Energy & Fuels 1993, 7, 185-190
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Microscale Determination of the Spectral Characteristics and Carbon-Isotopic Compositions of Porphyrins? Brian N. Popp*J and J. M. Hayes Biogeochemical Laboratories, Geology Building, Indiana University, Bloomington, Indiana 47405-5101
Christopher J. Boreham Australian Geological S u r v e y Organisation, GPO Box 378, Canberra 2601, ACT, Australia Received October 7, 1992. Revised M a n u s c r i p t Received December 14, 1992
Molar extinction coefficients for band I11 of Ni porphyrins are calculated from results of spectrophotometric and manometric analyses of individual etioporphyrins, DPEP, cyclic,and diDPEP porphyrins known to initially be pure from mass spectrometry, 'H NMR, and analytical HPLC studies. A method for determining carbon-isotopic compositions and purity of micromolar quantities of individual porphyrins using combined spectrophotometric and manometric techniques is presented.
Introduction Metalloporphyrins in sedimentary rocks are thought to derive from chlorophyll and are useful indicators of maturity and of depositional Recently, carbon- and nitrogen-isotopic analyses of biomarkers, including porphyrins and chlorophylls, have been used to identify diverse sources for individual compounds and have allowed more thorough reconstructions of complex microbial ecosystemsand features of global environments.%l' Isotopic analyses of pigments are especially important because structures of these compounds can provide taxonomic and diagenetic information. Isotopic analysis of pigments requires isolation of pure compounds and can be facilitated by high-performance liquid chromatography (HPLC).12 We show in this paper that ultraviolet/visible (UV/vis) spectrophotometric analyses of porphyrins can be an effective, nondestructive method for quantifying the purity of these compounds when prepared for stable isotopic analysis. SOEST contribution no. 3149. Present address: Department of Geology and Geophysics, University of Hawaii, 2525 Correa Road, Honolulu, HI 96822. (1) Baker, E. W.; Louda, J. W. In Biological Markers in the Sedimentary Record; Johns, R. B., Ed.; Elsevier, New York, 1986;pp 125-225. (2) Filby,R. H.;VanBerkel,G. J.,InMetalComplexesinFossilFuels; Characterization and Processing; Filby, R. H., Branthaver, J. F., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987; pp 2-39. (3) Hayes, J. M.; Takigiku, R.; Ocampo, R.; Callot, H. J.; Albrecht, P. Nature 1987,329, 48-51. (4) Hayes, J. M.; Popp, B. N.; Takigiku, R.; Johnson, M. W. Geochim. Cosmochim. Acta 1989,53, 2961-2972. (5) Popp, B. N.; Takigiku, R.; Hayes, J. M.; Louda, J. W.; Baker, E. W. Am. J . Science, 1989,289, 436-454. (6) Boreham,C. J.;Fookes,C. J. R.;Popp,B. N.; Hayes, J. M. Geochim. Cosmochim. Acta 1989, 53, 2451-2455. (7) Freeman, K. H.; Hayes, J. M.; Trendel, J-M.; Albrecht, P. Nature 1990,343, 254-256. (8) Ocampo, R.; Callot, H.; Albrecht, P.; Popp, B. N.; Horowitz, M.; Hayes, J. M. Naturwissenschaften 1989, 76,419-421. (9) Boreham, C. J.; Fookes, C. J. R.; Popp, _ _ B. N.; Hayes, J. M. Energy -_ Fuels 1990,4, 658-661. (10) Kennicutt, M. C.; Bidigare, R. R.;Macko, S. A.; Kenney-Kennicutt, W. L. Chem. Geol. (Isotope Geosci.) 15, 235-245. (11) Chicarelli, M. I.; Hayes, J. M.; Popp, B. N.; Eckhardt, C. B.; Maxwell, J. R. Geochim. Cosmochim. Acta, in press. (12) Bidigare, R. R.; Kennicutt, M. C.; Keeney-Kennicutt, W. L.; Macko, S. A. Anal. Chem. 1991,63, 13e133. +
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Spectrophotometric analyses have been of limited value in the quantitative analysis of geoporphyrins. The problem arises because, although many different species have been isolated and their absorption spectra recorded,13-15 the quantities available are often too small to provide accurate weights. Consequently, molar extinction coefficients cannot be determined and quantitative analyses are hindered. In the present work, porphyrin quantities have been determined using manometric techniques together with isotopically based blank corrections. As a result, new values have been obtained for extinction coefficientsof numerous geoporphyrins previouslyisolated and characterized structurally by mass spectrometry, lH NMR, and analytical HPLC studies. Further consideration of the interplay between spectrophotometric and combustion-basedisotopic analyseshas allowedrefinement of systematic approaches to the determination of 13C contents of porphyrins and to estimation of the purities of porphyrin isolates.
Samples and Methods Samples for this study are from immature sediments of the Cretaceous (Aptian-Albian) marine Toolebuc Formation'G and the early Tertiary lacustrine Condor Oil Shale,I7 both from Australia. Nickel and vanadyl porphyrins were isolated18 from samples of the Toolebuc Formation, whereas only nickel porphyrins were isolated from samples of the Condor Oil shale. Briefly, crude nickel and vanadyl porphyrin fractions were separated by column chromatography on silica. Vanadyl fractions were demetallated with methanesulfonic acid (80 OC for 90 min) and nickel was inserted into the free-base porphyrin using Ni(acetylacetonate)2.18Thin layer chromatography on all nickel (13) Gouterman, M. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. 111, pp 1-165. (14) Adar, F. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. 111, pp 167-209. (15) Weiss, C. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. 111, pp 211-223. (16) Ozimic, S. Geol. SOC.Aust. Spec. Publ. 1986, 12, 119-137. (17) Green,D. A.;McIver,R. G.;O'Dea,T.R.Proc.2ndAust.Workshop Oil Shales, Lucas Heights Research Laboratories, NSW 1984, 33-37. (18) Boreham, C. J.; Fookes, C. J. R. J . Chromatogr. 1989,467, 195208.
0 1993 American Chemical Society
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186 Energy &Fuels, Vol. 7, No. 2, 1993
blank) was determined using an electronic manometer (MKS Instruments, Baratron Model 122A) connected to the glass vacuum distillation line. The uncertainty for this measurement was found to be less than 20 nmol COZ(1standard deviation). Isotopic abundances were measured using a Delta-E mass spectrometer and are reported in the standard h o t a t i o n in permil relative to PDB.
Results and Discussion Molar Extinction Coefficients. Molar extinction coefficients at maximum wavelengths near 550 nm were determined using Beer's law rearranged as follows
diDPEP
diDPEP
4a;(C33)
5,&. R, = CHlCH,. R1 =CHI (C3,) ~
R I = CH2CH3,Ri * H (Cg) R , = CH,, R2 = H (C32)
BUTANO f& R = CHiCH, 4b;R=CHi(CiJ 4r; R = H (Cid
Figure 1. Chemical structures of porphyrins isolated from the Julia Creek and Condor Oil Shale. porphyrins yielded subfractions from which single species (Figure 1;la, Id, li, 2a-c, 3a, 4a, 5a-c, and 6a-c) or mixtures of species (lb+c, le+f, lg+h) were obtained in high purity after several semipreparative reverse-phase HPLC steps. Purity of these compounds at this stage was assessed by mass spectrometry, 'H NMR, and analytical HPLC and was generally about 95% with respect to other porphyrins.ls Each sample was further purified by TLC prior to acquisition of a UV/vis spectrum. Contributions of carbon from solvent impurities and handling were monitored by carrying a standard (Ni octaethylporphyrin, Ni-OEP) through TLC procedures in parallel with samples. Briefly, 3-4 porphyrin samples and the Ni octaethylporphyrin standard (containing 0.3-2.0 pmol of C) were spotted on TLC plates (E. Merck, Silica Gel 60). After elution (14/10/1 v/v hexane/toluene/ethyl acetate, R, 0.8 or 713 v/v hexane/CHzClz,R f 0.4), individual spots were transferred to a vial using 2 mL of CHzCl2and the solvent removed under a stream of N2 a t 25 "C. T o determine the mass of the analytical blank associated with TLC chromatography and transfers of the sample, a clean portion of each TLC plate with an area and an Rf approximately equal to that of the samples was collected in the same manner and carried through the subsequent procedures as a sample. Spectral analysis (Hewlett-Packard 8450A) was performed on eachsample in0.5 or 1.0 mL of CHzClzusing a quartz-glass cuvette witha path length of 5 mm. In addition to recording the maximum absorbance of the major peaks (determined using the "PeakFind" option in the software provided by Hewlett-Packard), the absorbance a t X = 305 nm was also monitored. The wavelength of maximum absorbance of Ni porphyrin peak I11 ranged from 548 to 556 nm. Uncertainty in absorbance as determined by repeated measurements and variations in volume of solvent as determined by measurements of mass of solution were found to be less than 0.005 absorbance units and less than 1 X L (1 standard deviation), respectively. After spectra were obtained, individual samples were transferred to a quartz tube (-200 mm x 9 mm, sealed a t one end) using an additional -2 mL of CHzCl2,the solvent was evaporated a t 25 "C using a rotary evaporator,
