2398
Anal. Chem. 1983,55,2398-2404 i.010
1 L
Oggo
o
i t
0001
001
0 i
i o
io
io0
RC
Flgure 6. Ratio of the isotope ratio (R,) obtained by the bead-disk extraction method to the correspondlng isotope ratio (R,) obtained by a conventional method ( 7 ) vs. the magnitude of the isotope ratio. Samples were irradiated UAI fuels of various enrichments: (0) 2341238;(0)2351238;(A)2361238;( 0 )2351236. Average value of R,IR, is 0.9996 f 0.003. In the above series of analyses, the U concentration was depleted by 30% for numbers 4,5, and 6 (Table 11). The T h concentration did not change significantly, thus the T h to U ratio was 30% higher for the last three analyses. The rate of fractionation did not show a dependence on this change. However, if Th to U ratios that are greatly different from those used in this work are used, the dependence of fractionation on T h to U ratio should be investigated in more detail. For example, in the absence of Th, the U fractionation rate was usually lower; it was usually indiscernible from the normal scatter of the data. A further test of the accuracy of the procedure for U was conducted on a series of UA1 fuels. These fuels were chosen for the test because (a) they represented a fuel matrix for which results could be compared with those obtained by a conventional procedure, (b) they spanned a wide range in isotope ratio, and (c) there was interest in the applicability
of the procedure to uranium fuels. The conventional procedure involved chromatographic separation of U followed by deposition of -30 fig of U on the side filaments (I). The isotopic ratios (R,) obtained by the bead-disk extraction method divided by the corresponding ratios (R,) obtained by the conventional method, are plotted in Figure 6 and show that good agreement was obtained between the two methods. The mean and standard deviation of the ratios of the isotopic ratios are 0.9996 and 0.003, respectively, and are typical of the accuracy and precision of the conventional technique. This demonstrates the applicability of the bead-disk extraction to uranium fuels. Registry No. UOz, 1344-57-6;Al/U, 11149-83-0;U, 7440-61-1; 'W, 13966-29-5;235U,15117-96-1;'W, 13982-70-2;Th, 7440-29-1; 230Th,14269-63-7.
LITERATURE CITED (1) Green, L. W.; Knight, C. H.; Longhurst, T. H.; Crocker, I. H. Report AECL-7686, 1982. (2) Walker, R. L.; Carter, J. A.; Smith, D. H. Anal. Lett. 1981, 14 (A19), 1603- 1612. (3) Smith, D. H.; Carter, J. A. Int. J. Mass Spectrom. Ion f h y s . 1981, 40, 211-215. (4) Faris, J. P.; Buchanan, R. F. Anal. Chem. 1964, 3 6 , 1157-1158. (5) Bruce, F. R., Fletcher, J. M., Hyman, H. H., Katz, J. J., Eds. "Progress in Nuclear Energy, Series 111, Process Chemistry"; Pergamon Press: London, 1956; Voi. 1. (6) Green, L. W.; Knight, C. H.; Longhurst, T. H.; Cassidy, R. M., submitted for publication. (7) Smith, F. J.; Mesmer, R. E.; McTaggart, D. R. J. Inorg. Nucl. Chem. 1981, 43, 541-547. (8) Braun, T.; Ghersini, G. "Extraction Chromatography"; Elsevier Scientific: New York, 1975; Chapter 4. (9) Smith, D. H.; Christie, W. H.; Eby, R. E. Int. J . Mass Spectrom. Ion fhys. 1980, 36, 301-316. (10) Dobretsov, L. N.; Gomoyunova, M. V. "Emission Electronics"; translated by I. Shecktam, Keter Press Binding: Jerusalem, 1971.
RECEIVED for review March 2, 1983. Accepted September 6, 1983.
Characterization of CH,-Homologous Azaarenes in Petroleum by Capillary Gas Chromatography and Mass Spectrometry Gernot Grimmer,* Jurgen Jacob, and Klaus-Werner Naujack Biochemisches Institut fur Umweltcarcinogene, Sieker Landstrasse 19, 2070 Ahrensburg, Federal Republic of Germany
I n Arablan Light about 150 nitrogen-containing polycyclic aromatic compounds of the azaarene fraction were separated by capillary gas chromatography characterized by mass spectrometry and could be attributed to flve CH,-homologous serles. Three of them were ldentlfled as methyl derivatives of 4-azaphenanthrene by comparlson with reference materlals. From the mass spectroscopic fragmentation patterns It may be assumed that the compounds derlve from 4-azaphenanthrene, azapyrene/azafluoranthene, azachrysenelazabenz[a]anthracene/azatrlphenyiene,azabenzopyrenes/azabenzofluoranthenes,and azadlbenzothlophenes, respectlvely. Mass concentrations of these azaarenes per kilogram of petroleum were recorded. The concentration of the main constituents (di- and trlmethyl-4-azaphenanthrene, respectlvely, range from 0.2 to 0.8 mg/kg (in comparison to benzo[a]pyrene, 2.7 mg/kg)).
Polar nitrogen-, sulfur-, and oxygen-containing compounds present in crude oil play an important role in the deactivation
of catalysts. Due to their adsorption on the surface of catalysts they are concentrated in the coke deposits. This is confirmed by the high N/C ratio of the coke layer during hydrotreatment of gas oils containing high concentrations of nitrogen-containing polycyclic aromatic compounds (N-PAC) (1). Apart from this, N-PAC cause instabilities of mineral oil products during storage (2-4). Azaarenes are formed during incomplete combustion or pyrolysis as well as during coalification of nitrogen-containing material and hence they are found in crude oils (5-15, for review see ref 16). A mass spectrometric characterization of polycyclic aromatic hydrocarbons (PAH) and neutral N-PAC (alkylcarbazoles) in a Qatar crude oil has recently been published ( 1 7 ) . Ninety different N-PAC, 41 of which were derivatives of benz[a]- and benz[c]acridine, were tested for carcinogenic effects in animal experiments. Unfortunately, derivatives of benzo[h]quinoline (4-azaphenanthrene)have not been tested for carcinogenicity, whereas quinoline and all of the seven monomethylquinolines were found to be carcinogens (for review see ref 18). Quinoline, all isomeric methylquinolines, and
0003-2700/83/0355-2398$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
some azaphenanthrenes such as 4-azaphenanthrene (benzo[hlquinoline), l-azaphenanthrene (benzo[flquinoline), and 9-azaphenanthrene (phenanthridine) were found to be mutagenic in Salmonella typhimurium TA 98 and TA 100 in the presence of rat liver homogenate5 (19,20). However, both benzoisoquinolines (2- and 3-azaphenanthrene) were found to be negative in this test system (20). Our objective was an inventory of the basic N-PAC (azaarenes) from a crude oil. The hitherto published procedures for the enrichment of azaarenes from crudes separate N-PAC into subfractions by means of chromatography with acidic and basic alumina, ion exchange or thin-layer chromatography, and high-performance liquid chromatography (8,12,13,21). In this work azaarenes were concentrated for mass spectrometric characterization by using liquid-liquid partitioning and open column chromatography on Sephadex L H 20, SPSephadex C 25, and silica gel. The dimethylazaphenanthrene fraction was further separated by TLC and HPLC and the main constituents were characterized by means of on-line UV-Vis spectrophotometry.
EXPERIMENTAL SECTION Instruments and Operating Conditions. a. Gas Chromatography: Carlo Erba, Model 4160-02; fused silica column 30 m x 0.32 mm (95% polymethyl + 5% phenylsiloxane, J&W, DB 5); on-column injection 10 min at 80 "C followed by 80-130 "C with 20 "C/min, and subsequently 130-270 "C with 2 "C/min temperature program, then isothermally; carrier gas He flow, 20 m/s; integrator, Spectra Physics SP 4100-02. b. Thin-Layer Chromatography: Merck AG, Darmstadt, HPTLC plates RP-18 (without fluorescent indicator) precoated with concentrating zone; mobile phase, acetone. c. Mass Spectrometry: Varian MAT instrument, Model 112s directly coupled to a Perkin-Elmer Model F22 gas chromatograph; turbomolecular vacuum pumping system; glass column 25 m X 0.23 mm Chrompack CP si1 5; interface temperature, 250 "C; ion source temperature, 200 OC; ionization voltage, 80 eV; mass range scanned, 40-300 masses; scan rate, 200 masses/s. d. High-Performance Liquid Chromatography: Spectra Physics Model SP 8700 solvent delivery system adapted to a Model 8750 pumping system; column CP spher C-18, 250 mm; elution with acetonitrile/water (8/2), 0.7 mL/min; SP 8440 UVVis detector system and electronic integrator Model SP 4100-02. e. UV Spectrophotometry: Carl Zeiss Model PQM 2; scan rate, 1nm; resolution, 185-250 nm of 0.2 nm, 250-300 nm et 0.5 nm, and 300-400 nm at 1.0 nm; cell dimension, 1cm; reference, cyclohexane. Materials. a. Sample: Crude oil (Arabian Light). b. Soluents: Solvents (analytical grade) were redistilled before use in all-glass distillation apparatus (protected against laboratory atmosphere, pressure compensation through filter systems). c. Silica Gel: Woelm Pharma GmbH & Co., D-3440 Eschwege for adsorption chromatography, 7G.150 mesh (100-200 wm); water content, 10%. d. Sephadex LH 20: Pharmacia, Uppsala, Sweden. e. SP-Sephadex C 25: Pharmacia, Uppsala, Sweden; SPSephadex C 25 (50 g) was activated with aqueous hydrochloric acid (0.5 N, 1000 mL), washed with methanol/water (7/3, 1500 mL) and 2-propanol (1500 mL). The gel bed was shaken bubblefree and compacted by soft knocking. f. Reference Materials: Benz[a]- and benz[c]acridine were obtained from the Commission of the European Communities, Bureau of Reference (BCR), Rue de la Loi 200, B-1049 Brussels, Belgium, in better than 99.0% purity. The following dibenzacridines were synthesized according to literature references and were refined by sublimation in vacuo and repeated crystallization to better than 99.0% purity: dibenz[a,h]acridine (22), dibenz[ajlacridine (22, 23), dibenz[a,i]acridine (24). 2-Methyl-, 2,3dimethyl-, and 2,3,4-trimethyl-4-azaphenanthrene were kindly provided by J. Szafranek (University of Gdadsk, Poland). M. Zander (Rutgerswerke, Castrop-Rauxel) kindly supplied us with 1- and 4-azaphenanthrene. All other reference materials were obtained from commercially available products by the above raffination methods in better than 99.0% purity.
2399
Enrichment of Azaarenes from Crude Oil. a. Partition: 0.5 N NaOH/Cyclohexane. Crude oil (20 g) was dissolved in cyclohexane (600 mL) and extracted twice with a mixture of 1 N NaOH/methanol ( l / l , 300 mL). The aqueous layer was discarded and the cyclohexane phase washed twice with ammonia (0.01 N, 100 mL). b. Partition: Cyclohexane/Dimethylformamide.The cyclohexane phase from (a) was extracted twice with a mixture of dimethylformamide/O.Ol N ammonia (9/1, 600 mL). The dimethylformamide/ammonia mixture (600 mL) was diluted with ammonia (0.01 N, 1200 mL) and extracted twice with benzene (1800 mL). Both benzene phases were washed twice with 0.01 N ammonia (200 mL) and evaporated under reduced pressure to 100 mL. 2-Propanol (200 mL) was added to the remaining solution and the mixture then evaporated to about 100 mL. c. Chromatography on Sephadex LH 20. The 100 mL 2propanol fraction from (b) (above) is chromatographed on a LH 20 column (100 g) with 2-propanol (360 mL) and subsequently with acetone (2000 mL). Apart from PAH the acetone fraction contains N-PAC with more than two rings. This fraction was evaporated in vacuo to 20 mL, subsequently cyclohexane (100 mL) was added and evaporated again to 50 mL. d. Chromatography on Silica. The solution obtained under (c) was chromatographed on a silica column (20 g; 10% water content). Elution with cyclohexane (300 mL) resulted in a PAH fraction. Azaarenes were eluted with benzene (saturated with water)/ethyl acetate (9/1,200 mL). The fraction was evaporated under reduced pressure to about 20 mL. Then 2-propanol (100 mL) was added and the solution evaporated to about 50 mL. e. Chromatography on SP-Sephadex C 25. The sample dissolved in 2-propanol (50 mL) was chromatographed on a SPSephadex C 25 column (50 g). After the column was washed with 2-propanol (1500 mL) a fraction was eluted with 2-propanol/ aqueous sodium acetate (1M) (l/l,1250 mL). The fraction was diluted with 1N NaOH (50 mL) and extracted twice with benzene (750 mL). The benzene extracts were washed with 0.01 N ammonia three times (100 mL) and evaporated as described under (b). f. Chromatography on Sephadex LH 20. The residue obtained under (e),dissolved in 2-propanol (100 mL), was chromatographed on a Sephadex LH 20 column (100 8). The following fractions were collected: 0-380 mL; 380-500 mL; 500-700 mL; 700-900 mL; 900-1800 mL of 2-propanol; and subsequently 700 mL of acetone. Azaarenes were gas chromatographically detected only in the 380-500 mL 2-propanol fraction (N-PAC with two to four rings) and 500-700 mL 2-propanol fraction (N-PAC with more than four rings). g. High-Performance Liquid Chromatography. The 380-500 mL 2-propanol fraction was evaporated to 5 mL, diluted with acetonitrile (20 mL) and then again evaporated to about 0.1 mL. The solution was chromatographed on a CP spher C-18 column by elution with acetonitrile/water (4/ 1). Under these conditions 1,3-dimethyl-4-azaphenanthrene was eluted between 7.1 and 9.1 mL as checked by gas chromatography. h. Further Enrichment of 1,3-Dimethyl-l-azaphenanthrene (peak no. 15 of the chromatogram, Figure 2) from the above HPLC fraction by thin-layer chromatography). The above HPLC fraction was diluted with water (10 mL) and then extracted with benzene (12 mL). The benzene was evaporated and the residue dissolved in 100 MLof acetone. This fraction was separated by preparative thin-layer chromatography on HPTLC plates precoated with concentrating zone within a distance of 6 cm with acetone as elutant. The zone containing peak no. 15 was scraped off and the compound extracted with acetone/ethyl acetate (1/1). The extract was purified by chromatography on Sephadex LH 20. A schematic presentation of the enrichment and separation methods is given in the flow diagram (Figure 1). RESULTS AND DISCUSSION In the chromatogram 132 peaks were recorded (Figure 2), the quantification and mass spectrometric characterization of which are presented in Table I. Apart from some single components (M 233,234, 243,247, 248,261), five homologous series (A-E) could be mass spectrometrically characterized in the azaarene fraction of the
2400
ANALYTICAL CHEMISTRY, VOL. 55, Cvde
oil
NO. 14,DECEMBER 1983
__
12Og1
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Sephndex LHZO 1100g1
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2000ml Ac Silica+lO% w a t e r 120g1
Bz ie t h y l acetote19/1,20Omll SP-Sepodex C 2 5 15Ogl N-PAC 1250mI IP+sodium a c e t a t e l l m o l a r l l l / l l
1
Sephodex LH20 llOOg1
N-PACl2-4ringsl 380-500ml IP
* PAC.0-300ml CH
-
0-1500ml IP
.
0-380ml
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HPLC , CP-Spher (18, a c e t a n i t r i l l v o t e r 14/11
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Aclethyl o r e t a t e 11/11 b e p h n d e x LH20 IlOgI 38-50ml IP HeOH D M 01 IP
cn IC
-
-
0 - 3 8 m l IP
mdhonoi
- dimalhylformamide -benzene -,IoprIpI"oi
- iymamre - NOtO"l
Figure 1. Schematic presentation of the enrichment and separation of azaarenes (1,3-dimethyl-4-azaphenanthrene) from crude oil.
crude oil investigated (Arabian Light). Series A (M 193,207, 221,235,249,263,277,291,305): This series may be derived from the base compound M 179, which, however, could not be detected itself. The series corresponds to C,-C9-substituted individuals, which according to their UV and GC data, might be considered to be more or less highly substituted, preferentially polymethyl-substituted 4-azaphenanthrenes (benzo[h]quinolines). The main component of this series was obtained in a gas chromatographically consistent form by means of a combination of HPLC and TLC. According to the similarities of the UV spectra, mass spectra, and the gas chromatographic retention time of synthetic 2,3-dimethyl-4-azaphenanthrene (2,3-dimethylbenzo[h]quinoline) and of this compound, the latter is likely to be a dimethyl-4-azaphenanthrene,preferentially 1,3-dimethyl-4-azaphenanthrene. Series B (M 245; 259; 273; 287; 301): This series derives from the base compound M 203 which might be azapyrenes
or azafluoranthenes. The base compound as well as its C1and C2-substituted derivatives were not detected. Series C (M 257; 271; 285; 299; 313): This series is derived from the base compound M 229, the C1-homologue of which and itself were not found in the sample. Azabenz[a]anthracenes (e.g., benzacridines), azachrysenes, and azatriphenylenes could be the base compounds. Series D (M 267; 281; 295; 309): M 253 may be considered to be the base compound of this series, corresponding to the class of azabenzopyrenes and azabenzofluoranthenes. Series E (M 213; 227; 241; 255; 269): No base compound of PAH isosteric azaarenes with the mass 185 can be attributed to this series. According to the short GC retention times in comparison to their molecular masses these compounds might be methyl-substituted azathiophenes of the azadibenzothiophene-type recently detected in anthracene oil (25). Characteristic mass spectrometric fragments and their intensities for some selected compounds from the series A-E are presented in Table 11. Azaarenes may be primarily recognized from their oddnumbered molecular ions. Characteristic fragments observed in oxygen- or sulfur-containing polycyclic aromatic compounds such as elimination of the heteroatom, CHX-, and CHCHCHX- units or corresponding fragments by one mass unit higher in methyl derivatives (26), though present in azaarenes also, are less prominent. In most of the unsubstituted azaarenes M - 14 cannot be observed. However, the elimination of CHN (=M - 27) may be used for the recognition of cyclic nitrogen. This fragment appears less or at most equally intense as the (M - 28) ( = M - CHNH) or the (M 29) fragment. In addition an elimination of the unit NCHCHCH (= M - 53) is observed in the spectra of unsubstituted azaarenes such as acridine, 1-,4-, 9-azaphenanthrene, l-azapyrene, benz [a]- and benz [e ]acridine, 10-azabenzo[a ]pyrene, and dibenz[a,h]-, -[a,c]-,- [ a i ] - ,and -[c,h]acridine (Table 111). Intense (M - 1) and (M - 28) fragments are found in the mass spectra of monomethyl-substituted azaarenes. Although it has been claimed that the (M - 15) elimination originates from the loss of CH3- (1.9, it is obvious that it derives partially also from elimination of NH. According to the increasing probability of the elimination of methyl groups, the intensity of (M - 15) is enhanced and exceeds that of (M - 28) in higher alkylated azaarenes. The ratio (M - 15)/(M - 28) is found to be about 0.5 in 3methyl-4-azaphenanthrene (2-methylbenzo[h]quinoline), whereas a ratio of about 2 was observed in dimethylazaphenanthrenes, e.g., in 2,3-dimethyl-4-azaphenanthrene and several homologues of the series A-E. In these compounds the fragments (M - 27), (M - 28), (M - 29), and (M - 30)
9
P'
Basic nitrogen-containing aromatic polycyclic compounds from crude oil (Arabian Light) (peak numbering corresponds to Table I). GC conditions were as follows: column 30 m X 0.32 mm coated with DB 5;on-column injection: detector temperature 270 OC; carrier gas He 20 cm/s. Figure 2.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
Table I. Azaarenes from Crude Oil (Arabian Light) peak numbersa mass 193 L 4 , 5, 6
207
8,9, 10, 12,13,14,15, 16,20,21, 24
compound
m/kg
C,-179 2-methyl-4-azaphenanthrene(peak no. 4) C,-179 1,*3-dimethyl-4-azaphenanthrene(peak no. 15) 2,3-dimethyl-4-azaphenanthrene (peak no. 16) C,-185 C,-179 2,3,4-trimethyl-4-azaphenanthrene(peak no. 36) C,-185 C,-203
0.236
2401
1.480
0.273 7,9,17 1.724 11,18,19,23, 26,21, 28, 29,30,33, 36 0.133 17,20, 22,25 227 0.065 231 58 0.078 55,60 233 0.090 diazaaren 32,34 234 0.660 C,-179 37,38,40,42,44,47,48, 51, 54,56 235 0.546 C,-185 31,35,39,41,43,45,46,48 241 0.072 243 2, 3 0.528 C,-203 245 64,66,67,68 0.100 247 50,54,59, 61 0.021 49 248 0.368 C,-179 249 52,57,62,63,68 0.145 C,-185 255 53, 56,65 0.489 C,-229 251 78,79,81,83 0.335 C,-203 71,74,75 259 0.167 261 64,65,69 1.233 C,-179 70,72,73,76,77,78,80,82,84 263 0.068 C,-253 91 267 0.008 C,-185 269 89 1.336 C,-229 271 91,96,98,100,102,103 0.268 C,-203 27 3 84,86,89 277 82, 85,87,88,90,92,93,94,95, 3.275 C,-179 97,99,101,103,105,113 0.028 C,-253 281 115 0.608 109,111,113,115,117,119 C,-229 285 0.034 C,-203 109 287 2.284 291 102,103,104,106,107,108, 110, C,-179 112,114,115,117,120,126 0.079 295 129,130,132 C,-253 0.062 299 122,123,131 C,-229 0.004 301 129 C,-203 305 116,118,121,124,125,127,128 0.352 C,-179 unnumbered in Figure 2 309 n.d. C,-253 unnumbered in Figure 2 313 C,-229 n.d. a Peak numbering corresponds to that in Figure 2. Peak numbers which appear under two different masses in the table indicate an inhomogeneity of the G C peak recorded. 213 221
m
I -
*-
, ( I . , .
Flgure 3. Mass spectrum of synthetic 2,3dimethyCQazaphenanthrene.
exhibit almost equal intensities and (M - 42) as well as (M - 54) fragments originating from a partial elimination of the substituted ring, e.g., HN=CCH3, CH3CCH3,or CH3C=CCH3 are also found (Figures 3 and 4). For the base compound of series A (mass 179)eight isomeric structures are possible, since the substitution of one carbon atom by nitrogen results in three isomers in the case of anthracene (acridine, benzo[g]quinoline, benzo[g]isoquinoline) and five isomers in the case of phenanthrene (benzo[h]- and
Figure 4. Mass spectrum of 1,3dimethyC4azaphenanthreneisolated from crude oil (Arabian Light).
benzo- [flquinoline, benzo[h]- and benzomisoquinoline, phenanthridine). After separation of the azaarene fraction by TLC and HPLC the main component of series A (peak no. 15 of Table 11) was characterized by its UV spectrum by means of a coupled UV-Vis spectrophotometer. In the same way a spectrum of the synthetic 2,3-dimethyl-4-azaphenanthrene was recorded
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
Table 11. Intensities of Characteristic Fragments in the Mass Spectra of Some Selected Individual Azaarenes from the Series A-E (% of the Molecular Ion) fragment (relative intensity) peak no. compound series M - 1 M - 1 5 M - 2 7 M - 2 8 M - 2 9 M - 3 0 M - 4 2 M - 5 3 M - 5 4 15 1,3-Me2-4-azaphenanthrene A 59.7 30.7 13.6 10.0 11.4 12.9 33.6 5.0 5.7 27 C3-179a A 21.1 20.3 4.5 2.3 1.5 9.8 6.8 1.5 B 19.4 54.2 6.5 5.2 2.6 9.0 8.4 2.6 1.0 67 C3-203b 71 C,-203b B 26.1 69.6 4.3 13.0 26.1 6.5 4.3 4.3 C 20.3 15.2 4.3 7.2 5.1 14.5 11.6 2.9 5.1 79 C,-229' 100 C,-229' C 26.0 19.9 2.7 3.4 8.2 9.6 1.4 4.1 6.8 130 C,-253d D 23.1 71.4 22.5 15.4 19.8 19.8 12.1 4.4 20.9 7 C,-18Se E 56.9 33.1 6.2 18.5 4.6 23.8 16.9 6.2 6.9 a Basic structures: azaanthracene, azaphenanthrene. Azapyrene, azafluoranthene. Azabenzanthracene, azachrysene, azatriphenylene. Azabenzopyrenes, azabenzofluoranthenes, azaperylene. e Azadibenzothiophenes. Table 111. Intensities of Characteristic Fragments in the Mass Spectra of Some Selected Individual Azaarenes (% of the Molecular Ion) fragment [relative intensits) M - 29 M - 53 compound M-1 M - 27 M - 28 14.7 7 .O 14.9 1-azaphenanthrene 5.9 27.6 6.6 9.0 12.3 18.5 4-azaphenanthrene 32.7 6.6 9.5 13.0 9-azaphenanthrene 20.8 21.7 4.3 3.3 1.6 31.1 4.9 benztc ]acridine 4.3 3.2 6.8 5.7 benz[a]acridine 23.9 4.3 9.7 9.8 6.5 10-azabenzo[aIpyrene 8.5 2.0 5.5 2.5 22.5 4.5 dibenz[c,h]acridine 2 .o 3.0 5.4 14.3 4.0 dibenz[a,clacridine 2 .o 7.5 5.5 26.9 9.5 dibenz[u,h]acridine 3.8 5.6 5.2 4.5 9.8 dibenz[aj]acridine
13-Dimethyl-l-ataphenanthrene (2.L-Dimethylbenzo (hl9uinolinc)
2.3-Oimethyl-4-nznphennnthrenc l2.3-Dimethylbenzo~hlquinoline)
I
250
2W
250
3iO
350 nm
I
300
1
350 nm
Flgure 6. UV spectrum of synthetic 2,3-dimethyl-4-azaphenanthrene recorded in the same way as in Figure 5.
methyl-6azaphenanthrene (2,4-dimethylbenzo[h]quinoline) from a gas oil fraction (boiling range 355-365 OC) of California petroleum and confirmed the structure by degradation reactions as well as by comparison with reference materials. The occurrence of both isomeric dimethyl-4-azaphenanthrenes in crude oils recently was confirmed by Schmitter et al. (15). He also reported on the occurrence of 3-methyl-4-azaphenanthrene (2-methylbenzo[h]quinoline) as well as 2,4,6- and 2,3,4-trimethylbenzo[h]quinoline identified by comparison of their GC retention times and UV spectra with authentic reference materials. From the first derivative (dA/dX) of the UV spectra the authors tried to exclude the presence of a
ANALYTICAL CHEMISTRY, VOL. 55,
log E
l-Azoohenanthrenc
NO. 14, DECEMBER
1983
2403
Acridine
249.5
4.0
33
2 00
I 200
250
360
250
350 nm
300
Flgure 9. UV spectrum of synthetic acridine.
350 nm
logE
248
9-Azophenanthrene (Benzo (0quinoline) 1 Phenanthridinel
+ 4-Azophenonthrene 1Benzolh)quinolincl
L.0
3.0
I I
I
250
300
350 nn
Figure 10. UV spectrum of synthetic 9-azaphenanthrene (benzo[ clquinoline, phenanthridine). 250
3M)
350m
Flgure 8. UV spectrum of synthetic 4-azaphenanthrene (benzo[h]quinoline).
1-azaphenanthrene structure in the above compounds.
CONCLUSIONS The 150 compounds characterized as basic nitrogen-containing aromatic substances can be attributed to five homologous series (A-E). According to the findings of Schenck and Bailey (6) it seems likely that compounds of series A derive from 4-azaphenanthrene (benzo[h]quinoline). The mass spectra indicate that polymethyl rather than other alkylated derivatives are present in all series. From their mass spectra it cannot be concluded whether the basic structure of the compounds of series B is azapyrene or azafluoroanthene.
The same holds true for series C for which azachrysene, azatriphenylene, and azabenz[a]anthracene and for series D for which azabenzopyrenes and /or azabenzofluoranthenesand azaperylene can be considered as base structures. The mass spectra of compounds from the series E might indicate azadibenzothiophene as base structure with the nitrogen atom located in another ring than the sulfur atom. Characterization of the main components by their GC retention time and their mass spectra are expected to allow a determination of their concentration in other petroleum and mineral oil products. Registry No. 2-Methyl-4-azaphenanthrene, 37062-82-1; 1,3dimethyl-4-azaphenanthrene,605-67-4; 2,3-dimethyl-4-azaphenanthrene, 37069-37-7; 1,2,3-trimethyl-4-azaphenanthrene, 63042-66-0.
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Anal. Chem. 1983, 55,2404-2407
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RECEIVED for review June 28,1983. Accepted September 14, 1983. The present study was supported by “Deutsche Gesellschaft fur Mineralijlwissenschaft und Kohlechemie* (“German Society of Mineral oil Scknce and Chemistry of Coal”).
Spectrophotometric Determination of Complex Formation Constants by Estimation of Free Ligand Concentration Emiko Ohyoshi Yatsushiro College of Technology, Hirayamashinmachi 2627, Yatsushiro, Kumamoto 866, J a p a n
A spectrophotometrlc method has been developed to deterboth promine the formation constants of complexes (l:l), tonated and normal types, without a knowledge of any molar absorptlvlties, and applied to study the Zn( 11) and La( 111) complexes of 4 4 2-pyrdylazo)resorclnol (PAR or H,R). This method Is based on indirect estimation of the complexes, MHR and MR, by measuring the absorbance at the peak of the ligand which decreases wlth an Increase In the metal concentration at a constant pH. The resuits showed that both MHR and MR complexes for Zn( 11) and La(II1) were formed at pH around 6. Their formation constants were determined by a graphlcal analysts. This method Is slmple and applicable to study many other complexes of slmilarly colored reagents.
For the study of complex formation with a polybasic acid, it has been useful to estimate the free metal ion in equilibrium with a large excess of the ligand (1-4). This suggests that it will also be available for such studies to estimate the free ligand in equilibrium with a large excess of the metal ion. It is well-known that many metal ions simultaneously form protonated and normal complexes with 4-(2-pyridylazo)resorcinol (PAR), both having intense color. This makes it difficult to determine their formation constants by spectrophotometry. Benesi and Hildebrand (5) and McConnell and Davidson (6) earlier introduced the method in which both the molar absorptivity and the formation constant were treated as unknowns. Recently, Sokol, Ochrymowycz, and Rorabacher (7) extended its applicability to the system involving the side reactions by means of the a coefficients (8). However, it seems to be rather complicated to apply the method (7) when the system involves several colored species, either complexes or ligand, such as some metal complexes of PAR. In the case
where the ligand itself has an intense absorption peak, the intensity of this peak indicating free ligand concentration in equilibrium would enable us to estimate the complexes formed. In the present work, we have developed a method to determine the formation constants of complexes by estimating free ligand concentration spectrophotometrically and have applied it to study the PAR complexes of Zn(I1) and La(II1).
THEORY When a metal, M, reacts with a reagent, HzL, under conditions where the concentration of the metal (C,) is in large excess over that of the ligand (CL),the 1:l complexes MHL and ML with the formation constants KMHL = [MHL]/ ([M][HL])and Km = [ML]/([M][L]) are formed (the charges are omitted for simplicity). The total concentration of the ligand, CL,and the absorbance at the peak of the ligand, A , are expressed as C L = [MHL] [ML] + [L’] (1)
+
A = ~ M H L [ M H L ] €ML[ML]+ €L,[L’]
(2)
where L’ is free ligand not combined with any metal, and E-, cML, and cLt are the molar absorptivity of MHL, ML, and L’ at the wavelength of maximum ligand absorbance. With the acid dissociation constants of H2L,Kl = [HI [HL] / [HZL] and K z = [H][L]/[HL], and a coefficient (81, ay~= [L’l/[Ll, eq 1 and 2 can be brought into the following expressions: [M](KMHLKI[H]-k K M L K I K ~ ) / ( ~ & I K z=) CL/[L’] - 1 (3) A / € L I = [L’](1 + (EMHLKMHLK1[Hl + tMLKMLK1K2) [ M I / ( C L ( ~ L K I K Z(4) )) where t ~ l =C L / ~ L+ ~HLKI[HI/(~LFIKz) + ~H,L[HI~/(~LK~KZ), which is a fixed quantity at a fixed pH. Combining eq 3 with eq 4 we obtain
0003-2700/83/0355-2404$01.50/00 1983 American Chemical Society
