul-Hasan. Fuel Sci. Technol. Int. 1989, 7, 1179-1208. (9) Later, D. W.; Lee, M. L.; Bartle, K. D.; Kong, R. C.; Vassilaros, D. L. Anal. Chem. 1981, 53, 1612-1620. (10) Nishioka, M. Energy Fuels 1988, 2, 214-219. (11) Nishioka, M.; Campbell, R. M.; Lee, M. L.; Castle, R. N. Fuel 1986, 65, 270-273. (12) Nishioka, M.; Whiting, D. G.; Campbell, R. M.; Lee, M. L. Anal. Chem. 1986, 58, 2251-2255. (13) Nishioka, M.; Tomich, R. S. Fuel 1993, 72, 1007-1010. (14) Nishioka, M.; Lee, M. L.; Castle, R. N. Fuel 1986, 65, 390395. (15) Milenkovic, A.; Schulz, E.; Meille, V.; Loffreda, D.; Forissier, M.; Vrinat, M.; Sautet, P.; Lemaire, M. Energy Fuels 1999, 13, 881887. (16) Rudzinski, W. E.; Sassman, S.; Watkins, L. M. Prepr. Pap.s Am. Chem. Soc., Div. Pet. Chem. 2000, 45, 564-565.

10.1021/ef0400666 CCC: $30.25 © 2005 American Chemical Society Published on Web 06/18/2005

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method was applied to a coal liquid and petroleum heavy ends, and the PASH fraction was examined by capillary column gas chromatography with flame ionization and flame photometric detection as well as GC/ MS. The results revealed the presence of PASH having two to six aromatic rings.11 Andersson and Schmid2 used the chloroperbenzoic acid oxidation method for 2-ring and the Nishioka method for 3-ring organosulfur compounds. Using gas chromatography with sulfur specific atomic emission detection (AED), and GC/MS, they were able to obtain quantitative data for 22 organosulfur compounds including alkylated benzothiophenes, dibenzothiophenes, and naphthothiophenes. Wise and coworkers extended the analysis of PASH up to 80 compounds using GC/MS and a variety of stationary phases.17 Although GC and GC/MS are powerful analytical tools for the characterization of volatile and semivolatile hydrocarbons and PASH, for high molecular weight, difficult to volatilize compounds, GC has limitations. The high temperature employed for volatilization may cause decomposition. In addition, if electron impact ionization is used, the parent ion may be absent due to fragmentation, thus hampering sample identification. Degradation of the front end of the capillary column can also occur as nonvolatile components from the sample accumulate over time. HPLC can obviate some of the problems associated with GC. For example, Matsunaga applied HPLC to the separation of aromatic compounds differing in ring size, the separation of polar compounds (nitrogen- and oxygencontaining species) from polynuclear aromatic hydrocarbons, and the separation of polar compounds with different functional groups.18 Other workers also applied HPLC techniques for the analysis of polyaromatic hydrocarbons with varying degrees of success.19-23 Unfortunately, unless HPLC is coupled with MS and MS2, it is often difficult to determine the identity of compounds for which standards are not available. One obvious approach for the separation and identification of PASH compounds would seem to be HPLC coupled with atmospheric pressure ionization/mass spectrometry. Unfortunately, PAH and PASH compounds are not amenable to the normal mechanisms of ion formation that occur during the ionization process. PAH and PASH compounds are not easily protonated or deprotonatedsthe primary means of inducing charge. This means that these species are not readily ionized in the source. One solution is coordination-ion spray. Here positively or negatively charged metal complexes are formed by the addition of a suitable metal ion to the sample prior to detection by MS. Metals of first and eighth transition groups such as Cu+, Ni2+, Pd2+, Pt2+, and Ag+ are employed. These form stable π-π or π-allyl complexes with unsaturated compounds.24 In a specific case, Ag+ (17) Mossner, S. G.; Lopez de Alda, M. J.; Sander, L. C.; Lee, M. L.; Wise, S. A. J. Chromatogr. 1999, 841, 207-228. (18) Matsunaga, A. Anal. Chem. 1983, 55, 1375-1379. (19) Wise, S. A.; Sandra, L. C.; May, W. E. J. Chromatogr. 1993, 642, 329-349. (20) Thomson, J. S.; Grizzli, P. L. Anal. Chem. 1982, 54, 1071-1078. (21) Robbins, W. K J. Chromatogr. Sci. 1998, 36, 457-466. (22) McKerrell, E. H. Fuel 1993, 72, 1403-1409. (23) Hayes, P. C.; Anderson, S. D. Anal. Chem. 1985, 57, 20942098.

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has been used to analyze heavy aromatic hydrocarbons in petroleum fractions by electrospray ionization/mass spectrometry (ESI/MS). The aromatic compounds react with the silver ion to form abundant adduct ions such as [M + Ag]+ and [2M + Ag]+.25 Van Berkel and co-workers in several reports have demonstrated that electron transfer reactions are also an efficient means of generating radical cations of aromatic, heteroaromatic, and other highly conjugated systems.26 For example, they ionized neutral PAH, thianthrene, a substituted aromatic, and the highly conjugated molecule buckminsterfullerene (C60) in solution via reactions with the chemical electron-transfer reagents: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and antimony pentafluoride and then detected these as their respective radical cations in the gas phase by ESI/MS. In yet another approach, Rudzinski et al. used Pd2+ to produce radical cations of PASH that were identified by ESI/MS. The process involved an electron transfer between the metal ion and the PASH compound.27 They further studied the solvent and matrix effects in the previous MS analysis. It was found that in a hydrogenated oil matrix, PASH compounds in the presence of PdCl2 in (50:50) CH3OH/CH2Cl2 gave an enhanced response when compared to a PASH mixture prepared in (50:50) CH3OH/CH3CN. This suggested that CH2Cl2 is an important solvent for radical cation stabilization.28,29 Recently, Airiau and co-workers analyzed PAH using HPLC/ESI/MS/MS after reaction with the tropylium cation, which produces positive ions.30 The tropylium cation is a strong π-acceptor, and it recognizes PAH by π-π interactions and almost quantitatively forms a 1:1 cation complex.30 This complex, which is formed in solution, can also undergo a charge transfer to form [PAH]+• in the electrospray interface. In this research, an established method for separating crude oil fractions has been extended to exploit the capabilities of high-performance liquid chromatography/ atmospheric pressure chemical ionization/tandem mass spectrometry (HPLC/APCI/MS/MS). Although gas chromatography/mass spectrometry (GC/MS) exhibits superior resolution,23 detection is ultimately limited by the volatility of the sample. Postcolumn addition of tropylium cations prior to APCI/MS enhances the formation of charged PASH compounds. Ultimately, the technique can detect 3- and 4-ring organosulfur compounds as well as generate a mass spectrum yielding a parent ion and separate fragmentation ion spectrum. It is anticipated that the technique can also be applied to the detection of polyaromatic sulfur heterocycles containing five or more rings or to polyaromatic sulfur heterocycles containing additional functional groups within the ring (24) Bayer, E. G.; Gfrorer, P.; Rentel, C. Angew Chem., Int. Ed. 1999, 38, 992-995. (25) Roussis, S. G.; Prouix, R. Anal. Chem. 2002 74, 1408-1414. (26) Van Berkel, G. J.; Asano, K. G. Anal. Chem. 1994, 66, 20962102. (27) Rudzinski, W. E.; Zhang, Y.; Luo, X. J. Mass Spectrom. 2003 38, 167-173. (28) Rudzinski, W. E.; Luo, X. Fuel Chem. Div. Preprints 2003, 48, 12. (29) Rudzinski, W. E.; Zhou, K.; Luo, X. Energy Fuels 2004 18, 1621. (30) Airiau, C. Y.; Brereton, R. G.; Crosby, J. Rapid Commun. Mass Spectrom. 2001, 15, 135-140.

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Figure 1. Representative organosulfur compounds.

structure, but these complex heterocycles would require a higher resolution mass analyzer (quadrupole/time-of flight/MS or Fourier transform/MS).

Figure 2. Scheme for the fractionation of the Arabian crude oil.

Experimental Procedures Reagents and Chemicals. Methanol(CH3OH), acetonitrile (CH3CN), hexane, chloroform (CH3Cl), dichloromethane (CH2Cl2), toluene, and diethyl ether were obtained from EM Science (Gibbstown, NJ). Arabian crude oil was obtained from Motiva Enterprises LLC. Alumina (Neutral, Brockmann Activity 1, 150 mesh), silica gel (70/270), tropylium tetrafluoroborate, and the organosulfur compounds (dibenzothiophene (DBT), 2-methyl dibenzothiophene (2-MDBT), 4,6-dimethyl-dibenzothiophene(4,6-DMDBT), thianthrene (TAN), and benzonaphthothiophene (BNTP)) were obtained from Sigma-Aldrich (Milwaukee, WI). Figure 1 shows the structure and formula weight (FW) for each of the organosulfur standards. Preparation of Standard Solutions. A total of 7.12 mg of tropylium tetrafluoroborate was dissolved in 2 mL of a solution of CH2Cl2/CH3CN (80:20). After 1 h, the solid dissolved and turned into a clear solution. A total of 50 µL of the 20 mM solution was taken and diluted to 1 mL in CH2Cl2. The resulting solution had a concentration of 1 mM. A total of 1.84 mg of DBT, 1.98 mg of 2-MDBT, 2.12 mg of 4,6-DMDBT, 2.13 mg of TAN, and 2.34 mg of BNTP were each dissolved in 10 mL of hexane. The concentration of each organosulfur standard was 1 mM. To prepare the standard mixture, 100 µL of each of the standard 1 mM solutions was mixed together and diluted with hexane to a final volume of 1 mL. Each of the organosulfur standards had a final concentration of 10-4 M. Fractionation of Arabian Crude Oil. The Arabian crude oil was distilled. A light distillate boiling up to 200 °C and a middle distillate boiling between 200 and 310 °C were collected under atmospheric pressure. The heavy distillate was obtained under reduced pressure (2.67 × 103 Pa), keeping the temperature below 260 °C. The residue was the remaining nonvolatile fraction. The heavy distillate was then fractionated using column chromatography (SARA method).6 The alumina was activated at 220 °C for 96 h. A total of 10 g of activated alumina was pre-wet with hexane and packed into a glass column (1.1 × 12.5 cm). A total of 0.325 g of heavy distillate was dissolved in 5 mL of hexane and loaded at the top of the column. The

saturates were eluted with 40 mL of hexane, the aromatic hydrocarbons with 80 mL of toluene, and the resin with 20 mL of toluene/methanol (80:20). The asphaltenes remained on the column. The fractions were evaporated to dryness. A total of 100 mL of distilled water was added to 1 g of PdCl2 and mixed thoroughly. This was then added to 20 g of silica gel. The supernatant was discarded, and the wet gel was dried overnight at 95 °C. This was then activated at 200 °C for 24 h. A total of 6 g of activated gel was pre-wet by a mixture of hexane/chloroform (50:50) and packed in a column (1.1 × 12.5 cm). A total of 50 mg of the aromatic fraction was dissolved in 5 mL of hexane/chloroform (50:50) and loaded on top of the column. The polyaromatic hydrocarbons (PAH) were eluted with 30 mL of hexane/chloroform (50:50) and the polyaromatic sulfur heterocycles (PASH) with an additional 50 mL of hexane/chloroform (50:50), while the sulfur containing polycyclic aromatic compounds (SPAC) were eluted with 100 mL of chloroform/diethyl ether (90:10). Fractions were then evaporated to dryness. See Figure 2 for the fractionation scheme. Solid-phase extraction on a LC-NH2 cartridge (Supelco LCNH2, 3 mL) removed excess PdCl2 from the PASH fraction. The tube was pre-wet with 2 mL of hexane/chloroform (50: 50). One mg of the PASH sample was then dissolved in 2 mL of hexane/chloroform (50:50) and loaded on top of the extraction cartridge. Five mL of solvent mixture was added to clean the sample. Six mL of CH2Cl2 was finally added to elute all of the PASH sample. The excess palladium stayed on the cartridge, resulting in a PASH sample that was colorless. The sample was evaporated to dryness. A total of 0.7 mg of PASH remained and was redissolved in 0.7 mL of hexane. This procedure is a modification of a method reported by Andersson31 for removal of PdCl2. Instrumentation. The LC system consisted of two Gilson 306 solvent delivery systems attached to a Gilson 506C Interface Module connected to a HCS (Hometown Computing, San Marcos, TX) computer with Unipoint version 1.71 software. The interface module was connected to a Rheodyne (31) Andersson, J. T. Anal. Chem. 1987, 59, 2209-2211.

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Figure 3. HPLC profile for a mixture of organosulfur compounds; LC-NH2 column. 7125s 100 µL injector. A LC-NH2 column (250 × 3.0 mm; dp ) 5 µm; Supelco, Bellefontaine, PA) and a polystyrene divinyl benzene (PRP) column (150 × 4.6 mm; dp ) 10 µm; Parker) were used for the sequential HPLC separations. The Gilson Model 118 UV-vis detector was set at 254 nm. All mobile phases were filtered through a 0.45 µm nylon filter prior to use to remove particulates and dissolved gases. A total of 50 µL of 10-4 M organosulfur standard mixture or 25 µL of a solution containing 1 mg of PASH/1 mL of hexane was injected into the LC-NH2 column and separated using 100% hexane mobile phase at a flow rate of 1 mL/min. Two separate fractions (3- and 4-ring compounds) were collected in the case of the standards as well as the oil. The solvent was evaporated and reconstituted in 25 µL of hexane/CH2Cl2 (70:30). Mass spectra were obtained on a Finnigan LCQ ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source. A Hometown Computing System computer with Excalibur 1.2 software was used for system control as well as collection and analysis of the data. Mass spectrometry (MS) was performed in full scan as well as single ion monitoring (SIM) modes. MS2 and MS3 were performed, and peak fragments were monitored over the m/z range of 100-300. Each scan was the average of three microscans, and spectra were obtained continuously. Instrumental parameters for the APCI source were as follows: nitrogen sheath gas flow-rate, 70 (arbitrary units); ion mode, positive; corona discharge current, 5 µA; vaporizer temperature, 450 °C; capillary temperature, 150 °C; capillary voltage, 17 V; and tube lens offset voltage, 15 V. All LC/MS experiments were performed with the PRP column attached to the MS, using (70:30) hexane/CH2Cl2 as the mobile phase at a flow rate between 0.30 and 0.33 mL/ min. All LC/MS runs involved a postcolumn reaction with a tropylium cation. Before entering the probe of the mass spectrometer, the mobile phase from the HPLC system was combined at a standard tee with 1 mM tropylium solution in 100% dichloromethane added at a flow rate of 15 µL/min.

Results and Discussion Analysis of the Organosulfur Standard Mixture. The LC-NH2 column separates aromatic compounds based on ring size rather than substituents. The column was used to separate the organosulfur standard mixture (see Figure 3). Through spiking with authentic standards, it was found that the first two peaks (tR ) 3.28 and 3.51 min) correspond to the 3-ring compounds (DBT, 2-MDBT, 4,6-DMDBT, and TAN), while the third peak (tR ) 4.51 min) corresponds to the 4-ring compound (BNTP). Injection of the first two combined peaks (fraction 1) from the LC-NH2 column into a PRP column yielded the chromatogram in Figure 4. Chromatographic peaks, once separated, are usually not recombined for further separation, but, in this case, the two were recombined

Figure 4. HPLC of fraction 1; PRP column.

Figure 5. HPLC/APCI/MS scan of (A) organosulfur standard mixture in the absence of tropylium and (B) tropylium; PRP column. Flow rate ) 0.30 mL/min. MS full scan mode.

to validate a procedure that would be applicable to oil samples that do not yield separated peaks for 3- and 4-ring compounds but chromatographic envelopes. The standard addition of genuine standards determined the following respective retention times (tR) at a flow rate of 0.30 mL/min: 4,6 DMDBT at 6.88 min, 2-MDBT at 7.35 min, TAN at 7.6 min, and DBT at 8.0 min on the PRP column. Injection of the eluent associated with the peak at tR ) 4.51 min (fraction 2) yielded an elution time of 8.9 min, which corresponds to BNTP. Although the PRP column does not appear to sufficiently resolve the 3-ring compounds to justify the added complexity of isolating the eluent from the LC-NH2 column, the PRP column does have the advantage that it can be used in an HPLC/MS system that uses CH2Cl2 in the mobile phase. In contrast, the LC-NH2 column offers no resolution of 3- and 4-ring compounds when CH2Cl2 is added to the mobile phase as a modifier. Yet, CH2Cl2 is needed when using an APCI source to enhance the sensitivity.28,29 Figure 5A shows the MS full scan of the organosulfur standard mixture in the absence of tropylium cation; there is no appreciable signal intensity for any of the organosulfur standards. Figure 5B shows the MS full

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Figure 6. HPLC/APCI/MS of organosulfur standard mixture; PRP column. Flow rate ) 0.33 mL/min. MS full scan mode: (A) total ion chromatogram; (B) MS spectrum of chromatographic envelope 6.31-6.67 min, range 100-300; (C) MS spectrum of chromatographic envelope 6.31-6.67 min, range 194-217; (D) MS spectrum of chromatographic envelope 6.89-7.50 min, range 100-300; and (E) MS spectrum of chromatographic envelope 7.93-8.44 min, range 100-300.

scan spectrum for tropylium. A parent ion appears at an m/z of 149 that corresponds to an unknown product produced from the tropylium cation. Upon postcolumn addition of tropylium, peaks corresponding to each of the standards may be observed. Figure 6A shows the total ion chromatogram (TIC) obtained at a flow rate of 0.33 mL/min for a standard mixture containing 4,6-DMDBT, 2-MDBT, TAN, DBT, and BNTP. Integrating the envelope appearing between 5.00 and 6.00 min did not yield any appreciable signal above the background. Integrating the peak envelope appearing between 6.31 and 6.67 (peak labeled 6.60) yielded two triplets centered around the nominal m/z (212.2, 198.3) of 4,6-DMDBT and 2-MDBT (see Figure 6B,C). These triplets designated M - 1, M, and M + 1 correspond to (M - H)+, the molecular ion peak [M•]+, and (M + H)+, respectively. The inset (Figure 6C) shows a relative intensity of M - 1> M > M + 1 for the two alkylated polyaromatic sulfur heterocycles. Figure 6D represents the MS spectrum for the chromatographic peak envelope labeled 7.30 in the TIC of the standards mixture. The MS spectrum reveals ions at 184.2, 197.2, 211.2, and 216.1. DBT with [M•]+ at 184.2 and TAN with [M•]+ at 216.1 are not resolved chromatographically and elute between 6.89 and 7.50 min. The smaller ions at 211.2 and 197.3.2 correspond to late eluting 4,6DMDBT-H and 2-MDBT-H. Figure 6E illustrates the MS spectrum for the peak labeled 8.07 in the TIC of the standards mixture. An MS ion at 234.3 corresponds to [M•]+ for BNTP. For the nonalkylated polyaromatic sulfur heterocycles, [M•]+ is the most intense. All of the standards generated ions with a signal intensity at or

above 105 at a concentration of 10-4 M in the presence of 1 mM tropylium. Figure 7A-D shows the chromatograms obtained for four of the standards when run in single ion monitoring (SIM) mode. 4,6-DMDBT and 2-MDBT essentially coelute between 6.4 and 7.5 min, while TAN and DBT coelute between 7.2 and 8.4 min. The data confirm the order of elution and coelution patterns observed previously; 4,6-DMDBT and 2-MDBT elute first, then TAN and DBT. Figure 8 illustrates the APCI/MS2 spectra of DBT, TAN, BNTP, 2-MDBT, and 4,6-DMDBT. DBT, TAN, and BNTP have a prominent precursor ion that is a stable radical cation, whereas, 2-MDBT and 4,6-DMDBT exhibit a precursor ion and a product ion missing one hydrogen. The signal intensity for the M - H species increases with collision energy. DBT and TAN exhibit neutral losses of 15 and 42 Da, which are assigned to [M H - CH2], and [M - H - C3H6], respectively, while 2MDBT and 4,6-DMDBT exhibit neutral losses of 16, 29, 43, and 57, which are assigned to [M - H - CH3], [M H - C2H4], [M - H - C3H6], and [M - H - C4H8], respectively. TAN had the previous losses as well as a neutral loss of 32, which corresponds to a loss of a sulfur atom.27 APCI/MS and MS/MS of Arabian Crude Oil Fractions. Once the Arabian crude oil had been fractionated as described in the Experimental Procedures, the PASH fraction (representing 0.3% of the total mass) was then separated using the LC-NH2 column. From the chromatogram (see Figure 9), it can be observed that the first peak comes out at 3.28 min, which is identical to the retention time of the first peak

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Figure 9. HPLC profile for PASH fraction from Arabian crude oil; LC-NH2 column.

Figure 7. HPLC/APCI/MS of organosulfur standard mixture. SIM mode. m/z set at (A) 212; (B) 198; (C) 216; and (D) 184. Figure 10. HPLC of PASH fraction 1 from Arabian crude oil; PRP column.

Figure 8. HPLC/APCI/MS2 for organosulfur standards. Collision energies are 38, 41, 41, 41, and 43% for DBT, 2-MDBT, 4,6-DMDBT, TAN, and BNTP, respectively.

in the LC-NH2 separation of the standards, while the second peak is around 4.7-4.8 min, which is also close to the retention time of the second peak in the standard chromatogram (4.51 min), suggesting that the PASH fraction contains both 3- and 4-ring compounds. Al-

though the integrated areas of the chromatographic peaks attributed to 3- and 4-ring compounds were approximately 95 and 5%, respectively, generally 4-ring compounds have higher molar absorptivities than 3-ring compounds so that the total concentration of 4-ring compounds is probably lower than 5%. The PASH fraction 1 collected from the LC-NH2 column when sent through a PRP column gave the chromatogram shown in Figure 10. There is a broad envelope between 6 and 8 min. This time interval is within the range of retention time for the 3- and 4-ring organosulfur standards. There are no individual peaks. This was expected as the oil is a complex mixture, and under the experimental conditions, individual components cannot be resolved. HPLC/APCI/MS of PASH fraction 1 gave the mass spectra in Figures 11 and 12. In Figure 11A, molecular ions are present at m/z 184.2, 198.2, 212.2, and 226.2. The first three molecular ions are isobaric with DBT, 2-MDBT, and 4,6-DMDBT, whereas, the m/z of 226.2 does not correlate with the m/z of any of the standards. A closer look at Figure 11B shows four ion envelopes that have a characteristic pattern of [M - 1]+•, [M]+•, and [M + 1]+•. A difference of 14 u appears between 184.3, 198.3, 212.2, and 226.2, which may be attributed to a -CH2 group. MS2 on the ion at 225.2 (M - 1) yielded a neutral loss of 42 and an ion at m/z 183.1, which indicates that the original ion may correspond to a derivative of DBT. MS3 on the ion at 183.1 yielded a fragmentation pattern identical to that of standard DBT (M - 1), confirming that 226.2 is a trimethylated DBT.

Polyaromatic Sulfur Heterocycles in Crude Oil

Figure 11. HPLC/APCI/MS scan of PASH fraction 1 between 6.5 and 8.2 min. MS full scan mode; mass range: (A) 100300 and (B) 180-230.

Figure 12. HPLC/APCI/MS scans of PASH fraction 1. MS full scan mode acquired at tR: (A) 6.88 min, (B) 7.34 min, (C) 7.60 min, and (D) 8.0 min.

Figure 12 shows the full-scan HPLC/MS of PASH fraction 1 at 6.88, 7.34, 7.60, and 8.0 min, respectively. It can be seen that at 6.88 min, which is the elution time of 4,6-DMDBT, molecular ions with m/z 212.2, 198.3, 226.2, and 184.3 appear with signal intensity of 105. The 212.2 peak has a mass/charge ratio that is isobaric with 4,6-DMDBT. Peaks 198.3 and 184.3 are isobaric with the molecular ions of 2-MDBT and DBT, respectively, and 226.2 is isobaric with DBT with three methyl groups. At 7.34 min, which corresponds to the elution time of 2-MDBT, molecular ions with m/z 212.3

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Figure 13. HPLC/APCI/MS scan of PASH fraction 2 between 6.5 and 9 min. MS full scan mode; mass range: (A) 100-300 and (B) 230-280.

and 198.2 are observed. Peak 198.2 is isobaric with the molecular ion of 2-MDBT. At 7.6 and 8.0 min, which are the elution times of TAN and DBT, respectively, there are no ions corresponding to the previous compounds. This shows the absence of these two compounds in this fraction of the oil. Figure 13A shows the full-scan HPLC/MS of PASH fraction 2 between 6.5 and 9.0 min. Figure 13B illustrates the mass spectrum of PASH fraction 2 in the mass range of 230-280; molecular ions at m/z 248.3, 262.3, and 276.3 appear. At 8.9 min, which is the elution time of BNTP, a low-intensity signal appears at m/z 233.2, which does not correspond to the molecular ion normally observed for BNTP. At 6.35, 6.70, and 7.0 min, molecular ions appear with m/z 276.3, 262.3, and 248.3, respectively. They have a similar characteristic fragmentation pattern as observed for the PASH compounds in fraction 1. There is a constant difference of 14 between m/z 248.3, 262.3, and 276.3, which corresponds to a -CH2 group. Also, the mass spectrum at 6.35 min contains the ion at m/z 276.3, the mass spectrum at 6.70 min contains the ion at m/z 262.3, and the mass spectrum at 7.0 min contains the ion at m/z 248; this evidence suggests that these ions correspond to methylated BNTP compounds since the more alkylated the PASH, the earlier the elution time (vide supra). MS2 on the ions at m/z 248.3, 262.3, and 276.3 generated product ions at m/z 234.1 and 233.1, which correspond to BNTP and BNTP-H. MS3 on the peaks at m/z 233.1 and 234.1 yielded a fragmentation pattern identical to that of standard BNTP, confirming that m/z 248.3, 262.3, and 276.3 are isobaric with mono-, di-, and trimethyl BNTP. Conclusions An established method was extended to HPLC/APCI/ MS2 for the isolation and detection of polyaromatic sul-

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fur heterocycles in a crude oil. The protocol utilizes distillation, alumina and ligand exchange chromatography, and HPLC to separate the complex mixture. Postcolumn addition of tropylium cations prior to APCI/MS leads to an increase in the sensitivity of the detector toward 3- and 4-ring organosulfur compounds. Nonalkylated species primarily generate radical cations in the APCI source, whereas alkylated species exhibit both radical cation and product ions resulting from the addition and loss of hydrogen and loss of alkyl groups. MS2 and MS3 fragmentation patterns confirmed the presence

Rudzinski and Rai

of dibenzothiophene and benzonaphthothiophene type derivatives in an Arabian crude oil. Acknowledgment. W.E.R. thanks the Environmental Protection Agency (Grant R825503) and the Texas Higher Education Coordinating Board through the Advanced Technology Program (Grant 003615-00392001) for support of this project. W.E.R. also thanks the Institute for Industrial and Environmental Science (IEIS) of Southwest Texas State University. EF0400666