Energy & Fuels 1990, 4, 741-747
rearrangement processes. FABMS and especially FABMS/MS offer a means of structural assignment of sedimentary chlorins when amounts or purities are too low for characterization using 'H NMR spectroscopic techniques. Furthermore, since a number of ions are common to the spectra of all of the components examined, possibilities exist for selecting the components from relatively crude mixtures by using specific fragment ions (e.g., parent ions of m / z 433). In particular, interpretation of the FABMS fragmentation processes through the use of MS/MS provides a basis for understanding the fragmen-
741
tation behavior of these components under LC/MS conditiom20 Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. We also thank the Natural Environment Research Council for MS facilities (GR3/2951 and GR3/3758) and Mr. J. F. Carter for technical assistance. (20) Eckardt, C. B.; Carter, J. F.; Maxwell J. R. Energy Fuels, in this issue.
Combined Liquid Chromatography/Mass Spectrometry of Tetrapyrroles of Sedimentary Significance C. B. Eckardt,* J. F. Carter, and J. R. Maxwell Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, U.K. Received April 27, 1990. Revised Manuscript Received J u n e 13, 1990
The on-line coupling of a high-performance liquid chromatograph to a mass spectrometer (LC/MS) has allowed the analysis of a number of tetrapyrrole pigments of sedimentary significance. Mass spectra were obtained under conditions of discharge ionization and are characterized by the presence of protonated ion species (positive-ion mode) or molecular ions (negative-ion mode), respectively. Fragmentation processes were found to be restricted to the periphery of the structures. In the positive-ion mode the extent of fragmentation could be manipulated by alteration of the repeller voltage in the ion source, allowing molecular weight information to be obtained, as weli as specific fragmentation patterns. These findings have suggested a number of applications of LC/MS in the investigation of sedimentary tetrapyrrole distributions. In particular, the assignment of individual components in mixtures without resort to their isolation is possible from comparison of mass spectra with those of authentic standards and from coinjection with the standards.
Introduction The investigation of sedimentary tetrapyrrole distributions can yield information relating to the assessment of biological input, depositional paleoenvironment, and thermal maturity of sedimentary organic matter.' Among other techniques, high-performance liquid chromatography (HPLC) of porphyrin free bases, and of chlorophylls and their derived intermediates on the degradative pathway to the porphyrins, has proved to be a rapid and reliable method for separation of complex mixtures with reproducibly good resolution.24 Typically, the analysis provides a UV/visible-monitored chromatogram, allowing assignments from relative retention time comparisons and coinjections with authentic standards. The method has also been applied extensively to the isolation of individual components for full structure determination, for example, by 'H NMR experiment^.^ (1) Baker, E. W.; Louda, J . W. In Biological Markers in the Sedimentary Record; Johns, R. B., Ed.; Elsevier: Amsterdam: 1986, pp 125-155. (2) Barwise, A. J. G.; Evershed, R. P.; Wolff, G. A.; Eglinton, G.; Maxwell, J. R.J . Chromatogr. 1986, 368, 1-9. (3) Chicarelli, M. I.; Wolff, G. A.; Maxwell, J. R. J. Chromatogr. 1986, 368,ll-19. (4) Keely, B.J. Ph.D. Thesis, University of Bristol, UK, 1989. (5) Chicarelli, M. I.; Maxwell, J. R. Trends Anal. Chem. 1987, 6, 158-164, and references therein.
The mass spectrometric detection of tetrapyrroles, performed via direct insertion of a mixture or a single compound, is frequently applied using electron impact (EI/MS),6v7reactant gas plasma (CI/MS),g'o or fast atom bombardment (FAB/MS)4JJ1ionization. Under EI/MS or FAB/MS conditions, the mass spectra are generally characterized by abundant molecular or pseudomolecular ions, respectively, and fragmentation appears to occur mainly within the substituents on the macrocycle and to depend on the degree of functionalization. More extensive structural information, based on cleavage of the macrocycle at the meso positions, can be obtained using chemical ionization (DCI/MS) conditions with certain reactant gases such as a m m ~ n i a . ~ J ~ J ~ The use of chromatographic separation directly prior to mass spectrometric analysis in the investigation of complex ~~
~~~
~~
~~~
( 6 ) Baker, E. W. J . Am. Chem. Soc.1966, i%, 2311-2315. (7)Castro, A. J.; van Berkel, G. J.; Doolittle, F. G.; Filby, R. H. Org. Geochem. 1989, 14, 193-202. (8) Shaw, G. J.; Edinton. G.; Quirke, J. M. E. Anal. Chem. 1981.53, 2014-2020. (9) Tolf, B. R.;Xiang Yu-Yiang;Wegmann-Szente, A.; Kehres, L. A.; Bunnenberg, E.; Djerassi, C. J. Am. Chem. SOC.1986, 108, 1363-1374. (10) van Berkel, G. J.; Glish, G. L.; McLuckey, S . A. Org. Ceochem. 1989, 14, 203-212. (11) Keely, B. J.; Maxwell, J . R. Energy Fuels, accompanying paper in this issue. (12) Eckardt, C. B. Ph.D. Thesis, University of Aachen, FRG, 1989.
0 1990 American Chemical Society
742 Energy & Fuels, Vol. 4, No. 6, 1990
Eckardt e t al.
TSP/TSQ 70 1 -
Table I. Operating Conditions Used for LC/MS Analysis of Tetrauyrrole Piuments component setting HPLC solvent flow 1.0 mL/min, composition as required for chromatographic resolution interface vaporizer temperature 65 "C source-block temperature 250 "C discharge electrode 1100-1300 V repeller voltage variable (see text) mass spectrometer scan parameters as required" "Recording at mass > ca. 200 Da (depending on the mobile phase) minimizes detection of chemical noise.
DATA SYSTEM
Figure 1. Schematic diagram of the LC/MS system: MS 600S, quaternary gradient HPLC system; I, Rheodyne 7125 injector valve; B, bypass valve allowing solvent flow to be directed via routes 1 or 2; C, HPLC column; UV/vis, variable-wavelength UV/visible detector; P, postcolumn HPLC pump; TSP, TSP-2 thermospray interface; TSQ 70, triple-stage quadrupole mass spectrometer; Sl-S4, S5, solvent reservoirs.
mixtures of sedimentary tetrapyrroles has so far only been applied semiroutinely in the case of GC/MS analysis of derivatized and underivatized alkylporphyrins.13J4 However, an early attempt to couple a HPLC system to a mass spectrometer for the analysis of porphyrin free bases was partially S U C C ~ S S ~ although U ~ , ~ ~ the chromatographic resolution was insufficient to resolve complex mixtures. With the recent improvements in the chromatographic resolution of free-base alkylporphyrins2v3 and their metallo c~unterparts,'~J' and of functionalized tetrapyrroles,"'J9 as well as the development of more reliable interfacing systems,20renewed attempts to investigate tetrapyrrole distributions by LC/MS seemed promising. In contrast to the GC/MS approach, both functionalized tetrapyrroles and alkylporphyrins are amenable to HPLC separation. Also, for the separation of porphyrin free bases, HPLC provides, at present, the highest chromatographic resolut ion .2,3 In this paper we report a preliminary study of tetrapyrrole pigments using LC/MS, with the aim of investigating both their mass spectrometric behavior under such conditions and the possibilities of developing a routine method for the identification of components in mixtures, without resort to the isolation of individual compounds.
Experimental Section Instrumentation. The LC/MS coupling was carried out with a WATERS MS 600 Silk quaternary delivery HPLC system and a FINNIGAN MAT TSQ 70 quadrupole mass spectrometer, linked via a FINNIGAN MAT TSP-2 thermospray interface. Figure 1 is a schematic diagram of the system. Prior to entering the ion source, the HPLC effluent is passed through a UV/visible variable-wavelength detector (WATERS 484), to allow monitoring (13)Gill, J. P.;Evenhed, R. P.; Eglinton, G.J. Chromatogr. 1986,369, 281-312,and references therein. (14)Blum, W.;Ramstein, P.; Eglinton, C. J . High Resolut. Chromatogr. 1990,13,85-93,and references therein. (15)McFadden, W . H.; Bradford, D. C.; Eglinton, G.;Hajibrahim, S. K.; Nicolaides, N. J . Chromatogr. Sci. 1979,17, 518-522. (16).Sundararaman, P.;Biggs, W. R.; Reynolds, J. G.;Fetzer, J. C. Geochrm. Cosmochrm. Acta 1988,52,2337-2341. (17)Boreham, C. J.; Fookes, C. J. R. J . Chromatogr. 1989, 467, 195-208. (18) Zapata, M.; Franco, J. M.; Garrido, J. L. Chromatographia 1987, 23,26-30. (19)Keely, B.J.; Brereton, R. G.;Maxwell, J. R. Org. Ceochem. 1988, 13,801-805. (20)Vestal, M. L. Science 1984,226, 275-294.
Table 11. Samples Investigated sample (structurea) originb vanadyl etioporphyrin I (1) synthetic standard desoxopyropheophorbide a (2)c synthetic standard synthetic standard desoxomesopyropheophorbide a (3a)c nickel desoxomesopyropheophorbide a (3b)C synthetic standard synthetic standard pyropheophorbide a (4)c isolated standard chlorophyll a ( 5 ) synthetic standard pyrochlorophyll a (6) synthetic standard pyropheophytin a (7) recent sediment4s21 SED 5-10/1 See Chart I. *See text.
Methyl ester.
of the chromatographic separation. A postcolumn HPLC pump (WATERS 510, P in Figure 1) allows addition of solvent and/or buffer solution to stabilize the aerosol in the ion source or to perform analyses under thermospray ionization conditions, respectively. On the MS 600s an additional two-way valve (B in Figure 1) is used in-line with a Rheodyne 7125 injector valve (I in Figure 1) to allow the chromatographic column (C in Figure 1) to be bypassed, for sample introduction via the liquid inlet alone (direct liquid insertion, DLI/MS, flow via route 2, Figure 1). The HPLC system chosen allows essentially pulse-free delivery of the LC effluent by way of a flow control device (WATERS Silk system) mounted on the main pump. The postcolumn pump was also fitted with an additional pulse dampener. Injector and bypass valves, as well as the detector cell, were chosen to withstand back pressures up to ca. 4000 psi. The spectrometer and interface are controlled by the TSQ 70 data system, whereas the HPLC pumps are operated by individual control units. The detector is linked to the data system as well as to a conventional chart recorder, to allow UV/visible-monitored chromatograms to be obtained. In the above system, the effluent from the HPLC flows through heated vaporizer tubing whose temperature can be varied to produce an aerosol, which enters the thermospray ion source. Ions can be formed by interaction of sample molecules with ions from a buffer solution (thermospray process) or by interaction with plasma ions generated in an electrical discharge. Driven by a repeller, the ions leave the source via a sampling cone. Excess evaporate is pumped out of the source and condensed with liquid nitrogen in a cold trap. For all applications, HPLC-grade solvents, filtered through 0.2-pm filters, were used. Samples were filtered through 0.22-pm Teflon filters (MILLIPORE Millex-13). Optimization of source parameters, with the aim of gaining a stable background and high signal-to-noise ratios, revealed that maximum stability and ionization yields were obtained in the discharge mode a t source temperatures between 200 and 250 "C. Discharge voltages between 1100 and 1300 V gave optimum results in all applications. For the alkylporphyrins (e.g., l), spectra recorded in the positive-ion mode gave the best results. In the case of functionalized compounds, the signal-to-noise ratio was found to increase by a factor of ca. 10 through the acquisition of negative ions (see below), presumably as a result of the greater efficiency of electron capture ionization, in comparison with protonation. For various ternary mixtures of acetone, methanol, and water, the optimum vaporizer temperature was found to be 65 "C. Higher temperatures appeared to lead to sample precipitation inside the vaporizer and subsequently to blockage. At lower temperatures, the ionization yields were extremely low, presumably due to the
LCIMS of Tetrapyrroles
Energy & Fuels, Vol. 4 , No. 6, 1990 743 chart I
0-c
\O-c20Ys
O-f0-QAp
high abundance of liquid particles in the aerosol, which prevented sufficient formation of plasma ions in the electrical discharge. The settings found to be most suitable for the analyses are summarized in Table I. Using the system as a liquid insertion device (DLIIMS, see above) typical back pressures were around 300 psi (vaporizer temperature 65 “C; acetonelmethanolfwater 80/10/10v/v/v, at 1.0 mL/min). For LC/MS runs, a WATERS Radial Compression Module fitted with a reversed-phase column (Novapak C18cartridge, 100 mm x 5 mm i.d.) was used. Typical back pressures were around 700 psi (conditions as above). Samples. A variety of standards and a gel permeation fraction of a methylated extract from a recent sediment (Priest Pot Lake, Cumbria, UK43*) were studied, in both the DLI/MS and LC/MS modes (Table 11). Etioporphyrin I was obtained from Prof. K. M. Smith (University of California, Davis) and metalated according to the standard procedure. Desoxopyropheophorbide a and desoxomesopyropheophorbidea and its Ni counterpart were prepared from chlorophyll a (5), isolated from spinach leaves,’ and methylated (2, 3a, 3b, courtesy of W. G. Prowse). Pyrochlorophyll a (6),pyropheophytin a (7), and pyropheophorbide ~~~
(21)
Robinson, N. Ph.D. Thesis, University of Bristol, UK, 1984.
z (methyl ester, 4) were prepared from chlorophyll a according ;o standard methods (4, 5, 6,7, courtesy of B. J. Keely).
Results a n d Discussion To exemplify the mass spectrometric behavior of alkylmetalloporphyrins and their free-base counterparts, the mass spectrum of vanadyl etioporphyrin I (C32N4H36V0, MW 543, 1) in the range 400-600 daltons (Da) is shown 1in Figure 2. The spectrum was obtained by DLI/MS with iischarge ionization in the positive-ion mode and with the 1repeller set to 40 V (cf. Table I). Under these conditions, 1the protonated molecular ion at mjz 544 is the base peak, with the carbon isotope peak a t m j r 545 in the expected 1relative abundance. The spectruii is also characterized 1by the absence of any significant fragmentation. Thus, 1metalloporphyrins and free-base alkylporphyrins can be 1readily analyzed by DLI/MS as an alternative to conven1tional probe EI/MS, whenever molecular ion information iabout individual compounds is needed and/or carbon Inumber distributions of porphyrin mixtures are to be investigated. As in the case of spectra acquired under electron impact conditions (probe EI/MS), which can show 1
Eckardt et al.
744 Energy & Fuels, Vol. 4, No. 6,1990 544
‘1 404
4
400
500
,
;,,;
414 ‘i‘
,;
600
mIz
Figure 2. Mass spectrum of vanadyl etioporphyrin I (1) obtained by DLI/MS. Conditions: source temperature 250 O C ; vaporizer temperature 65 O C ; discharge 1100 V; repeller 40 V. Scan range m / t 400-700 in 1.0 s.
Figure 3. Mass spectra of desoxopyropheophorbide a (2)obtained by DLI/MS at (a) 40, (b) 120, and (c) 180 V repeller voltage, respectively. Source conditions as for Figure 2. a considerable extent of &cleavage, especially a t higher electron energies (i.e., ca. 70 eV), the DLI spectra also show 0-cleavage a t higher repeller voltages. Spectra similar to those of the alkylporphrins were also obtained for a number of functionalized chlorins. The spectrum of desoxopyropheophorbide a, analyzed as the methyl ester (21, in the range 300-550 Da is shown in Figure 3a. The spectrum was obtained at 40 V repeller voltage and is characterized by the protonated molecular ion at mlz 535 and absence of fragmentation (see above). It was found, however, that the degree of fragmentation could be controlled by increasing the repeller voltage to ca. 100-200 V. Figure 3b and Figure 3c are the spectra recorded at 120 and 180 V repeller voltage, respectively. In comparison with the spectrum recorded at 40 V (Figure
Figure 4. Mass spectrum of desoxopyropheophorbide a (2) obtained by DLI/MS. The spectrum was obtained by switching the repeller voltage in alternating scans from 40 to 120 V. Source conditions as for Figure 2. 3a) the 120-V spectrum shows a lower relative abundance of the protonated molecular ion (ca. 8%) and abundant fragment ions in the mass range mlz 380-450. Although the exact fragmentation pathways of 2 under these conditions (i.e., DLIIMS, discharge ionization) have not yet been fully elucidated, the major fragment ions can be related formally to losses within the p-substituents. Thus, mlz 447 involves loss of the C-17 side chain (-CH2CH2COOCHJ, and ions a t mlz 389, 403, 419, and 433 reflect additional losses from the periphery of the macrocycle. It is apparent that the specificity in the molecular ion information and of the fragment losses in the spectra acquired a t 40 (Figure 3a) and 120 V (Figure 3b), respectively, is lost by applying a higher repeller voltage. Thus, in the spectrum recorded at 180 V (Figure 3c), the intensities in the molecular ion region are considerably reduced and the spectrum is comparatively complex. Fragment ions of lower mass occur and there is abundant clustering. It is noteworthy that, apart from mlz 447, most of the prominent fragments occur a t masses two or three units lower than in the 120-V spectrum, for example, mlz 4331431,4191416, and 3891387. From both spectra (Figure 3b,c), it is clear that a number of rearrangement processes occur, the nature of which is, however, not fully understood a t present. Clearly, further studies are required to investigate the fragmentation pathways under these conditions, using MSIMS experiments. Variation of the repeller voltage between 0 and 200 V afforded considerable control over the fragmentation of all of the compounds examined. As a rule, fragment ions become more abundant with increasing repeller voltage, leading to decreasing intenstities in the molecular ion region, but also to an increase in total ion current. In general, significant molecular ion information was obtained at low repeller voltages (0-40 V). Specific and reproducible fragmentation patterns were obtained between 100 and 120 V which, especially for the functionalized compounds, can provide information for their characterization. Repeller voltages > ca. 120 V usually led to nonspecific fragmentation patterns and abundant clustering (see above). Presumably, the repeller not only promotes the extraction of ions into the mass analyzer but also rejects ions against the aerosol flow, thus inducing further ionization or fragmentation by collision. In an attempt to optimize the structural information that can be obtained from the spectra, switching of the repeller voltage during the acquisition time (i.e., mass scans) was investigated. The spectrum of 2, acquired by switching the repeller setting from 40 to 120 V in alter-
LCIMS of Tetrapyrroles
Energy & Fuels, Vol. 4, No. 6, 1990 745 7
a 4
t I
Time
b
-
3a1
-
scan/t ime Figure 5. UV/visible (a) and RIC (b) chromatogramsfrom the LC/MS analysis of the methyl ester of desoxomesopyropheophorbide a (3a) and its nickel counterpart (3b). MS conditions: positive-ion LC/MS; scan range m/z 300-700 in 1.0 s. HPLC conditions: 100 mm x 5 mm WATERS RP C18 Radial Com-
pression Cartridge; acetone/methanol/water 70/15/15%, isocratic at 1.0 mL/min; UV/visible detection at 400 nm.
nating scans, is shown in Figure 4. The spectrum, resulting from the summation of the mass intensities of all the scans over the entire analysis time, corresponds to those in Figure 3a plus 3b, and gives both molecular weight information and the specific fragmentation pattern (Figure 4). The same information could be obtained in the LC/MS mode. Figure 5 shows the UV/visible-monitored chromatogram and the reconstructed ion current (RIC) chromatogram from LC/MS analysis of the methyl ester of desoxomesopyropheophorbide a (3a) and its nickel counterpart (3b) under reversed-phase HPLC conditions. The components were assigned from the spectra (not shown) obtained by using repeller voltage switching. Figure 5 also indicates that the chromatographic resolution was maintained in the interface. Figure 6a is the UV/visible-monitored chromatogram (400nm) obtained from the LC/MS analysis of a mixture of standards under reversed-phase HPLC conditions. The mixture contains pyropheophorbide a (as the methyl ester, 4), desoxopyropheophorbide a (as the methyl ester, 2), chlorophyll a (5), pyrochlorophyll a (6), and pyropheophytin a (7). The corresponding RIC trace is given in Figure 6b. Analysis was performed in the negative-ion
Time
---)
Figure 6. UV/visible (a) and RIC (b) chromatograms from the LC/MS analysis of a mixture of standards. For compound identification see Table 11. MS conditions: negative-ion LCJMS; scan range m / z 400-950 in 1.5 s; source temperature 250 "C, vaporizer temperature 65 O C ; discharge 1200 V; repeller 0 V. HPLC conditions: 100 mm X 5 mm WATERS RP C18 Radial
Compression Cartridge; linear gradient elution acetonelmethanoljwater 70/15/15% to 90/5/50/, in 15 min at 1.0 mL/min; UV/visible detection at 400 nm.
mode, with the repeller electrode switched off. As with the LC/ MS analysis of desoxomesopyropheophorbide a and its nickel counterpart (3a, 3b; Figure 5), loss of chromatographic resolution in the interface (cf. Figure 1) appears to be negligible. Peak assignments were based on retention time comparison with the individual compounds and mass chromatography using the respective molecular ions (see below). From comparison of the UV/visiblegenerated chromatogram (Figure 6a) and the RIC trace (Figure 6b), it is apparent that both traces do not closely resemble each other. For example, peak areas of 4 and 2 differ markedly. Presumably, this effect is the result of (i) variations in the absorbance of the compounds at 400 nm and/or (ii) differences in the ionization efficiencies. That differences in the absorbance are a contributing factor is indirectly indicated by the relative distributions of chlorophyll a (5) and pyrochlorophyll a (6), which are comparable in both chromatograms, both compounds having very similar extinction coefficients? Clearly, further
Eckardt et al.
746 Energy & Fuels, Vol. 4, No. 6, 1990
' "7
I
T
8
---
ml z
Figure 7. Negative-ion mass spectrum of chlorophyll a (5), obtained from LCIMS analysis (cf. Figure 6). MS and HPLC conditions as for Figure 6. For formal assignment of fragment ions see Table 111. Table 111. Formal Assignment of Fragment Ions in the Mass Spectrum of Chlorophyll a (5), Obtained from Negative-Ion LC/MS (cf. Figure 7) mlz
formal assianment
Time
-
Figure 8. Partial RIC chromatogram from the LCIMS analysis of an extract fraction (SED 5-loll), previously isolated from a recent sediment. For compound identification see text. MS conditions as for Figure 6. HPLC conditions: 100 mm X 5 mm WATERS RP CISRadial Compression Cartridge;linear gradient elution acetonelmethanollwater 50/25/25% to 901515% in 25 min at 1.0 mL/min; UVIvisible detection at 400 nm. I
a
892 870 834 812 628 614 592 570 556 538 534 516
investigations are required, involving UV/visible quantitation of the compounds a t various wavelengths prior to LC/ MS analysis. As an example of the spectra obtained from LC/MS analysis of the mixture of standards, the spectrum of chlorophyll a (5) is shown in Figure 7. It is characterized by M'- at m / z 892 and shows abundant fragment ions in the ranges m / t 500-650 and 810-870. As in the case of the positive-ion spectra, the exact fragmentation pathways are not yet fully known. The major fragment ions appear, however, to result from fragmentation within the substituents. For example, m / z 614 is a prominent fragment ion in the positive-ion FAB spectrum of chlorophyll a, where it results from fragmentation involving loss of the phytyl moiety and hydrogen rearrangementa4 Other fragment ions in the spectrum can be assigned formally to losses involving the Mg ligand, the C-132substituent, and fragments from the phytyl substituent, all with hydrogen rearrangement (Table 111). At present, we do not know whether the loss of Mg represents a genuine mass spectral fragmentation or occurs from chemical reaction prior to entry into the ion source, although the latter seems more likely. Again, MS/MS experiments are required for elucidation of the fragmentations. The preliminary results discussed above have been utilized to study the tetrapyrroles in a fraction from an extract, previously isolated from an immature sediment (Priest Pot Lake, Cumbria, UK4). Figure 8 is the partial RIC chromatogram of this sample (SED 5-10/1) obtained from negative-ion LC/MS. On the basis of relative retention times and mass spectrometric comparison with standards, the previously reported4 presence of pheophytin b (8), pyropheophytin b (91, pheophytin a (lo), and pyropheophytin a (7) could be confirmed. For example, Figure 9 contains the spectrum of pyropheophytin a (7)
516
I 5a4
b
Figure 9. Mass spectra of (a) peak 7 from LC/MS analysis of SED 5-10/1 (cf. Figure 8) and (b) authentic pyropheophytin a (7). MS and HPLC conditions as for Figure 8. from the sediment (Figure 9a) and the standard (Figure 9b). In both spectra, the molecular ion at m / z 812 is the base peak and the two major fragment ions at mlz 534 and 516 occur in comparable relative abundance and can be assigned formally to a rearrangement involving loss of CzoHS (m/z 534) and subsequent loss of H 2 0 (mlz 516). This example shows the potential of LC/MS for the investigation of the distributions of sedimentary tetrapyrroles, by way of assignment of compounds through comparison with authentic standards. Summary and Conclusions. This preliminary study shows the potential of LC/MS for the analysis of sedimentary tetrapyrroles. Alkylmetalloporphyrins and their free-base counterparts can be analyzed by DLI/MS as an alternative to conventional probe EI/MS. For these compounds, molecular weight information and carbon number distributions can be derived from positive-ion spectra
Energy & Fuels 1990,4, 747-748 characterized by the presence of protonated molecular ions. For functionalized tetrapyrroles, both porphyrins and chlorins, molecular weight information and characteristic fragment ions can be obtained in both positive and negative ionization modes. Thus, LC/MS should facilitate the identification of compounds without resort to the isolation of the individual component, through comparison with authentic standards. Hence, LC/MS provides a basis for the routine investigation of sedimentary tetrapyrrole
747
mixtures on the same level as GC/MS, which is used routinely to obtain, for example, sedimentary sterane or triterpane distributions. Acknowledgment. We thank the Deutsche Forschungsgemeinschaft (DFG Grant Ec 95/ 1-1) and the National Environmental Research Council (NERC Grant GR3/6619) for financial support. Samples were kindly provided by Dr. B. J. Keely and Mr. W. G. Prowse.
Communications Characterization of Geoporphyrins Using Plasma Desorption Mass Spectrometry
Sir: Plasma desorption mass spectrometry (PDMS), which utilizes a 252Cfionizing source with a time-of-flight mass spectrometer, has been shown to be useful for analyzing metalloporphyrins. The PDMS spectrum obtained from an Anna Shale indicated the presence of a vanadyl porphyrin series, Cza to CS2,which was consistent with isobutane chemical ionization (CI) results. The PDMS results had an improved signal-to-noise relative to the CI results and did not necessitate heating the sample to 350 OC, which was necessary in order to volatilize the metalloporphyrins to obtain CI results. The results suggest PDMS might be a viable screening method for metalloporphyrin analysis with minimal sample cleanup. The inability to distinguish deoxophylloerythroetioporphyrins(DPEP) and etioporphyrins (ETIO) due to the lack of mass resolution (on the PDMS instrument used in this study) is a limiting factor in assessing the potential of PDMS for determining the maturity of oils. Plasma desorption mass spectrometry (PDMS) has been utilized for obtaining molecular weight information on large proteins (to 30K d a l t ~ n s ) . ' - ~ Other compound classes, including oligonucleotides," have also been analyzed by PDMS. In this study we report the results of the relatively low molecular weight mass analysis (for PDMS) of metalloporphyrins in shale oil. A marine black shale, Anna Shale Member of the Carbondale Formation (Pennsylvanian), in the Illinois Basin: was used in this study. The metalloporphyrin containing polar fraction was obtained by extracting 25 g of pulverized shale with benzene in a modified Soxhlet extraction apparatus. The resulting extract was condensed and further separated into three fractions, aliphatic, aromatic, and polar, by use of silica gel and alumina column chromatography. These fractions were eluted with n-hexane, benzene, and benzene-methanol (1:l v:v), respectively. The polar fraction was dissolved in tetrahydrofuran (THF) prior to electrospraying onto a nitrocellulose-coated Mylar target. The sample target was inserted into the PDMS (1) Cotter, R. J. Anal. Chem. 1988, 60, 781A-793A. (2) Jardine, I.; Scanlon, G. F.; Tsarbopoulos, A. Anal. Chem. 1988,60, 1086-1088. (3) Nielsen, P. F.; Klarskov, K.; Hojrup, P.; Roepstorff, P. Biomed. Enuiron. Mass Spectrom. 1988, 17, 355-362. (4) Viari, A.; Ballini, J. P.; Meleard, P.; Vigny, P.; Dousset, P.; Blonski, C.; Shire, D. Riomed. Enuiron. Mass Spectrom. 1988, 16, 225-228. (5) Chou, M. M.; Chou, C. L.; Allen, R. A. Proceedings of the 1987
Eastern Oil Shale Symposium, Lexington, K Y , Nouember 1987; Kentucky Energy Cabinet Laboratory: Lexington, KY, 1988; pp 137-144.
2
C31
m O /
m/z
50 0
600
Figure 1. Plasma desorption mass spectrum of the polar fraction of an Anna Shale.
carousel for analysis. The plasma desorption mass spectrometer used in this study was the BIOION 20 (Bioion Nordic, Uppsala, Sweden). The acceleration potential used for these studies was 18000 V. Figure 1 is the mass spectrum of the Anna Shale polar fraction. As can be seen, the mass spectrum shows only slightly resolved peaks (the modest resolution is inherent in the BIOION 20 PDMS time-of-flight instrument) indicative of the DPEP/ETI06 doublet of vanadyl geoporphyrins. The most intense ions, mlz 486-542, correspond to the CZs through C32 vanadyl geoporphyrins. These results are in good agreement with the isobutane chemical ionization (CI) mass spectra of the polar fraction, which indicates the vanadyl geoporphyrin doublets. Of particular interest in this study was the improved signal-to-noise obtained in the PDMS results compared with the CI results obtained on a Finnigan 4000 mass spectrometer. In addition, the CI results were obtained by heating the probe to 350 "C, which caused rapid ion intensity deterioration due to the ion source becoming dirty. These results prompted investigating the viability for using PDMS for metalloporphyrin analysis with minimal sample cleanup. In a preliminary investigation the Anna (6) Gallegos, E. J.; Sundararaman, P. MQSSSpectrom. Reu. 1985, 4 , 55-85.
0007-0624/90/2~04-0747$02.50/0 0 1990 American Chemical Society
