Anal. Chem. 1982, 5 4 , 769-772
769
Identification of Triaromatic Nitrogen Bases in Crude Oils Jean-Marie Schmitter, Henri Colin, Jean-Louis Excoffier, Patrick Arpino, and Georges Guiochon* Ecole Polytechnique, Laboratoire de Chlmle Analytlque Physique, Route de Saclay, 9 7 120 Palaiseau, France
Determination of the location of the nitrogen atom in petroleum triaromatic azaareries was accomplished after recording UV spectra on-line during a reversed-phase liquid chromatographic separation. The identificationof individual compounds proceeded through comparison with reference compounds by combined gas chromatography and mass spectrometry. A small number of benzo[h]quinoline homologues represent the malor triaromatlc: nitrogen bases occurring in petroleum.
Petroleum nitrogen bases occur as complex mixtures of alkylazaarenes containing mainly one nitrogen atom ( I ) . One of the most challenging problems posed by the analysis of such substances consists in the determination of the precise location of the nitrogen atom in these polyaromatic molecules. The importance of the definition of model structures is related to various undesirable prolperties attributed to azaarenes: carcinogenic and mutagenic activity @), poisoning of re-forming catalysts (3),and, partly, also the instability of fuels during storage (4-6). In particular, it is well-known that only a few compounds among the large number of theoretical isomers for a given number of aromatic rings exhibit high carcinogenic activity which can be related to the location of the nitrogen atom (7, 8). Of particular interest for geochemists is the investigation of the formation pathway leading to the observed distribution of azaarenes in crude oils. However, the genesis of these compounds still remains an open question. Alkyl-substituted quinolines and benzoquinolines have been recognized as major nitrogen bases of crude petroleum and submitted to several investigations (9-12). However, except for the early identification of dimethylbenzo[h]quinolinesin a California crude oil by Schenck and Bailey ( I 3 ) , no unambiguous determination d the location of the nitrogen atom in triaromatic azaarenes and full identification of individual compounds could be achieved. The lack of reference substances is partly responsible for this situation since usually only unsubstituted com~poundsare available. The challenge was found even more interesting with the evidence of relatively simple distributions of liomologous alkylbenzoquinolines in some crude oils differing widely in their origins and geochemical histories ( I ) . 'Typically two prominent peaks corresponding to C2-alkylbenzoquinolines were detected by combined gas chromatog+aphy/mass spectrometry (GC/MS), and less than 10 peaks accounted for the Cs-alkylated species. This indication of the existence of a selective geochemical formation pathway (for instance, two major isomers were found among 350 theoretically possible), and the fact that benzoquinolines could represent key intermediates for the identification of higher inolecular weight azaarenes led us to a detailed investigation of these compounds. Because of the nature of crude oil basic extracts (i.e., low abundance, wide range of molecular weights and concentrations), no single analytical technique allows the identification of the position of the nitrogen atom in individual azaarenes. Even GC/MS cannot solve this problem because of the lack of structure-indicating fragments in the spectra of molecules having more than two fused aromatic rings. UV spectrometry makes possible the differentiation of various positions of the 0003-2700/82/0354-0769$0 1.25/0
nitrogen atom but is of restricted value when applied to complex mixtures because of drastic band broadening. In our previous report on the investigation of petroleum nitrogen bases ( I ) ,the UV spectrum of a fraction of di- and triaromatic azaarenes suggested the occurrence of benzo[h]quinolines as major compounds among petroleurr bases. The identification supof 2,4-dimethyl- and 2,4,6-trimethylbenzo[h]quinoline ported this hypothesis. The next stage in the characterization of triaromatic azaarenes was to determine if all the major homologues also belonged to the benzo[h]quinoline group. Thus, ths possibility of recording complete UV spectra on-line with a liquid chromatographic separation (LC/UV), which is a basic C F ~ pability of diode-array based spectrophotometers, seemed to be a promising solution. However, the total identification of individual compounds still has to proceed through an analytical sequence involving complementary chromatographic and spectroscopic techniques and the final comparison with authentic reference compounds. We used an analytical sequence which included a selective extraction step (trapping of strong nitrogen bases on HCImodified silica), followed by a purification of the total basic extracts by means of reversed-phase liquid chromatography (RPLC) ( I ) . Fractions containing azaarenes with sizes ranging from two to seven fused rings could be obtained in this way and characterized by a set of methods including GC, GC/MS, and LC fingerprinting. A further micropreparative RPLC fractionation was also used in order to concentrate the triaromatic nitrogen bases.
EXPERIMENTAL SECTION All solvents (analytical grade from Merck, Darmstadt, GFR) were glass-distilled before use. The details of the selective extraction method and preparative RPLC purification of total basic extracts were published in part in ref 1 and will be completed elsewhere. Micropreparative fractionations were performed on a 15 cm X 4 mm column, home packed with Lichrosorb RP 18 5 pm (Merck), using a methano1:water mixture (4:1, v/v) containing 0.1% (v/v) NH40H as the eluent. ",OH, also added for the analytical separations, was introduced in order to increase peak symmetry. After removal of methanol with a rotary evaporator, the basic fractions were extracted with chloroform, washed with water, and dried over Na2S04before concentration under a stream of dry nitrogen. Analytical LC separations were achieved with a Tracor Model 995 pumping system. Injections were made with a Rheodyne 7125 sampling valve. The column was from Merck (25 cm X 4 mm Hibar, 5 pm C18 packing). On-line UV spectra were recorded with a Hewlett-Packard 8450A spectrophotometer with a 8-pL flow cell connected directly to the column outlet. A Waters Model 400 detector was mounted in series to record chromatograms at a single wavelength of 254 nm. Glass capillary columns coated with various stationary phases (SE-52, OV-1, and Plrironic F-68) were used for the gas chromatographic separations and identifications of individual compounds by means of coinjection with authentic reference compounds; the gas chromatograph was a Perkin-Elmer Model Sigma 3. A Varian Model 2700 gas chromatograph was coupled witii a DuPont Model 21-492B mass spectrometer for GC/MS. A DuPont Model 094 B-2 disk-based data system was used for 0 1982 American Chemical :Society
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 C A
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380
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0
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Flgure 1. RPLC separation of reference azaarenes. The UV spectra were recorded at the maximum of peaks A (2,4dlmethylbenzo[h]quinoline) and C (phenanthrldine); the acquisition time was 20 ms. Acetonitrile with 0.1 % ",OH was used as the eluent.
L
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340 wavelength (nm) 260
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Flgure 2. The first derivative of the absorbance dA /dX plotted vs. the wavelength A allows the dlfferentiation of 2,4dimethyl-and-2,3,4-trimethylbenzo[h]quinolines (A and D) from 1,3-dimethylbenzo[f]quinoline (E). The spectra were recorded under the same RPLC conditions as in Flgure 1, during a separate analysis of compounds A, D, and E.
I
spectra acquisition and processing. Synthetic reference benzo[h]quinolines were prepared by condensation of the corresponding amines and ketones or aldehydes (14). RESULTS AND DISCUSSION Choice of a n LC System. The results of a previous investigation of the behavior of azaarenes in liquid chromatography oriented the choice of an LC system in order to achieve optimum separations of benzoquinoline homologues (15). In normal-phase chromatography (NPLC), the solute retention is mainly governed by the steric hindrance of the nitrogen atom. In RPLC, a methanol-water solvent and long chain packings allow a separation according to the number of carbon atoms, whereas an acetonitrile-water solvent affords higher selectivity for positional isomers. However, a normal phase system is of little practical value for the separation of petroleum azaarenes which almost always show a high degree of steric hindrance around the nitrogen atom (16). An RPLC system consisting of a CI8 packing with an acetonitrile-water eluent gave the best separations of reference alkylbenzoquinolines and was thus chosen for the investigation of triaromatic azaarenes. Combined LC/UV of Reference Compounds. Because of intrinsic features of UV spectrometry, LC/UV coupling is potentially a very useful tool for the structural elucidation of azaarenes in complex petroleum extracts, compensating for the lack of alkylated reference compounds. Indeed, alkylation and cycloalkylation produce only a bathochromic shift of the absorption bands, keeping the general appearance of the spectra of azaarenes unchanged and allowing the prediction of the location of the nitrogen atom in homologous compounds. Five alkylated and unsubstituted reference nitrogen bases were used for testing the LC/UV system, the capability of which is illustrated in Figures 1and 2. In general, several spectra could be recorded and stored during the elution of a single chromatographic peak. Compounds such as benzo[h]- and benzo[flquinolines, which are hardly differentiated from their UV spectra ( I ) , can be readily identified by plotting the first derivative of the absorbance (dA/dX) vs. the wavelength (Figure 2). This fingerprinting technique can greatly facilitate the recognition of the type of azaarene molecules. One of the major features is the ratio of the absorbances in the vicinity of 248 and 272 nm, which is close to 1for benzo[h]quinolines and larger than 3 for benzo[flquinolines. Isolation of Benzoquinoline Homologues. In order to optimize the analytical separations of the benzoquinoline
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Flgure 3. RPLClUV of C1,-CI7 azaarenes extracted from the Likouala crude oll sample. The UV spectrum was recorded at the maximum of the peak marked by an arrow. A 85:15 (v/v) acetonitri1e:water mixture with 0.1 % ",OH was used as the eluent.
homologues, the basic extracts of three different crude oils (Emeraude, Likouala (Congo) and Baliste (Guinea)) were submitted to micropreparative RPLC. Fractions highly enriched in CI4-Cl7 azaarenes were obtained in this way and reanalyzed. The LC/UV of such a concentrate allows the easy recognition of the major peak in the Likouala crude oil as a C2-alkylbenzo[h]quinoline derivative (Figure 3), the ring system being identified by its UV spectra. The analytical conditions, however, are insufficient for the separation of individual compounds, especially for higher homologues. Therefore, a cut containing only C2- and C3alkylbenzoquinolineswas separated from the Likouala crude oil sample in which these compounds are among the most abundant nitrogen bases (cf. Figure 3). Identification of Benzo[h ]quinolines. Under more favorable LC conditions, the group of isomeric C,-alkylbenzoquinolines could be partly resolved and six UV spectra of these compounds were recorded (Figure 4). All spectra are very similar in their general appearance and closely compare with known spectra of reference benzo[h]quinolines. The quasiidentity of the first derivative of absorbance, dA/dA, shown for three of these spectra, confirms the identification and allows us to rule out the occurrence of benzolflquinolines as major compounds, since the ratio of absorbances in the vicinity of 248 and 272 nm is close to 1 (vide supra and Figure 2). The occurrence of benzo[h] quinolines as the most prominent and almost unique structure of petroleum triaromatic azaarenes could be more firmly established after the identification of individual compounds. Several authentic reference
ANALYTICAL CHEMISTRY, VOL. 54, NO. 4, APRIL 1982 771
Table I. Identified and Detected Benzo[h]quinolines in Crude Oils identification method GC/MS RPLC/UV (M+) RPLC ( A , ~nm)
GC no.
structure
(KIU)
2-meth~ylbenzo [hlquinoline 2,4-dinnethylbenzo[hlquinoline 2,3-dinnethylbenzo[hlquinoline C,-alkyl benzo [hlquinoline C,-al kyl benzo [hlquinoline C,-alkyl benzo [hlquinoline 2,4,6-trlmethylbenzo [h]quinoline C,-alkylbenzo[ hlquinoline C,-alkylbenzo[ hlquinoline C,-alkylbenzo [hlquinoline 2,3,4-trimethylbenzo[h ]quinoline
t (1850) t (1978) t (1986)
synthetic ref
(193) C t 2 (207) t t (243, 266) t 3 (207) t t (244, 268) t 4 t t (221) 5 (221) t t (221) t 6 t 7 t t (2106) (221) t (247, 268) t t 8 (221) t 9 (221) t t 10 (221) t t 11 t (2159) (221) t (246, 268) t t Wavelengths of the two most intense absorption a Kova'ts indexes measured on SE-52; same conditions as for Figure 5. Data not available because of several impurities occurring with the standard bands measured for reference compounds. reference compound. .1
t t t t t t t t t t t
--
TableII. Mass Spectral Data on Reference Benzo[ h]quinolinesa
M.+ structure 2-methylbenzo [hliquinoline 2,4-dimethylbenzo [hlquinoline 2,3-dimethylbenzo [hlquinoline 2,3,4-trimethylbenzo/h]quinoline 2,4,6-trimethylbenzo[h]quinoline a
m/z
%
193 207 207 221 221
100 100 100 100 100
[M - HI+ m/z % 192 206 206 220 220
19 25 22 18 24
[M - CH:,]+ mJz %
[M - (H t HCN)]' m/z %
178 192 192 206 206
165 179 179 193 193
Spectra were taken under electron impact at 70 eV.
I
A
1
8
5 9 10
8
4 2 1 2
151 165 165 179 179
6 26 12 4 3
LIKOUALA crude oil
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[M - (CH, t HCN)]' m/z %
LIKOUALA crude oil
%?--G---G---d0
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Figure 4. RPLC/UV of C2-.and C3-alkylazaarenesfrom the Likouala crude oil sample. Six spectra, marked by arrows, were recorded on the C3-alkylated species. Three spectra (a, b, c) are shown in the normal mode and three others as the first derivative (dA /dX). A 70530 (v/v) acetonitrikwater mixture wlth 0.1 % ",OH was used as the eluent.
compounds were synthesized and identified in crude oils by means of coinjection followed by coelution on three different glass capillary columns (OV-1, SE-52, and Pluronic F68). This result is illustrated by the chromatogram shown in Figure 5. The structures of the benzo[h]quinolines identified and detected are listed in Table I, where GC retention data and UV maximum absorption bands of the reference compounds are also indicated. The major mass spectral fragmentations of these compounds are given in Table 11. The C3-alkylbenzo[h]quinolinescould not be further characterized, since no valuable information about the side chain (5)could be gaineld from their mass spectra. However,
Flgure 5. Gas chromatogram of a concentrate of di- and triaromatic azaarenes from the Likouala crude oil sample. SE-52 glass capillary column, 0.15 pm film thickness: temperature programmed from 65 to 200 "C at 2 "C/min. The compound numbering is the same as In Table I.
a-propyl, isopropyl, and ethyl side chains could be excluded, since the ratio of the [M - H]+/M'- ions and the absence of rearrangement ions suggest that all compounds are polymethylated (17-19),but the lower fragments are not specific enough to substantiate this hypothesis. Unfortunately the use of carbon and nitrogen NMR is practically ruled out a t this stage of our work since most compounds identified so far are at concentrations around or below 1 ppm in the crude oil and the chromatographic techniques cannot provide both the resolution and the sample capacity to prepare pure enough compounds. It is noteworthy that all identified compounds bear a methyl substituent in the a position with respect to the nitrogen atom. This fact, together with the evidence that the number of isomers of benzo[h]quinolines present in crude oils is still lower
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Anal. Chem. 1082, 54. 772-777
than the number theoretically expected for a particular location of the nitrogen atom, reinforces the hypothesis of the existence of a selective geochemical formation pathway, as discussed in another paper (20). All the benzo[h]quinolines that we have identified occur at variable concentrations in five crude oils previously studied by GC and GC/MS (1). This fact is noteworthy, considering the different origin and geochemical history of these samples. The knowledge of a formation mechanism, with precursor-product relationships which could explain the formation of the major nitrogen bases would considerably help the elucidation of more structures of this type. This aspect of the genesis of petroleum azaarenes needs to be further documented through the characterization of higher molecular weight nitrogen bases by means of the same LC/UV technique and by an investigation of azaarenes in organic matter from ancient and recent sediments.
ACKNOWLEDGMENT The authors thank Hewlett-Packard France, for the loan of a 8540 A spectrophotometer, J. Szafranek (University of Gdansk, Poland) for the synthesis of reference azaarenes 1, 3, and 11(Table I). Crude oils were kindly supplied by SociW Nationale Elf Aquitaine. LITERATURE CITED (1) Schmitter, J. M.; Vajta, 2.; Arpino, P. J. In "Advances in Organic Geochemistry 1979, Phys. Chem. of the Earth"; Douglas, A. G., Maxwell, J. R., Eds.; Pergamon Press: Oxford, 1980; Vol. 12, pp 67-76. (2) Ho, C. H.; Clark, B. R.; Guerin, M. R.; Ma, C. Y.; Rao, T. K. frepr. Pap.-Am. Chem. Soc., Div. FuelChem. 1979, 2 4 , 281-291.
Furimsky, E. Erdol Kohle 1979, 3 2 , 383-390. Dlnneen, G. U.; Bickei, W. D. Ind. Eng. Chem. 1951, 4 3 , 1604-1607. Frankenfeld, J. W.; Taylor, W. F. frepr. Pap.-Am. Chem. Soc., Dlv. Fuel Chem. 1978, 23, 205-214. Ford, C. D.; Holmes, S. A,; Thompson, L. F.; Latham, D. R. Anal. Chem. 1981, 53, 831-836. Dipple, A. In "Chemical Carcinogens"; Searle, C. E., Ed.; American Chemical Society: Washington, DC, 1976; p 258; ACS Monogr, No. 173. Andr6, J.; Buu-Hoi, N. P.; Jacquignon, P. J . Chem. Soc., ferkln Trans. 11972, 1261-1263. Jewell, D. M.; Hartung, G. K. J . Chem. Eng. Data 1984, 9 , 297-304. Snyder, L. R. Acc. Chem. Res. 1970, 3 , 290-299. McKay, J. F.; Weber, J. M.; Latham, D. R. Anal. Chem. 1978, 48, 89 1-898. McKay, J. F.; Amend, P. J.; Cogswell, T. E.; Harnsberger, P. M.; Erickson, R. B.; Latham, D. R. Adv. Chem. Ser. 1978, No. 170, 128-142. Schenck, L. M.; Bailey, J. R. J . Am. Chem. SOC. 1941, 6 3 , 2331-2333. Walls, L. P. In "Heterocyclic Compounds"; Elderfield, R. C., Ed.; Wiley-Interscience: New York, 1952; Vol. 4, pp 627-661. Colin, H.; Schmitter, J. M.; Gulochon G. Anal. Chem. 1981, 53, 625-631. Lochte, H. L. Ind. Eng. Chem. 1952, 4 4 , 2597-2601. Sample, S. D.; Lightner, D. A.; Buchardt, 0.; Djerassi, C. J . Org. Chem. 1987, 32, 997-1005. Draper, P. M.; McLean, D. 8. Can. J . Chem. 1988, 46, 1487-1497. Novotny, M.; Kump, R.; Merll, F.; Todd, L. J. Anal. Chem. 1980, 5 2 , 401-406. Schmitter, J. M.; Arplno, P. J. "Advances in Organic Geochemistry 1981", In press.
RECEIVEDfor review September 21,1981. Accepted December 14, 1981. Financial support of DBl6gation GBn6rale ?I la Recherche Scientifique et Technique (DGRST, Paris, France) for this work is gratefully acknowledged (Grant RAP-79-71306).
Binding of Pentachloroiridite to Plasma Polymerized Vinylpyridine Films and Electrocatalytic Oxidation of Ascorbic Acid J. Faccl and Royce W. Murray" Kenan Laboratories of Chemistty, University of North Carollna, Chapel Hill, North Carolina 275 14
Coordination of [IrCi,( acetonato)]*- In acetone/methylene chloride to a fllm of vlnyipyrldlne radio frequency plasma polymerlred on a carbon electrode results in an electrode surface wave at +0.40 V vs. SCE in 1 M H,S04. This potential Is more negatlve than expected for -pyIrCi;coordlnatlon. Charge transport In the fllm Is very fast and the fllm catalyzes the oxldatlon of ascorblc acld at a dlffuslon controlled rate. The fllm electrochemistry is sensltlve to the choke of supportlng electrolyte catlon but not anion.
Investigations of electron-transfer-mediated electrocatalytic reactions evoked by electrodes coated with molecularly designed redox-active films have been of considerable recent interest (1-11). Efforts have been directed at theoretical and quantitative kinetic descriptions of such reactions (9, 12-16). The electrocatalysis described here grew out of our investigations of electrodes coated using radio frequency (RF) plasma polymerization reactions (15, 17, 18) and our interest in binding iridium complexes to electrode surfaces (19). Vinylferrocene, for instance, can be plasma polymerized to form stable films with expectable ferrocene-ferricenium electrochemical reactivity (15, 17, 18). Having used the solvento 0003-2700/82/0354-0772$0 1.2510
complex [IrCl&acetonato)]*- for coordination reactions to pyridine and to vinylpyridine monomers, a natural extension was the use of plasma polymerization to prepare "poly(vinylpyridine)" films from vinylpyridine and the reaction of the solvento iridium complex with this film (in a manner akin to that of Oyama and Anson (20)). As described here the plasma film chemistry proves to be not simply that of pyridine since the ligand site binding the iridium is unknown. The iridium metalated film is, however, exceptionally stable and also catalyzes the oxidation of ascorbic acid at a diffusion controlled rate.
EXPERIMENTAL SECTION Electrodes. Teflon shrouded glassy carbon disk electrodes (0.071 cm2)were constructed as described elsewhere (21) except that conductive silver epoxy was used to seal the glassy carbon rod (Atomergic) concentrically within the brass holder, and the heat shrunk Teflon shroud was made concentric with the brass holder by gentle latheing. See Figure 1. Electrodes were mirror polished with 1 pm diamond paste (Buehler) and washed with distilled water prior to use. Equipment. Cyclic voltammetry was done with a PAR Model 175 signal generator and a locally designed potentiostat, rotated disk voltammetry with a PIR rotator (Pine Instrument Co., Grove City, PA), and potential step chronoamperometry with a PAR Model 173 potentiostat/galvanostat in conjunction with a TekCI 1982 American Chemlcal Society
