Anal. Chem. 1987, 59, 339-343 (23) Chen, H. H. L. W.D. Thesis, University of Iowa, Iowa CW, I A , 1975. (24) Fredriksson, S. A; Cedergren, A. Anal. Chem. 1081, 53, 614-618. (25) Brent, D. A.; Chandrasurin, P.; Ragouzeos, A.; Huribert, 8. S.; Burke, J. T. J . Phafm. Scl. 1080, 89. 906.
RECEIVED for review June 18,1986. Accepted September 9,
339
1986. This paper was presented in part a t the 9th InternaLiquid Chromatography, ~ d tional ~~~~~i~~ on column inburgh, July 1985. The work was supported by the National Swedish Environment Protection Board and the Swedish Natural Science Research Council.
High-Efficiency Microcolumn Liquid Chromatography Separation and Spectral Characterization of Nitrogen-Containing Polycyclics from Fossil Fuels Claudio Borra, Donald Wiesler, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Very large polycycilc compounds, with one or more nitrogen atoms In their structures, were separated from fossil fuel samples and characterized spectroscoplcaily. High chromatographic efficiencies (around 200 000 theoretical plates) of microcolumn liquid chromatography were essential to separate such complex mixtures: within structures of 3-10-rings, almost 170 peaks were resolved and over 600 nitrogen poIycycuCS were spectrally characterized. Most of the structural data were obtained from mass spectroscopy; however, fluorescence emission spectra yielded additional Information concerning the shape of molecules.
All fossil fuels appear to contain some nitrogen, which is believed to be present almost exclusively in the organic portion of the crude materials. Its levels usually vary from approximately 0.5% in crude petroleum samples to 1-2% in shale oils and coal. Distillation, extraction, and combustion produda contain nitrogen compounds as minor components. Although these compounds have not been investigated as extensively as hydrocarbons and the neutral aromatic components, the compositional information is important for several reasons. Nitrogen polycyclic aromatic compounds (NPAC) are deemed responsible for some problems during the refining process because of their association with color, odor, corrosive power, and the formation of gums and deposits during storage ( 1 , 2 ) . In addition, they deactivate the catalysts during the cracking and re-forming process (3). Numerous basic nitrogen-containing substances are toxic. Several azaarenes and primary aromatic amines have been reported to be tumorigenic in experimental animals,while additional compounds of this type are suspected carcinogens or cocarcinogens (4-6). Furthermore, several neutral nitrogen aromatics, such as dibenzocarbazoles, show carcinogenic activity (7). Capillary gas chromatography (GC) and its ancillary techniques have been, over the last decade, used extensively to investigate various fossil fuel samples. However, the gas-phase analytical methods fail when the compounds to be analyzed are either too large or highly polar. Concurrently, conventional high-performance liquid chromatography (HPLC) lacks the necessary separation efficiency to provide adequate component resolution of the mixtures with increasing molecular weight and the number of possible isomers. TWOprevious reports from this laboratory (8,9)have demonstrated the ability of
microcolumn liquid chromatography (LC) to separate high molecular weight neutral polycyclic molecules of up to ninering structures. The present report describes the techniques that expand the analytical scope for nitrogen polyaromatics. When microcolumns with more than 200 000 theoretical plates were used, nearly 170 NPAC peaks (up to ten-ring structures) were resolved. Moreover, since up to several microgram amounts can be injected into slurry-packed LC capillary columns (10) without serious overloading,the individual peaks were trapped and investigated by mass spectrometry (MS). To further aid the structural characterization of such compounds, the recently developed intensified photodiode array (IPDA) fluorescence detector (11,129 was coupled with the capillary LC columns. On-line fluorescence emission spectra from the NPAC eluted peaks were thus obtained.
EXPERIMENTAL SECTION SRC-I1 fuel oil blend in a 5.75:l ratio of middle-to-heavy distillate (Pittsburgh and Midway Coal Mining Co., DuPont, WA; code no. 1701),COED syncrude oil (FMC Corp., Princeton, NJ; code no. 1106),and crude shale oil (code no. 4601), obtained from the Fossil Fuel Research Matrix Corp. repository (administered by the Oak Ridge National Laboratory), were used in this study. These materials were of a pilot plant origin and should not be considered as representative of products that may eventually be produced on a commercial scale. NPAC fractions were separated from the crude sample according to the procedure of Later et al. (13),by using a column packed with neutral alumina. The adsorbent was activated at 300 "C for 15 h prior to use. To further fractionate the NPAC samples, a silicic acid column was employed. With a slight deviation from the procedure of Later et al., the following fractions and compound types were recovered: (a) fraction S-1, containing secondary nitrogen polycyclic aromatic heterocycles (2OPANH), was eluted with 50 mL of 1/1(v/v) hexane/benzene; (b) fraction S-2, containing amino polyaromatic hydrocarbons (APAH) and tertiary nitrogen polycyclic aromatic heterocycles (3OPANH),was eluted with 40 mL of benzene. Each fraction was evaporated to dryness and then redissolved in an appropriate amount of tetrahydrofuran (THF). HPLC was employed to eliminate low molecular weight substances. A 4.6 mm X 25 cm, 5 pm particles, Techsphere Ultra CI8 column (Phenomenex, Rancho Palos Verdes, CA) was used. The flow rate was set at 1.5 mL/min. Solvent A was THF/acetic acid/triethylamine 100/0.1/0.2 (v/v/v); solvent B was acetonitrile/water/acetic acid/triethylamine 55/45/0.1/0.2 (v/v/v/v). The initial mobile phase contained 0.1% A. Fifteen minutes after
0003-2700/67/0359-0339$01.50/00 1987 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
i NO OF RIHCS
0
1
’1 j
2
3
4
5
6
7
8
9
TIME,HRS
Flgure 1. SRC-I1 fuel oil blend fraction H-1, as separated by microcolumn LC and detected fluorometrically. Stepwise gradient elution was as follows: 240 PL, 2 0 % HZO, 80% CHSCN; 300 /IL, 1 0 % H20, 9 0 % CHBCN; 360 PL, 1 0 0 % CHBCN; 120 /IL, 9 0 % CHSCN, 1 0 % THF; 120 /IL, 8 0 % CH,CN, 20% THF; 360 PL, 7 0 % CH3CN, 30% THF. All mobile phases contained 0 . 1 % acetic acid and 0.2% triethylamine. The elution intervals for 2’PANH of different numbers of rings are reported at the top of the chromatogram.
the sample injection, the percentage of A was linearly increased to 9.0% over 5 min, then to 99.9% over 3 min, and this composition was maintained for 17 min. During the last 20 min, the eluent was collected. A UV detector a t 254 nm was employed to monitor this elution process. The collected fractions, H-1 and H-2 from the S-1and S-2 samples, respectively, containing high molecular weight NPAC, were evaporated to dryness and weighed. The fractions were reconstituted in chloroform, a t a concentration of 50 mg/mL, and subsequently chromatographed on a slurry-packed capillary column (200 cm X 250 pm, i.d.) and detected with a fluorescence detector (Aex = 365 nm, , X, = 418 nm cutoff filter). The capillary columns were packed with ODS 5 pm Spherisorb (Phase Separation, Norwalk, CT) as previously described (IO). Three-hundred-nanoliter samples were injected by the stop-flow technique, and a stepwise gradient was further employed (14). The initial flow rate was set at 1.4 pL/min (the corresponding inlet pressure was about 200 atm during the run). The separated substances were manually trapped into small capillaries and analyzed by direct probe mass spectrometry. A Model 5982A dodecapole mass spectrometer (Hewlett-Packard Corp., Palo Alto, CA), in conjunction with an INCOS 2300 data system (Finnigan MAT Sunnyvale, CA), was employed at the electron energy of 70 eV to obtain and record the mass spectral data. In order to record fluorescence emission spectra, the microcolumn was connected to the photodiode array detector (11, 12),developed in this laboratory. The excitation wavelength was set a t 366 nm. The separations involving conventional packed columns were performed on a series 3B liquid chromatograph with a LC-55 UV monitor (both from Perkin-Elmer, Norwalk, CT). The miniaturized system, described previously (I4),and a fluorometric detector (Model 950, Kratos Analytical, Ramsey, NJ), with an in-house modified 20-nL cell were used for work with the mi-
Table I. Weight Percentage of H-l and H-2 Fractions from Different Samples
fossil fuel SRC-I1 fuel oil blend no. 1701
Syncrude oil no. 1106 crude shale oil no. 4601
H- 1
H-2
fraction, wt %
fraction,
1.3 1.9 6.8
0.5
wt %
0.3 3.6
crocolumns. Tetrahydrofuran, UV grade, was supplied by Burdick and Jackson (Muskegon, MI). All other solvents (Fisher, Fair Lawn, NJ) were used as purchased.
RESULTS AND DISCUSSION Different fossil fuel samples were analyzed in this work. The results of fractionation are shown in Table I as the weight percentages of the crude materials. Similarly to the previously described procedure (9),the samples were first “profiled” for their approximate molecular weight distribution by microcolumn LC. As a general observation, the chromatograms from crude shale oil fractions showed a Gaussian-like concentration distribution of their nitrogen-containing constituents, with poor peak resolution. Conversely, the syncrude oil and the SRC-I1 fuel oil blend fractions exhibited irregular distribution profiles and a better component resolution. As the primary purpose of this study was to establish, within the limits of techniques currently available to us, a system for the characterization of large nitrogen-containing polycyclics, the SRC-I1 fuel oil blend was chosen as a model,
ANALYTICAL CHEMISTRY, VOL. 59, NO. 2, JANUARY 1987
Table 11. Representative Polycyclic Structures: 2'PANH d e c r e a s i n g "molecular compactness"
-
no. of r i n g s 4
5
-
Table 111. Representative Polycyclic Structures: 3OPANH decreesing " m l r c u l a r CYupmctness'
t y p e Cil
341
t y p e Et:
type AN
69 @ M'hl
203
Mk'
221
Mh
211
KW
t y p e CN
220
@ ' *
H
MY
2'5
I
