High-pressure ammonia chemical ionization mass spectrometry and

High-Resolution Mass Spectrometric Analysis of a Vanadyl Porphyrin Fraction Isolated from the >700 °C Resid of Cerro Negro Heavy Petroleum. R. D. Gri...
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Energy & Fuels 1990,4, 720-129

High-pressure Ammonia Chemical Ionization Mass Spectrometry and Mass Spectrometry/Mass Spectrometry for Porphyrin Structure Determination Gary J. Van Berkel,* Gary L. Glish, and Scott A. McLuckey Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365

Albert A. Tuinman Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996 Received April 27, 1990. Revised Manuscript Received July 27, 1990

High-pressure ammonia chemical ionization mass spectrometry (CI-MS) and chemical ionization mass spectrometry/mass spectrometry (CI-MS/ MS) methods for porphyrin analysis are discussed in this paper, focusing on the utility and information content of the spectra obtained while operating under different ion source conditions. Adjustment of ammonia CI parameters, in particular the ion source temperature, is used to alter the appearance of the mass spectra and, therefore, their information content. At high ion source temperatures (>523 K), protonation predominates and little fragmentation is observed. The daughter ion MS/MS spectra of the protonated porphyrins is shown to provide information regarding the nature of the peripheral macrocycle substituents. Operating at low ion source temperatures (typically 423 K) promotes a surface-assisted reduction and decomposition of the porphyrin macrocycle resulting in a relatively complex spectrum that consists of mono-, di-, and tripyrrolic fragment ions. From the masses and pattern of the pyrrolic fragments in the mass spectrum, the pyrrole sequence of the porphyrin can, in many cases, be determined. This technique can be used to sequence only individual porphyrin isomers, and the spectra are often difficult to reproduce and/or complicated by the presence of structurally uninformative peaks. A superior pyrrole sequencing method, developed recently in our laboratory, which is based on CI-MS/MS, is discussed. In this method, the daughter ion spectrum of the reduction product, (M + 7H)+ (i.e., the protonated porphyrinogen), produced in situ is used to pyrrole sequence the porphyrins. In addition to the analysis of single porphyrin isomers, the ability to pyrrole sequence the porphyrins within simple mixtures of nonisobaric porphyrins using the CI-MS/MS technique is demonstrated.

Introduction The potential use of geoporphyrins as biomarkers in fossil fuel and the study of the origin and evolution of ge~porphyrins,~-~ have provided the stimulus for determining complete structures of both geoporphyrin precursors and geoporphyrins. With the development of efficient HPLC methods for separation of individual porphyrin isomers on a preparative scale (i.e., milligram q ~ a n t i t i e s ) , ~nuclear ~' magnetic resonance (NMR) has become the most widely used method for complete structural assignment.8 However, other techniques, in particular mass spe~trometry,~-'~ still play an important role in geoporphyrin analysis and structure determination. Mass spectrometry is especially useful when insufficient sample is isolated for analysis by NMR (Le., less than several hundred micrograms) or when structural information is desired from porphyrins within porphyrin mixtures. In sediments, shales, petroleum and the associated petroleum source rocks, the two most commonly encountered porphyrin skeletal types have been etioporphyrins (etio, 1) (see Chart I) and deoxophylloerythroetioporphyrins (DPEP or DPEP-5, 2), but variable amounts of at least seven other skeletal types (e.g., DPEP-5', 3; DPEP-6, 4; DPEP-7, 5 ; tetrahydrobenzo (THB), 6; tetrahydrobenzoDPEP (THBD), 7;benzo-DPEP, 8; and benzo, 9) have also * Author

to whom correspondence should be addressed.

0887-0624/90/2504-0720$02.50/0

been identified.'J'J6 More recently, porphyrins bearing functionalized components have been extracted from these

(1) Baker, E. W.; Louda, J. W. In Biological Markers in the Sedimentary Enuironment; Johns, R. B., Ed.; Elsevier: Amsterdam, 1986; pp 125-225. (2) Branthaver, J. F.; Filby, R. H. In Metal Complexes in Fossil Fuels; Filby, R. H., Branthaver, J. F., Eds.; ACS Symposium Series 344; American Chemical Society: Washington, DC, 1987; pp 84-99. (3) Mackenzie, A. S.; Quirke, J. M. E.; Maxwell, J. R. In Advances in Organic Geochemistry 1979; Douglas, A. G., Maxwell, J. R., Eds.; Pergamon Press: Oxford, 1980; pp 239-248. (4) Baker, E. W.; Louda, J. W. In Advances in Organic Geochemistry 1981; Bjoroy, M., e t al., Eds.; Wiley: Chichester, 1983; p p 401-421. (5) Van Berkel, G. J. Ph.D. Dissertation, Department of Chemistry, Washington State University, Pullman, WA, 1987. (6) Barwise, A. J. G.; Evershed, R. P.; Wolff, G. A,; Eglinton, G.; Maxwell, J . R. J . Chromatogr. 1986, 368, 1-9. (7) Chicarelli, M. I.; Wolff, G. A.; Maxwell, J. R. J. Chromatogr. 1986, 368, 11-19. (8) Chicarelli, M. I.; Kaur, S.; Maxwell, J. R. In Metal Complexes in Fossil Fuels; Filby, R. H., Branthaver, J . F., Eds.; ACS Symposium Series 344; America1 Chemical Society: Washington, DC, 1987; pp 40-67. (9) Jackson, A. H.; Kenner, G. W.; Smith, K. M.; Alpin, R. T.; Buzikiewicz, H.; Djerassi, C. Tetrahedron 1965,21, 2913-2924. (10) Baker, E. W. J . Am. Chem. SOC.1966,88, 2311-2315. (11)Baker, E. W.; Yen, T. F.; Dickie, J. P.; Rhodes, R. E.; Clark, L. F. J . Am. Chem. SOC.1967,89, 3631-3639. (12) Smith, K. M. In Porphyrins and Metalloporphyrins; Smith, K. M., Ed.; Elsevier: Amsterdam, 1975; pp 381-398. (13) Budzikiewicz, H. In The Porphyrins, Vol. III; Dolphin, D., Ed.; Academic: New York, 1982; p p 395-461. (14) Eglinton, G. In Mass Spectrometry in the Health and Life Sciences; Burlingame, A. L., Castagnoli, N., Eds.; Elsevier: Amsterdam, 1985; pp 47-64.

0 1990 American Chemical Society

CI-MS and CI-MSIMS for Porphyrin Analysis

Energy & Fuels, Vol. 4, No. 6, 1990 721

Chart I RQR

R

R

R

R

R

R

R

\

R

R

R

R

2

1

3

R

4

5

7

8

6

9 \ / /

\

CWH

10

CWH

11

sources (e.g., 10 and ll).17 In the geologic environment these porphyrins occur mainly as nickel (Ni(I1)) and vanadyl (VO(I1))complexes,lJ6but free-base porphyrins and a variety of other metal chelates have been identified in various samples.1J&20 Electron ionization mass spectrometry (EI-MS) has been the most used technique in the mass spectrometric analysis of porphyrins isolated from these samples, for the analysis of both single geoporphyrin isomers and geoporphyrin m i x t ~ r e s . ~ ~ The J ~ J E1 ~ J mass ~ spectra for single isomers of these types of porphyrins are dominated by the molecular ion, Me+with smaller abundances of fragment ions (15) Gallegos, E. J.; Sundararaman, P. Mass Spectrom. Reu. 1985,4, 55-85. (16) Filby, R. H.; Van Berkel, G. J. In Metal Complexes in Fossil Fuels; Filby, R. H., Branthaver, J. F., Eds.; ACS Symposium Series 344; American Chemical Society: Washington, DC, 1987; pp 2-39. (17) Ocampo, R.; Callot, H. J.; Albrecht, P. In Metal Complexes in Fossil Fuels: Filby, R. H.; Branthaver, J. F., Eds.; ACS Symposium Series 344; Americal Chemical Society: Washington, DC, 1987; pp 68-73. (18) Bonnett, R.; Burke, P. J.; Czechowski, F. In Metal Complexes in Fossil Fuels; Filby, R. H., Branthaver, J. F., Eds.; ACS Symposium Series 344; Americal Chemical Society: Washington, DC, 1987; pp 173-183. (19) Bonnett, R.; Burke, P. J.; Czechowski, F.; Reszka, A. Org. Geochem. 1984, 6 , 177-182. (20) Eckardt, C. B.; Wolff, M.; Maxwell, J. R. Org. Geochem. 1989, 14, 659-666.

formed via benzylic-like cleavage of the peripheral subs t i t ~ e n t s . ~ JOn ~ J the ~ basis of the nominal mass of the molecular ion, the carbon number, skeletal type, and metal chelated can usually be determined and the fragment ions provide formation regarding the nature (but not location) of the peripheral substituent groups on the macrocycle. The inability to fragment the porphyrin macrocycle, a necessary step to determine the location of the substituent groups on the porphyrin macrocycle, limits the amount of structural information obtainable. A remedy for this lack of macrocycle fragmentation was found, quite paradoxically, by Eglinton and co-workers when they used chemical ionization mass spectrometry (CI-MS), first with methane21as the reagent gas and later with for porphyrin analysis. When obtained under the appropriate conditions, these CI mass spectra contained tri-, di-, and monopyrrolic fragment ions from which the pyrrole sequence of the porphyrin could be determined. The paradox arises because CI is typically intended to be a “soft” ionization technique used to minimize, not enhance, fragmentation. Therefore, the spectra obtained are not, at first glance, readily explained. Djerassi and c o - w o r k e r ~have ~ ~ ~shown ~ ~ that ammonia CI-MS can be used to produce similar, even more structurally informative, mass spectra. Even though the phenomena responsible for the extensive fragmentation observed in these CI mass spectra had not been elucidated until re~ e n t l y ,the ~ ~ammonia ,~~ and hydrogen CI-MS techniques had proven quite useful for obtaining porphyrin pyrrole sequence information. Considerable effort by our group has been spent on understanding the phenomena responsible for the fragmentation observed in the ammonia CI mass spectra of porphyrins to better control the processes involved and, thereby, increase the utility of the t e ~ h n i q u e . ~ - We ~ l have shown that a series of complex gas-phase and surface-assisted reactions in the CI plasma results in the reduction and subsequent decomposition of the porphyrin macrocycle.2s Adjustment of ammonia CI parameters, in particular the ion source temperature (also noted by Eglint ~ n l ~ can , ~ ~be) used, , however, to alter the appearance of the mass spectra and, therefore, their information content. For example, a t relatively high ion source temperatures (>523 K), protonation of the porpyrin predominates and little fragmentation is observed. This contrasts with low ion source temperature (ca. 423 K) operation which promotes the reduction and decomposition of the porphyrin (21) Eglington, G.; HajIbrahim, S. K.; Maxwell, J. R.; Quirke, J. M. E.; Shaw, G. J.; Volkman, J. K.; Wardroper, A. M. K. Philos. Trans. R. SOC.London, A 1979,293,69-91. (22) Shaw, G. J.; Eglinton, G.; Quirke, J. M. E. Anal. Chem. 1981,53, 2014-2020. (23) Wolff, G. A.; Chicarelli, M. I.; Shaw, G. J.; Evershed, R. P.; Quirke, J. M. E.; Maxwell J. R. Tetrahedron 1984, 40, 3777-3786. (24) Evershed, R. P.; Wolff, G. A.; Shaw, G. J.; Eglinton, G. Org. Mass Spectrom. 1985, 20, 445-453. (25) Jiang, X.; Wegmann-Szente, A.; Tolf, B.; Kehres, L. A.; Bunnenberg, E.; Djerassi, C. Tetrahedron Lett. 1984,25,4083-4086. (26) Tolf, B.; Jiang, X.; Wegmann-Szente, A.; Kehres, L. A.; Bunnenberg, E.; Djerassi, C. J . Am. Chem. SOC.1986, 108, 1363-1374. (27) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Proceedings of the 36th ASMS Conference on Mass Spectrometry and Allied Topics, Sun Francisco, CA, June 5-10, 1988 ASMS: East Lansing, MI; pp 302-303. (28) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A.; Tuinman, A. A. J . Am. Chem. SOC.1989, 111,6027-6035. (29) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Org. Geochem. 1989, 14, 203-212. (30) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Tuinman, A. A. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, May 21-26,1989; ASMS: East Lansing, MI; pp 796-797. (31) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A., Tuinman, A. A. Anal. Chem. 1990,62, 786-793.

Van Berkel et al.

722 Energy & Fuels, Vol. 4, No. 6, 1990 Mass Dispersion of Parent Ions

Mass Spectrum

Mass Dispersion of Daughter Ions

MS/MS

Spectrum

u u

n n

UUQ Figure 1. Illustration of the principle of the MS/MS technique and a schematic representation of the hybrid BEqQ mass spectrometer used to acquire the data presented in this paper. S = source, B = magnetic sector, E = electric sector, DECEL = deceleration lenses, q = collision quadrupole, Q = analyzer quadrupole, and D = detector.

macrocycle, resulting in a relatively complex spectrum that consists of mono-, di-, and tripyrrolic fragment ions. In this paper, we discuss the utility and information content of' the mass spectra obtained under high-pressure ammonia CI conditions a t both high and low ion source temperatures. The use of mass spectrometry/mass spectrometry (MS/MS)32-34to obtain additional structural information from the ions formed in both temperature regimes is also discussed. The majority of the discussion overviews a CI-MS/MS pyrrole sequencing technique we have recently de~eloped.~* In this technique, the MS/MS daughter ion spectrum of the reduction product, (M + 7H)+(Le., the protonated porphyrinogen), formed in situ at low ion source temperatures, is used to pyrrole sequence the porphyrin. In addition to pyrrole sequencing single porphyrin isomers, the ability to pyrrole sequence the porphyrins within simple mixtures of nonisobaric porphyrins using the CI-MS/MS technique is demonstrated.

150 eV, an emission current of 1.0 mA, and an accelerating potential of 8 kV. The ion source was operated at a temperature of 523 K to obtain spectra containing minimal porphyrin reduction and decomposition products and at a temperature of 423 K to promote the reduction/decomposition process. Porphyrin was introduced into the ion source under both temperature regimes using a desorption chemical ionization (DCI) probe fitted with a platinum DCI Typically, 1-5 pg of porphyrin was dissolved in chloroform and applied to the DCI coil in stages using a microsyringe. At a source temperature of 523 K no heating of the DCI coil was necessary, as the porphyrin desorbed rapidly from the coil into the gas phase, owing to heating from the hot CI plasma. In the experiments run under reducing conditions (423 K source temperature), the DCI current was ramped from 0 to 1.4 A at 3.75mA s-l and then held at 1.4 A for several minutes. In both experiments, mass spectra were acquired by scanning the magnetic sector from 700 to 70.u at a rate of 6 s/decade. Daughter ion MS/MS spectra were acquired under ion source conditions similar to those used to obtain the respective CI mass spectra. A larger sample amount (typically 20-50 pg), as well as a different DCI probe current ramp, were both essential in generating sufficient intensity of (M + 7H)+ for MS/MS31 Parent ions were selected with the sector portion of the instrument (BE) and then decelerated to 30, 70, or 100 eV. Collision-induced dissociation (CID)was performed in the rf-only collision quadrupole (4)using argon as the collision gas. Argon pressure, measured with an ionization gauge outside the 254-mm-long collision quadrupole, was 1.5 X Torr (corresponding to an estimated pressure of 3 mTorr inside the quadrupole). The analyzer quadrupole (Q)was scanned from 30 to 600 u at 4 s/scan to analyze the daughter ions formed by CID. Approximately 16 quadrupole scans were acquired in a typical MS/MS experiment.

Results and Discussion

In using high-pressure ammonia CI for porphyrin analysis, conditions can be established to optimize either reduction and decomposition of the porphyrin macrocycle or protonation of the porphyrin with little or no macrocycb fragmentation. Our previous workz8 has shown that reduction and decomposition of the porphyrin macrocycle is the result of a series of gas-phase and, more importantly, surface-assisted reactions in the CI plasma. Several experimental parameters affect the reduction/decomposition process, the most important being the nature of the ion source surfaces, the composition of the CI plasma, and the ion source temperature.28 We found, as did Eglinton and co-workersZ1when using methane as the CI reagent gas, that the shift from protonation and little fragmentation to macrocycle reduction and decomposition can be easily achieved by varying the ion source temperature, while Experimental Section holding all other ion source parameters constant. That is, Samples. 2,3,7,8,12,13,17,18-0ctaethyl-21H,23H-porphine at low source temperatures (ca. 423 K) reduction/decom(free-baseoctaethylporphyrin),vanadyl octaethylporphyrin, and position is promoted while a t high source temperatures dimethyl 3,7,12,17-tetramethy1-2IH,23H-porphine-2,18-di- (1523 K) protonation is predominant and little fragmenpropionate (free-basedeuteroporphyrin IX dimethyl ester) were tation occurs. This observation is readily explained once obtained from Aldrich Chemical (Milwaukee,WI). 15,17-Butait is understood that the reduction/decomposition process no-3,8-diethyl-2,7,12,18-tetramethyl-21H,23H-porphine was obis surface-assisted. At low source temperatures, interaction tained from P. S. Clezy (University of New South Wales). All of the relatively involatile porphyrins with the surfaces samples were used as received from their respective supplier. within the ion source is enhanced, thereby facilitating Mass Spectrometry. All spectra were obtained on a hybrid reduction and decomposition. At high ion source temmass spectrometer of BEqQ geometry (B = magnetic sector, E = electric sector, q = rf-only collision quadrupole, and Q = anperatures, the porphyrins remain in the gas phase during alyzer quadrupole) located at the University of Tennessee: the the majority of their residence time in the ion source, ZAB-EQ from VG Analytical. A schematic representation of this minimizing porphyrin-surface interactions, and therefore, instrument is shown in Figure 1. High-pressure ammonia CI mass little or no reduction/decomposition is observed.2s The spectra were acquired using a reagent gas (ammonia; Nationanalytical utility of, and structural information within, the al/Bower, Philadelphia, PA) pressure of (1.3-2.0) x Torr mass spectra of porphyrins obtained under high-pressure (measured in the vacuum source housing),an electron energy of ammonia CI-MS and MS/MS conditions at both high and low ion source temperatures are discussed below. (32) Cooks, R.G.;Glish, G. L.Chem. Eng. News 1982,59 (48),412-52. MS/MS is a technique for ion structure analysis in (33) McLafferty, F. W. Tandem Mass Spectrometry; Wiley: New which a mass-selected ion undergoes a change in mass York, 1983. and/or charge (usually via metastable ion dissociation or (34) Busch, K. L.; Glish, G . L.; McLuckey, S. A. Mass Spectrometry/Mass Spectrometry; VCH Publishers: New York, 1988. collision-induced dissociation (CID)) prior to a second stage

C I - M S and CI-MSIMS for Porphyrin Analysis

Energy & Fuels, Vol. 4, No. 6, 1990 723 (M+H)+

O

mh fM+M+ loo.

(b)

p -3 !n

. MW = 599 u

518

572

I

1

mh

Figure 2. High-pressure ammonia CI mass spectrum of (a) free-base octaethylporphyrin and (b) vanadyl octaethylporphyrin obtained at an ion source temperature of 523 K. mass a n a l y s i ~ . ~Recent ~ - ~ ~ MS/MS work applied to porphyrin analysis has employed triple quadrupole systems35 for the analysis. For example, Johnson et al.36used EIMS/MS to determine the nature of the substituent groups on selected high carbon number geoporphyrins within a complex mixture and Brodbelt et al.,’ investigated the use of isobutane CI-MS/MS for direct determination of vanadyl porphyrins in petroleum. To perform the MS/MS experiment on the present instrument (Figure l), ions formed from the sample in the ion source (S) are separated by the first stage of mass analysis (BE) and these selected ions (parent ions) are decelerated (DECEL) to the appropriate kinetic energy (30-100 eV) and are fragmented by collision with a target gas (argon) in the rf-only quadrupole (q). A second stage of mass analysis, employing the analyzer quadrupole (Q), is used to obtain the mass spectrum of the resulting fragments ions (daughter ions). This mode of MS/MS operation is termed a daughter ion scan. It should be pointed out that although a BEqQ instrument was used in this study, a number of other MS/MS instrument^,^^ including the triple quadrupole, that can perform low-energy CID and record daughter ions with unit mass resolution, could have been used to acquire similar spectra. Nonreducing Conditions, Ammonia CI Mass Spectra. Figure 2 is the ammonia CI mass spectra of free-base octaethylporphyrin (FBOEP; Figure 2a) and vanadyl octaethylporphyrin (vanadyl-OEP; Figure 2b), respectively, both of which were obtained at an ion source (35) Yost, R. A.; Enke, C. G. Anal. Chem. 1979, 52, 1251A-1264A. (36) Johnson, J. V.; Britton, E. D.; Yost, R. A.; Quirke, J. M. E.; Cuesta, L. L. Anal. Chem. 1986,58, 1325-1329. (37) Brodbelt, J. S.;Cooks, R. G.; Wood, K.V.; Jackson, T. J. Fuel Sci. Tecnnol. Int. 1986,4 (6), 683-698.

temperature of 523 K to minimize reduction and decomposition of the porphyrin macrocycle. In the case of FBOEP (Figure 2a), the protonated molecule ( m / z 535) is the major ion in the spectrum with little or no substituent group fragmentation. Although some monopyrrolic ions are observed at low mass (e.g., m / z 138), decomposition of the macrocycle is not occurring to any significant extent. From this spectrum, the molecular weight of the porphyrin can be determined and, therefore, the carbon number, skeletal type, and nature of the metal (if any) chelated. Similar spectra were obtained from other free-base porphyrins analyzed under these ion source conditions. Because fragmentation is minimized, this method could probably be used for the analysis of free-base porphyrin mixtures. The mass spectrum of vanadyl-OEP obtained under nonreducing ion source conditions (Figure 2b) is more complex than that of FBOEP. In this case, fragment ions resulting from substituent group cleavage as well monopyrrolic fragment ions are observed in the spectrum. The most abundant fragment ion resulting from substituent group cleavage is observed a t m / z 572, corresponding to a loss of 28 u from the protonated molecule. This fragment ion, and the other major fragments resulting from substituent group cleavage, are observed at m / z values corresponding to consecutive losses of 14 u from the protonated molecule. As discussed below, this fragmentation limits the utility of the analysis to single isomers. The appearance of monopyrrolic fragments in the spectrum, as well as the isotope pattern of the protonated molecule (which is different than would be theoretically predicted for this p~rphyrin,~), demonstrates that some reduction and decomposition of the macrocycle takes place. Similar fragmentation was noted for iron and nickel porphyrins run under identical ion source conditions. In the cases we have examined, the ammonia CI mass spectra of metalloporphyrins obtained under “nonreducing” conditions are suitable for determining the molecular weight of the porphyrin (and therefore skeletal type and metal chelated) and the substituent group fragments reveal some structural information. The problems associated with using this technique for the analysis of metalloporphyrin mixtures are demonstrated in Figure 3, which is a comparison of the ammonia CI mass spectrum (obtained under the same conditions as the spectrum in Figure 2) with the E1 (70 eV electrons) mass spectrum of a mixture of vanadyl porphyrins isolated from the New Albany oil hale.^,,^ The E1 mass spectrum (Figure 3a) shows the sample to consist of a mixture of vanadyl-DPEP and etio-type porphyrins, with both skeletal types extending over a considerable carbon number range (Le., C28-C34-etioand C2,-C35-DPEP). DPEP is used here to refer to porphyrins of skeletal types 2-6 which are structural isomers and, therefore, for the same carbon number have the same exact mass. The distribution of DPEP skeletal types in the New Albany sample has not been determined. Also visible in the spectrum are three peaks of low abundance that correspond to the molecular ions of C,,-C,,-THBD-type prophyrins. The molecular ion region of the ammonia CI mass spectrum of this same sample, obtained under “nonreducing”conditions, is shown in Figure 3b, and the total mass range of this spectrum is shown in Figure 3c. The distribution of porphyrins identified in this spectrum (as the protonated molecules) is (38) Van Berkel, G. J.; Joubert Castro, A.; Filby, R. H. Applied Geochem., in press. (39) Van Berkel, G. J.; Quirke, J. M. E.; Filby, R. H. O g . Geochem. 1989, 24, 119-128.

Van Berkel et al.

724 Energy & Fuels, Vol. 4 , No. 6, 1990

c,

etio 515

mh 535

I

miz , C DPEP

1/11

II 1 \

mh

- THBD 5

mlz

0

mlz

mh Figure 3. Molecular ion region of (a) the E1 (70 eV) mass spectrum and (b) the high-pressure ammonia CI mass spectrum, obtained at an ion source temperature of 523 K, of the total vanadyl porphyrin mixture isolated from the New Albany oil shale bitumen. (c) The total mass range of the CI mass spectrum. Details regarding this geoporphyrin sample are given elsewheree53 similar to the distribution in the E1 mass spectrum, with two notable exceptions. First, the abundances of the THBD porphyrins is much greater in the CI mass spectrum. This difference might be attributed to a greater propensity for fragmentation under E1 conditions and/or a greater ionization efficiency under CI conditions of this skeletal type, relative to DPEP or etioporphyrins. The second difference is that the carbon number range of the porphyrins, as determined from the CI mass spectrum (i.e., C2,-C3,-etio and C,,-C,,-DPEP), extends to lower carbon number than the distribution determined by E1 (Le., Cz8-Cs4-etioand C29-Cg5-DPEP). A t first glance, this difference might be attributed to better S / N in the lower mass region of the ammonia CI mass spectrum than in the E1 mass spectrum. On the basis of the observed substituent group fragmentation of vanadyl-OEP (Figure 2b), and the similar low mass fragments in both the CI mass spectrum of vanadyl-OEP and the New Albany vanadyl

Figure 4. MS/MS daughter ion spectra of (M + H)+from free-base octaethylporphyrin obtained at collision energies of (a) 30, (b) 70, and (c) 100 eV. (M + H)+was produced under nonreducing ion source conditions. porphyrin mixture (Figure 34, we can conclude that these newly observed species in the lower mass region of the spectrum are more probably fragment ions from higher molecular weight porphyrins in the mixture, rather than lower molecular weight homolgues in the mixture undetected by EI-MS. Even though fragmentation of the porphyrins also occurs under E1 conditions, the fragment ions are formed via benzylic-like cleavage (Le., (M - 15)+, (M - 29)+, etc.) and, therefore, are not of the same mass as the lower molecular weight homologues. These data indicate that using ammonia CI-MS under nonreducing conditions to determine the distribution of metallogeoporphyrins in mixtures might skew the distribution to lower mass and erroneously indicate the presence of lower molecular weight homologues in the mixture. MS/MS Spectra. A CI-MS/MS study by Brodbelt et al.37indicated that the daughter ion spectrum of a protonated porphyrin might be of utility in determining the nature of the substituent groups on the macrocycle. We report here our initial results aimed at more fully exploring this potential application. Shown in Figures 4 and 5 are daughter ion MS/MS spectra of protonated FBOEP and vanadyl-OEP, respectively, obtained at collision energies of 30,70, and 100 eV. The daughter ion spectra of these two porphyrins are similar, but do show some important differences. At the lowest collision energy (30 eV), multiple daughter ions are observed in the case of both porphyrins, and as the collision energy increases, the degree of fragmentation increases. Each of the daughter ions observed in these spectra results from either single or multiple cleavages of the ethyl substituent from the macrocycle, expressed as single and combined loss of CH3', C2H5*, and/or CHI, as summarized in Table I. Daughter ions resulting from cleavage of the stable aromatic macrocycle are not observed. Overall, the degree of fragmentation appears to be greater in the case of FBOEP than vanadyl-OEP. Apparently, fewer fragmentation pathways, and

CI-MS and CI-MSIMS for Porphyrin Analysis

Energy & Fuels, Vol. 4, No. 6,1990 725

Table I. Daughter Ions Observed in the MS/MS Spectra of Protonated Free-base Octaethylporphyrin and Protonated Vanadyl Octaethylporphyrin daughter ions parentions ( M + H (M+H(M+H(M+H(M+H(M+H(M+H(M+H(M+H(M+H(M + H)+ 15)' 16)' 29)+ 30)+ 44)+ 59)+ 60)' 74)+ 88)+ 891+ m l z 535

520 -CH,'

m/z 600

585 4H3'

519 -CH,

506 -C2H5'

505 -2CH3'

491 -CH3' -C2H5'

476 -2CH3' -C,H,'

475 -4CH3'

461 -3CH3' -CZH5'

447 -2CH3' -2C2H,'

526 -3CH3' -C2H5'

512 -2CH3' -2CzHS'

446 -4CH3' -C,H,'

VOOEP 571 -CZH5'

556 -CH3' -C2H5'

571 585

.1

I

460

480

500

520

541 556 540 560

580

600

mlz

mh

mh

Figure 5. MS/MS daughter ion spectra of (M + H)' from vanadyl octaethylporphyrin obtained at collision energies of (a) 30, (b) 70, and (c) 100 eV. (M + H)+was produced under nonreducing ion source conditions. some different pathways, are available to the vanadyl porphyrin relative to the free-base porphyrin. However, studies of additional standards will be needed before more firm generalizations can made. For the MS/MS spectra of the protonated porphyrins to be of greatest utility, only one daughter ion representative of each of the different substituents on the macrocycle should be observed. Data of this type would indicate the nature and relative numbers of the different substituents on the macrocycle. The MS/MS spectrum in Figure 5b comes closest to providing this type of data. In this spectrum only one major daughter ion, m / z 571, originating from a-cleavage of an ethyl group, the only type of substituent present, from the macrocycle (i.e., (M + H - 29)+). Multiple collision conditions in the analyzer quadrupole might account for the multiple fragmentations observed in most of the MS/MS spectra in Figures 4 and 5. This severely impedes extraction of information regarding the nature and number of the different substituent groups on the macrocycle. It must be noted that this problem exists even for a porphyrin containing only one type of peripheral substituent (ethyl, in the case of OEP). The spectra become even more complex for porphyrins

54 1 -2CH3' -CZH,'

bearing two or more different substituent groups. However, on the basis of low-energy-CID studies of protonated porphyrins carried out with a quadrupole ion trapto we anticipate that, under single-collision conditions a t the appropriate collision energy, the daughter ion spectra obtained will contain only those daughter ions resulting from single cleavage processes. In any case, the pattern of daughter ions obtained from a porphyrin under the present experimental conditions might be used as a fingerprint for the molecule. In that way, the daughter ion spectrum could be used in targeted analysis studies to identify the presence of a particular porphyrin structure in other samples. Reducing Conditions. Ammonia CI Mass Spectra. High-pressure ammonia CI-MS, carried out under conditions that promote the reduction and decomposition of the porphyrin macrocycle, is of great utility in determining the pyrrole sequence of single porphyrin isomers. The only experimental change necessary to produce such spectra is to operate the ion source a t a low temperature (typically 423 K), relative to the ion source temperatures normally used in the analysis of porphyrins (typically >473 K). An ammonia CI mass spectrum of FBOEP obtained a t a source temperature of 423 K is shown in Figure 6a. The major ions in this spectrum are the pseudomolecular species (M + nH)+ and tri-, di-, and monopyrrolic fragment ions. The most abundant of the pyrrolic fragment ions are those corresponding to the protonated forms of the respective stable pyrrolic compounds formed by cleavage of the macrocycle at the appropriate meso carbons.%l% Each of the possible pyrrolic units (mono-, di-, and tripyrrole) is manifest in the spectrum as a triad of peak clusters. For a particular mono-, di-, or tripyrrole triad, the peaks within each of the three respective peak clusters that make up the triad correspond to that pyrrolic unit containing either 0, 1, or 2 terminal meso carbons.26 In ideal cases, the pyrrole sequence of the porphyrin can be determined from the masses and pattern of pyrrolic fragment ions as is described elsewhere.22,26Although spectra similar to those of the free-base porphyrins can, in some cases, be obtained for the corresponding metal chelates,z4the most readily interpretable CI mass spectra, in terms of determining the pyrrole sequence, are usually obtained when analyzing free-base porphyrins. As such, the analysis of only freebase porphyrins is discussed here. Although the ammonia CI mass spectrum of a porphyrin obtained under these reducing conditions is potentially quite useful in porphyrin pyrrole sequencing,. general application of the technique can be problematic. Because the reduction/decomposition process is highly dependent (40) Van Berkel, G. J.; Glish, G.L.; McLuckey, S. A. Proceedings of the 36th ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, June 5-10, 1988; ASMS: East Lansing, MI; pp 639-640.

Van Berkel et al.

726 Energy & Fuels, Vol. 4 , No. 6, 1990

(a) Mass Spectrum

monopyrrole A

”1

\A‘

#

(M+H)’

A/

11%

I 1141,m

eE

dipyrrole )

5

...

4

0

! I

MS/MS of (M+7H)+ dipyrrole AA 70 eV collision energy

I.e

(M+7H)’

Ah

100

+

271 269

541

monopyrrole

-I d

-.-

.550

450

Figure 6. Comparison of (a) the ammonia CI mass spectrum of free-base octaethylporphyrinobtained at an ion source temperature at 423 K and (b) the MS/MS daughter ion spectrum (70-eV collision energy) of in situ formed (M + 7H)+. Adapted from ref 31. Table 11. PorDhvrin Classification Scheme”

porphyrin structural type A.

AjB A2BZ AzBC ABCD

pyrrole sequence A..A-A-A._. AAAB AABB ABAB AABC ABAC ABCD ACBD ABDC 1 . 1 .

monopyrroles A

..

dipyrroles

A. .A_.

AA, AA, AB AA, AB, AB, AC, AB,

...

tripyrrolesb

AAA

AB AB, B B AB, BC, CA AC BC, CD, DA CB, BD, DA BD, DC, CA

AAB,AAB*, ABA* AAB, ABB ABA, BAB AAB, ABC*, BCA*, CAA ABA, BAC, ACA ABC, BCD, CDA, DAB ACB, CBD, BDA, DAC ABD, BDC, DCA, CAB

Classification scheme ignores structural and positional isomers of the substituent groups on the individual pyrroles. porphyrin pyrrole sequence tripyrroles marked with an asterisk have the same mass but a different pyrrole sequence.

on ion source parameters, the appearance of the mass spectrum obtained from a porphyrin can be highly variable and is often complicated by an abundance of structurally redundant and/or uninformative peaks. The utility of the CI mass spectrum of pyrrole sequencing a porphyrin a priori, especially an asymmetrical substituted porphyrin, is therefore quite limited. MS/MS Spectra. We recently developed an ammonia CI-MS/MS technique30v31 in which the daughter ion spectrum of the protonated porphyrinogen, (M + 7H)+ produced in situ from the porphyrin, is used to determine the pyrrole sequence of the porphyrin. This CI-MS/MS pyrrole sequencing technique overcomes the major problems associated with using the CI mass spectrum for this purpose. The discussion and figures that follow are derived from our earlier publication31 in which we introduced this sequencing method. Figure 6b is the daughter ion spectrum, obtained at a collision energy of 70 eV, of the in situ generated (M + 7H)+ species from FBOEP. This spectrum, consisting of an even-mass peak “triplet”, an oddmass peak “quadruplet”, and an even-mass peak “quadruplet”, is much simpler than the ammonia CI mass

For a particular

spectrum shown in Figure 6a. As demonstrated below, each of the peaks in the MS/MS spectrum is structurally significant. Determining the pyrrole sequence of a porphyrin from the MS/MS spectrum of (M + 7H)+ requires assignment of the individual peak “triplets” and “quadruplets“ as either tri-, di-, or monopyrroles. The pattern of pyrrolic units observed characterizes the porphyrin as having one of the nine possible pyrrole sequences shown in Table 11. The peak triplets and quadruplets can be easily identified as mono-, di-, or tripyrroles based on the nitrogen rule. For each porphyrin discussed here, (M + 7H)+ is an evenelectron ion with an even number of nitrogens, and, therefore, and odd mass. The daughter ions produced are even-electron species such that the pyrrolic fragments with an even number of nitrogens (i.e., the dipyrroles) have an odd mass and the species with an odd number of nitrogens (Le., the tripyrroles and monopyrroles) have an even mass. Since octaethylporphyrin has an AAAA pyrrole sequence, the three sets of daughter ion peaks observed in the daughter ion spectrum should represent the monopyrrole A, dipyrrole AA, and tripyrrole AAA portions of the

CI-MS and CI-MSIMS for Porphyrin Analysis

Energy & Fuels, Vol. 4, No. 6,1990 727

(a) Mass Spectrum

HN

N

B

cH2chzc-=Y II

%y%CH2

100

0

--

0

BB

AAB

275

,

347 3 1 350

l 4 5 4 ‘ 440 . 4 6 8 450

-

*.

545; ’-5391 )I 550

(b) MS/MS of (M+7H)+ AB

8 5

A7

x

185 285

Figure 7. Comparison of (a) the ammonia CI mass spectrum of free-base deuteroporphyrin IX dimethyl ester obtained at an ion source temperature of 423 K and (b) the MS/MS daughter ion spectrum (70-eV collision energy) of in situ formed (M + 7H)+.Adapted from ref 31.

macrocycle (see Table 11). Therefore, the even-mass peak “triplet” ( m / z 124, 136, and 150) is assigned as monopyrrole A, the odd-mass peak “quadruplet” ( m / z 257,269, 271, and 283) as dipyrrole AA, and the even-mass peak “quadruplet” ( m / z 392, 404, 406, and 418) as tripyrrole AAA. In general, monopyrroles are observed as even-mass peak “triplets”, dipyrroles as odd-mass peak ”quadruplets”, and tripyrroles as even-mass peak “quadruplets”. Assignment of the daughter ion peaks to specific portions of the macrocycle is trivial for FBOEP and other A4-type porphyrins. In the case of porphyrins with an asymmetrical pyrrole sequence, the peaks must be assigned to specific portions of the macrocycle by assembling the expected di- and tripyrroles from their constituent monopyrroles. This can be done by defining a “monopyrrole unit” for each of the monopyrrole peak triplets observed in the MS/MS spectrum and assigning each a different letter designation, either A, B, C, or D. The respective “monopyrrole unit” for each triplet is defined as having a mass (Mx) one unit less than the mass of the center peak (Mx 1) in the respective monopyrrole triplet (e.g., MA = 135 u for FBOEP). The center peak in each triplet corresponds to that monopyrrole with one meso carbon attached. The lower and high mass peaks in each triplet, which occur a t m/z values of Mx - 13 and Mx + 15, are the monopyrrole with no meso carbons and two meso carbons, respectively. A “dipyrrole unit” can then be viewed as being comprised of two monopyrrole units with a mass equal to the sum of the masses of its constituent monopyrrole units (e.g., Mu = 270 u for FBOEP). The peaks of each dipyrrole quadruplet occur at m / z values of M x y - 13, Mxy - 1, MXY+ 1 and Mxy + 13. The peaks at m/z MXY- 1 and Mxy + 1 are comprised of two monopyrrole units (Le., two pyrrole rings and two meso carbons), while the peaks a t m / z MXY- 13 and Mxy + 13 contain one less and one more meso carbon, respectively,

+

than the basic dipyrrole unit. Similarly, a “tripyrrole unit” can be defined as consisting of three monopyrrole units with a mass equal to the sum of the masses of those monopyrrole units (e.g., MAAA= 405 u for FBOEP). The peaks of each tripyrrole quadruplet occur at m / z values of Mxyz - 13, MXYZ- 1, and MXYZ+ 1, and MXYZ 13. The peaks at m/z MXYZ- 1 and MXYZ+ 1 are comprised of three monopyrrole units (i.e., three pyrrole rings and three meso carbons), while the peaks at m/z MXY- 13 and MXY+ 13 contain one less and one more meso carbon, respectively, than the basic tripyrrole unit. The advantages of the CI-MS/MS technique over CIMS for pyrrole sequencing of porphyrins are best illustrated by comparing the two techniques for the analysis of more complex porphyrins made up of pyrroles of different masses. Figure 7 compares the ammonia CI mass spectrum of deuteroporphyrin IX dimethyl ester (AABB pyrrole sequence) with the MS/MS spectrum of the protonated porphyrinogen ((M + 7H)+) formed from this porphyrin. For a porphyrin of this pyrrole sequence, two monopyrrole, three dipyrrole, and two tripyrrole triad peak clusters are expected in the CI mass spectrum (see Table 11). The CI mass spectrum in Figure 7a, however, is missing some of the structurally informative peaks, and others are only barely visible above background noise. This inhibits interpretation of the spectrum as does the mass overlaps of some of the triad peak clusters (e.g., B and AA). The daughter ion MS/MS spectrum of (M + 7H)+ (Figure 7b) is relatively simple and straightforward to interpret when compared with the CI mass spectrum (Figure 7a). The pattern of pyrrolic units observed (two monopyrroles, A and B; three dipyrroles, AA, AB, and BB; and two tripyrroles, AAB and ABB) matches that expected for an AABB pyrrole sequence. These peaks were readily assigned to the portions of the macrocycle which they represent by using the procedures described above.

+

Van Berkel et al.

728 Energy & Fuels, Vol. 4, No. 6, 1990

(M+7H)+ 483

X3.6

1

450

550

Figure 8. MS/MS daughter ion spectrum (70-eV collision energy) of in situ formed (M + 7H)+ from 15,17-butano-3,8-diethyl2.7.12.18-tetramethvl-21H.23H-~or~hine. Dashed lines indicate the location of peaks not expected to appear in the spectrum as described in the text. Adaptkd from rei311

It is also possible to determine the combined mass of the substituent groups on the individual monopyrroles from the information in the daughter ion spectrum of (M + 7H)+. As we have defined it, a “monopyrrolic unit” devoid of substituent groups has a mass of 77 u. Therefore, the mass of a monopyrrolic unit observed in the daughter ion spectrum minus 77 will be equal to the combined mass of the substituent groups on that monopyrrole (i.e., Msx = Mx - 77). For FBOEP this corresponds to 135 - 77, or 58 u as expected for two ethyl groups. In the case of deuteroporphyrin IX dimethyl ester, the mass calculated for the substituent groups on the monopyrroles (MSA = 16 u and MsB = 102 u) is consistent with the methyl/hydrogen and methyl/methyl propionate substituent pairs on the respective monopyrroles. We have also investigated, on a preliminary basis, the utility of the CI-MS/MS technique for pyrrole sequencing porphyrins containing an exocyclic ring. Porphyrins containing exocyclic rings, such as DPEP porphyrins (skeletal types 2-61, comprise a substantial fraction of the geoporphyrins encountered. Therefore, the general application of the CI-MS/MS method for pyrrole sequencing geoporphyrins also hinges on its ability to sequence prophyrins of these skeletal types. Our investigations of these types of porphyrins has been hampered, however, by difficulties in acquisition of suitable standard compounds. The only porphyrin of this type that we have investigated (15,17-butano-3,8-diethyl-2,7,12,18-tetramethyl-21~,23~porphine) has an AABC pyrrole sequence and contains a seven-membered exocyclic ring that bridges a pyrrole ring with a meso carbon. The daughter ion spectrum of (M + 7H)+ from this porphyrin is shown in Figure 8. Previous ammonia CI-MS studies of these types of porphyrinsz3 have indicated that the presence of an exocyclic ring complicates spectral interpretation due to a preferential retention of the substituted meso carbon on the pyrrole containing the exocyclic ring, resulting in the absence or reduced abundance of certain peaks in the mass spectrum. If the presence of the exocyclic ring affects the fragmentation of (M + 7H)+,there should be distinct differences between the daughter ion MS/MS spectrum obtained and the spectrum we would predict (using the procedure described above) for a porphyrin with the same pyrrole sequence and combined mass of the substituents,

but without an exocyclic ring. We would expect to observe from this porphyrin three monopyrrole peak triplets (A, MA = 121 u; B, MB = 93 u; C, Mc = 147 u), four dipyrrole peak quadruplets (AA, MAA = 242 u; AB, MAB = 214 u; BC, MBc = 240 u; CA, MCA = 268 u) and three tripyrrole peak quadruplets (AAB, M- = 335 u; ABC, Mc, = 361 u; CAA, MCAA = 389 u). Preferential retention of the ring-substituted meso carbon on monopyrrole C, if it occurs, would translate into the absence from the spectrum of the MB + 15 species ( m / z 108; two terminal meso carbons on B), the Mc - 13 species ( m / z 134; no terminal meso carbons on C), the MCA - 13 species ( m / z 255; no terminal meso carbons on CA), the MAB + 13 species (m/z 227; two terminal meso carbons on AB), the MMB + 13 species (m/z 348; two terminal meso carbons on AAB), and the MCCA - 13 species (m/z 376; no terminal meso carbons on CAA). The locations of these unexpected peaks are indicated by the dashed lines in Figure 8. Unfortunately, except for the absence of a peak at m / z 376, these predictions cannot be verified or refuted since peaks from other pyrrolic ions fall at these same masses. In addition to the absence of the peak at m/z 376, the MS/MS spectrum in Figure 8 differs from what we predict by the presence of the peaks a t m / z 120, 146, and 160. These peaks, as well as the peaks at m/z 134,148, and 162, can be attributed to monopyrrole C, which contains the exocyclic ring. As discussed above, a peak at m/z 134, corresponding to Mc - 13, is not expected if there is preferential retention of the ring-substituted meso carbon on monopyrrole C. Both the species a t m / z 120 and 134 might, however, be products of exocyclic ring opening followed by losses of ethylene (i.e., (& + 1- 28)’ and (& + 15 - 28)+, respectively). Dehydrogenation of the exocyclic ring might account for the unexpected peaks at m/z 146 and 160 (i.e., (Mc + 1 - 2)’ and (Mc + 15 - 2)+, respectively). Although the peaks that appear to result from dehydrogenation and fragmentation of the exocyclic ring add complexity to the MS/MS spectrum, these ions serve to indicate the presence of an exocyclic ring on the macrocycle and also serve to identify the pyrrole on which the ring is located. Thus, the CI-MS/MS method appears to be useful for pyrrole sequencing of porphyrins with an exocyclic ring. Once the appropriate standards become available, a more comprehensive study of the extent of the

CI-MS and CI-MSIMS for Porphyrin Analysis

Energy & Fuels, Vol. 4 , No. 6,1990 729

M2 124

138 16a 162

* ,

experiment. This information can be acquired from the E1 mass spectrum run before the CI experiments). An ammonia CI mass spectrum of this mixture obtained at a source temperature of 423 K is shown in Figure 9a. This spectrum is quite complex and virtually impossible to interpret as it is a convolution of the CI mass spectra that would be obtained from the individual compounds (see Figures 6b and 7b). As can be seen, however, the protonated porphyrinogen from both porphyrins (i.e., (M, + 7H)+and (M2+ 7H)+) is generated in sufficient abundance to enable the MS/MS spectrum of each to be obtained. The MS/MS spectra of these (M + 7H)+ species in the mixture, which are shown in Figure 9b and 9c, respectively, are virtually identical with the MS/MS spectra of the respective (M + 7H)+ species that were obtained when analyzing the individual compounds (Figures 6b and 7b, respectively). Conclusions

1oh

273

Figure 9. Analysis of a mixture (approximately 1:l w/w) of

free-base octaethylporphyrin (MI, MW = 534 u) and free-base deuteroporphyrin IX dimethyl ester (M2,MW = 538 u). (a) The ammonia CI mass spectrum obtained at a source temperature of 423 K, (b) the MS/MS daughter ion spectrum of (M + 7H)+from octaethylporphyrin ((MI + 7H)+),and (c) the MS/MS daughter ion spectrum of (M + 7H)+from deuteroporphyrin IX dimethyl ester ((M2+ 7H)+). Both MS/MS spectra were acquired at a collision energy of 70 eV. Adapted from ref 31. low-energy-CID fragmentation of porphyrins containing an exocyclic ring can be undertaken. A very significant advantage of the CI-MS/MS technique over CI-MS for pyrrole sequencing porphyrins lies in its capability to provide for pyrrole sequencing of individual porphyrins within a simple mixture of nonisobaric porphyrins. Sequencing of the porphyrins within a porphyrin mixture is not possible from the CI mass spectrum, because the origin of the individual pyrrolic ions observed cannot be specified. The problem is overcome with the CI-MS/MS method by obtaining the daughter ion spectrum of each (M + 7H)+ species produced from the porphyrins in the mixture. This capability is demonstrated by analyzing a 1:l (w/w) mixture of FBOEP (M,, 2) and deuteroporphyrin IX dimethyl ester (M2, 3). (Note: knowledge of the mass of the porphyrins present in the mixture is essential to selecting the appropriate reduced porphyrin species in the CI mass spectrum for the MS/MS

As shown in this paper, the data obtained from highpressure ammonia CI-MS and CI-MS/MS analysis of geoporphyrins is of potential analytical utility to the geochemist. This is particularly true in cases in which ion source parameters are adjusted to promote reduction and decomposition of the porphyrin macrocycle such that the resulting mass spectra provide pyrrole sequence information. While the CI-MS/MS pyrrole sequencing technique we developed expands the capability of mass spectrometry for the structural analysis of porphyrins, further work is still needed to fully exploit the potential mass spectrometry holds in this application. Clearly, additional standards need to be analyzed to obtain a better understanding of the fragmentation of (M 7H)+, especially in the case of porphyrins containing exocyclic rings. While mass spectrometry will not replace NMR for complete structure determination of geoporphyrins, future developments of mass spectrometric methods that provide information regarding the structure and position of the macrocycle substituents, along with the already developed pyrrole sequencing capability, will make mass spectrometry an even more powerful supplement to NMR and, in some applications, a viable and attractive alternative.

+

Acknowledgment. We thank P. S. Clezy (University of New South Wales) for providing a sample of 15,17-butano-3,8-diethyl-2,7,12,18-tetramethyl-21H,23H-porphyrin. Research at ORNL was sponsored by the US. Department of Energy, Office of Basic Energy Sciences, under Contract No., DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc. The UTK Chemistry Mass Spectrometry Center is funded by the Science Alliance, a State of Tennessee Center of Excellence. The NSF Chemical Instrumentation Program also contributed to acquisition of the ZAB-EQ (Grant No. CHE-86-09251).