Energy & Fuels 1993, 7, 411-419
411
Geoporphyrin Analysis Using Electrospray Ionization-Mass Spectrometry+ Gary J. Van Berkel’ Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 -6365
Miguel A. Quinofies and J. Martin E. Quirke Department of Chemistry, Florida International University, Miami, Florida 33199 Received December 22, 1992. Revised Manuscript Received February 11, 1993
The utility of electrospray ionization combined with mass spectrometry (ES-MS) for the analysis of free-base, nickel and vanadyl geoporphyrins is demonstrated. In positive ion mode, free-base alkyl-substituted porphyrins are detected as the protonated molecule, (M + H)+, while the nickel and vanadyl chelates of these same porphyrins are observed as their radical cations, M*+,as is chlorophyll a. Detection of free-base octaethylporphyrin (OEP) using continuous infusion at 1-5 pL/min required sampling as little as 1fmol of material, while at these same flow rates 18fmol of OEP could be detected via flow injection. Detection of 500 fmol of mesoporphyrin IX dimethylester injected is demonstrated using on-line reverse-phase microbore-HPLC at a solvent flow rate of 40 pL/min. ES-MS is shown to be well-suited for geoporphyrin molecular weight and carbon number determination since no fragment ions are produced by this ionization process. Accuracy in determining the relative abundance5 of porphyrins within geoporphyrin mixtures is demonstrated using standard solutions containing known molar ratios of OEP and free-base etioporphyrin-I11 (etio-111)and a total free-base porphyrin mixture isolated from Gilsonite bitumen. On-line separation/mass spectrometric detection of free-base and nickel geoporphyrin mixtures using reverse-phase microbore-HPLC/ES/MS is demonstrated with Gilsonite porphyrins.
Introduction Geoporphyrins,both free-base and metal chelates, are found as complex pseudohomologous series of numerous structural types in sediments, shales, coals, petroleum, and petroleum source rocks.lt2 The potential use of geoporphyrins as biomarkers in fossil fuel exploration and interest in the origin and evolution of geoporphyrins has provided much of the stimulus for determiningthe relative distributions of the different carbon number species in such geological samples as well as the complete structures of the individual porphyrin species present.14 In this regard, mass spectrometry has proven to be one of the more powerful analytical techniques available. Electron ionization-mass spectrometry (ELMS), because of the molecular weight information supplied, has been the most used technique for identification of individual porphyrins and for characterization of the porphyrins within complex porphyrin mixtures.”ll Chemical ionization-mass spectrometry (CI-MS)is also often used because of the ability
* Address
correspondence to this author. Phone: 615-574-1922.
FAX: 615-576-8559. + Research at ORNL was sponsored by the United States Department of Energy, Office of Basic Energy Sciences, under contract DE-ACO5840R21400 with Martin Marietta Energy Systems, Inc. (1) Baker, E. W.; Louda, J. W. In Biological Markers in the Sedimentary Environment;Johns, R. B., Ed.;Elsevier: Amsterdam, 1986;pp
125-225. (2) Filby, R. H.; Van Berkel, G. J. In Metal Complexes in Fossil Fuels;
Filby, R. H.,Branthaver, J. F., Eds.; ACS SymposiumSeries 344;American Chemical Society: Washington, DC, 1987; pp 2-39. (3) Filby, R. H., Branthaver, J. F., Eds. Metal Complexes in Fossil Frcek;ACS Symposium Series 344; American Chemical Society: Washington, DC, 1987. (4) Energy Fuels 1990,4; Special Iseue: ACSSymposiumonPorphyrin Geochemistry-The Quest for Analytical Reliability.
using this technique topyrrole sequence a single porphyrin isomer or the porphyrins in a simple mixture of nonisobaric porphyrins.12-21 However, CI-MS is of limited value for the study of complex mixtures because of the convoluted spectra generated as a result of the extensive fragmentation of each porphyrin in the mixture. Many other ionization ~~
~
(5) Jackson, A. H.; Kenner, G. W.; Smith, K. M.; Alpin, R. T.; Buzikiewicz, H.; Djerassi, C. Tetrahedron 1965,21, 2913-2924. (6) Baker, E. W. J. Am. Chem. SOC.1966,88, 2311-2315. (7) Baker, E. W.; Yen, T. F.; Dickie, J. P.; Rhodes, R. E.; Clark, L. F. J. Am. Chem. SOC.1967,89,3631-3639. (8) Smith, K. M. In Porphyrins and Metalloporphyrins; Smith, K. M., Ed.; Elsevier: Amsterdam, 1975; pp 381-398. (9) Budzikiewicz, H. In The Porphyrins, Vol. IZfi Dolphin D., Ed.; Academic: New York, 1982; pp 395-461. (10) Eglinton, G. In Mass Spectrometry in the Health and Life
Sciences; Burlingame, A. L., Castagnoli, N., Eds.; Elsevier: Amsterdam, 1985; pp 47-64. (11) Gallegos, E. J.; Sundararaman, P. Mass Spectrom. Rev. 1985,4,
55-85. (12) Eglinton, 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. (13) Shaw, G. J.; Eglinton, G.; Quirke, J. M. E. Anal. Chem. 1981,53, 2014-2020. (14) Wolff,G.A.;Chicarelli,M.I.;Shaw,G.J.;Evershed,R.P.;Quirke, J. M. E.; Maxwell, J. R. Tetrahedron 1984,40,3777-3786. (15) Evershed, R. P.; Wolff, G. A.; Shaw, G. J.; Eglinton, G. Org. Mass Spectrom. 1985,20, 445-453. (16) Jiang, X.; Wegmann-Szente, A.; Tolf, B.; Kehres, L. A.; Bunnenberg, E.; Djerassi, C. Tetrahedron Lett. 1984,25,4083-4086. (17) Tolf, B.; Jiang, X.; Wegmann-Szente, A.; Kehres, L. A.; Bunnenberg, E.; Djerassi, C. J. J. Am. Chem. SOC.1986,108, 1363-1374.
(18)Van Berkel, G. J.; Glish, G. L.; McLuckey, S.A.; Tuinman, A. A.
J. Am. Chem. SOC.1989,111,6027-6035.
(19) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Org. Geochem. 1989,14, 203-212. (20) Van Berkel, G. J.; Glish, G. L.; McLuckey, S.A.; Tuinman, A. A. Anal. Chem. 1990,62, 786-793. (21) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A.; Tuinman, A. A. Energy Fuels 1990, 4 , 720-729.
0887-0624/93/2507-0411$04.00/00 1993 American Chemical Society
412 Energy & Fuels,
Vol. 7,No.3, 1993
Van Berkel
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Figure 1. Cross-sectionalview of the electrospray ionization/ion trap mass spectrometer combination. Sample introduction is possible using either continuous infusion, flow injection, or microbore-HPLC. Drawing is not to scale.
techniques,with various degrees of success, have been used for the mass spectrometric analysis of geoporphyrins, including, fast atom bombardment (FAB),22*23 thermospray i o n i z a t i ~ nand , ~ ~252Cf-plaema desorption (PD).25Tandem mass spectrometry (MS/MS) has often been used in conjunction with these ionization techniques to provide detailed information regarding porphyrin structure,1&21,23,24,26-29 A relatively new ionization technique, electrospray provides another means to analyze geoporphyrins by mass spectrometry. Electrospray is an atmospheric pressure ionization method that uses electrical energy to assist the transfer of analyte ions initially present in a liquid phase into the gas phase for analysis by the mass spectrometer. In a typical ES source (Figure 11, highly charged droplets of a solution containing the analyte are dispersed at atmospheric pressure through application of a high potential difference (typically 3-5 kV of the same polarity as the ion of interest) between a stainless steel capillary needle, through which the analyte solution is flowing (from 1 to 40 pL/min), and the atmospheric sampling aperture of the mass spectrometer. In the (22) Castro, A. J.; Van Berkel, G. J.;Doolittle, F. G.; Filby, R. H. Org. Ceochem. 1989,14,193-202. (23) Keely, B. J.; Maxwell, J. R. Energy Fuels 1990,4, 737-741. (24) Eckardt, C. B.; Carter, J. F.; Maxwell, J. R. Energy Fuels 1990, 4,741-747. (25) Wood, K. V.; Bonham, C. C.; Chou, M. M. Energy Fuels 1990,4, 747-748. (26) Johnson, J.V.;Britton,E.D.;Yost,R.A.;Quirke, J.M.E.;Cuesta, L. L. Anal. Chem. 1986,58, 1325-1329. (27) Quirke,J. M. E.;Cuesta, L. L.; Yost, R. A,; Johnson, J. V.; Britton, E. D. O g . Ceochem. 1989,14,43-50. (28) Beato, B. D.; Yost, R. A.; Van Berkel, G. J.;Filby, R. H.; Quirke. J. M. E. Org. Ceochem. 1991,17, 93-105. (29) Concha, M. A.; Quirke, J. M. E.; Beato, B. D.; Yost, R. A,; Mercer, G. E.; Filby, R. H. Chem. Ceol. 1991,91, 153-168. (30) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. K.; Whitehouse, C. M. Science 1989,246,64-71. (31)Fenn, J. B.;Mann, M.; Meng, C. K.; Wong, S. K. Mass Spectrom. Rev. 1990,9, 37-70. (32) Smith, R. D.; Loo, J. A.; Edmonds, C. G.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1990,62,882-899. (33) Huane. E. C.: Wachs.. T.:. Conbov. - . J. J.:. Henion. J. D. Anal. Chem. 1990,62, 713A-725.' (34) Mann, M. O g . Mass Spectrom. 1990,25, 575-587. (35) Smith, R.D.; Loo,J. A.; Light-Wahl, K. J.; Ogonalek-Loo, R. R.; Busman, M.; Udseth, H. R. Mass Spectrom. Rev. 1991,10, 359-451.
positive ion mode of operation, a partial separation of positive and negative ions present in the solution occurs leading to an excess of positive charges on the surface of the liquid at the needle tip. Conversely, in negative ion mode the separation of charge leads to an excessof negative charges in solution at the needle tip. In either case, this excess charge destabilizesthe surface and leads to emission of charged droplets, of the same polarity as the voltage applied to the needle, from the needle tip. Thisis followed by droplet evaporation and finally ion evaporation or desorption to yield gas-phase ions that can be sampled and analyzed by the mass spectrometer. The major focus for the application of ES-MShas been the analysis of high molecular weight biopolymers such as proteins and oligonucleotides. Electrospray ionization is, however, also suited for the determination of certain types of lower molecular weight species that are difficult to volatilize and ionize intact by other methods. Typically, best ES-MS results are obtained with analytas that are ionic in solution (e.g., metal or organic salts) and with analytes that can be ionized in solution via Bronsted or Lewis acid/base chemistry. Compounds of the latter type include, for example, basic amines, carboxylic acids, and polar compounds. In a recent paper,%we demonstrated the general utility of ES-MS for the analysis of a wide variety of free-base porphyrins and metalloporphyrins. The data in that paper indicated that ES ionization offered several potential advantages for geoporphyrin analysis when compared to some of the other ionization techniques currently in use. First of all, because of their nonvolatility, geoporphyrins are generally introduced to the mass spectrometer for analysis by E1 or CI typically via heating from a solids probe. Molecular weight and structural data can be obtained from pure compounds in this manner, but fragmentation, differential volatilities of the porphyrins in the mixture, and condensation of the porphyrins on the walls of the ion source inhibit the precise analysis of mixtures. Fragmentation is reduced in EI, allowing the (36) Van Berke1,G. J.; McLuckey, S. A.; Glieh, G. L. Anal. Chem. 1991, 63,1098-1109.
Geoporphyrin Analysis Using ES-MS
Energy & Fuels, Vol. 7, No. 3, 1993 413
analysis of mixtures, by lowering the electron energy (1214eV) used for i o n i z a t i ~ n However, . ~ ~ ~ ionizationefficiency is reduced resulting in poorer detection limits and the problem of differential volatilities remains. In addition to the volatility problem, mixture analysis is hindered with CI-MS because a plethora of molecular species and fragment ions, the abundances of which depend in a complicated and difficult to reproduce manner on ion source conditions, can be formed.18 While this fragmentation aids pyrrole sequencing of individual porphyrins, it thwarts mixture analysis. With ES ionization, porphyrin ions are transferred from a solution phase to the gas phase via electrically-assisted nebulization and thermal evaporation of the solvent. This means of forming gas-phase ions circumvents the need for sample heating and the problem of differential porphyrin volatilities. Also, in most cases only one molecular species is produced from each porphyrin by ES and no fragment ions are generated. It should be noted, however, that fragment ions can be generated during transport of the ions from atmosphere into the vacuum region of the mass spectrometer. In general, the voltages on the lenses in atmospheric sampling interface region of an ES source can be adjusted so that the molecular species generated by ES are fragmented through energetic ion/molecule collisions in the interface region or adjusted so that fragmentation is avoided.36 Thus, the technique may be used to determine geoporphyrin molecular weights and might possiblybe used to accurately determine the relative abundances of the porphyrins within complex geoporphyrin mixtures. In addition, structural information is available through the use of interface fragmentation. Second, a wide variety of solvents can be sprayed so that the solubility of the porphyrins, which can sometimes limit the applicability of FAB and thermospray to porphyrin analysis, is usually not a problem with ES-MS. And third, ES is readily coupled with any of several separation techniques, including m i ~ r o b o r e - and ~ ~ qpacked ~ capillaryH P L C , ~ Oand V ~ capillary ~ electrophore~is,4~~~ so that online separatiodmass analysis of the analytes of interest is possible with ES ionization. In this paper, we examine the utility of ES-MS for the analysis of geoporphyrin-like standards as well as geoporphyrins isolated from Gilsonite bitumen.46 The ES-MS detection limits, the use of ES-MS for determination of relative porphyrin abundances within geoporphyrin mixtures, and the use of on-line microbore-HPLC/ES/MSfor geoporphyrin analysis are demonstrated and discussed.
Experimental Section
(37)Lee, E. D.; Henion, J. D.; Covey, T. R. J. Microcolumn Sep. 1989, 1, 14-18.
(38)Huang, E.C.; Henion, J. D. J.Am. SOC.Mass Spectrom. 1989,I , 158-165. (39)McLuckey, S.A,; Van Berkel, G. J.; Glish, G. L.; Huang, E. C.; Henion, J. D.Anal. Chem. 1991,63, 375-383. (40)Huang, E.C.; Henion, J. D. Anal. Chem. 1991,63,732-739. (41)Griffin, P.R.;Coffman, J. A.; Hood, L. E.; Yates 111,J. R. Int. J. Mass Spectrom. Ion Processes 1991,111, 131-149. (42)Smith, R.D.;Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988, 60,1948-1952. (43)Lee, E.D.;Muck,W.; Henion, J. D.; Covey, T. R. Biomed. Enuiron. Mass Spectrom. 1989,18, 844-850. (44)Moseley, M. A,; Jorgenson, J. W.; Shabanowitz, J.; Hunt, D. F.; Tomer, K.G. J. Am. SOC.Mass Spectrom. 1991,3,289-300. (45)Tinke, A.P.;Reinhoud, N. J.; Niessen, W. M. A.; Tjaden, V. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 1992,6,560-563. (46)Quirke. J. M. E.; Eglinton, G.; Maxwell, J. R. J . Am. Chem. SOC. 1979,101,7693-7697.
A cross-sectional view of the ES-MS instrumentation used in these studies is shown in Figure 1. The mass spectrometer employed is a modified version of a Finnigan-MAT ion trap mass spectrometer (ITMS) adapted to sample ambient air?' Sample introduction into the system can take place via continuous infusion, flow injection, or an on-line separation method. A more detailed description of this ES/ITMS instrument and its operation can be found e l s e ~ h e r e . ~In~ reference * ~ ~ * ~ to ~ the analysis of geoporphyrins by ES-MS, the type of mass spectrometer used is not of primary importance, except that detection limits that can be obtained with the ion trap might, because of ita ion storage capability, be better than those obtainable with other types of mass analyzers. For the experiments employing continuous infusion,porphyrin solutions were pumped through a short length of 500-pm4.d. Teflon tubing at a rate of 1-5 pL/min with a syringe pump (Harvard Apparatus, Inc., Cambridge, MA) and then through a dome-tipped 120-pm4.d.stainless steel syringe needle. The outlet side of the needle was placed 0.5-1.0 cm from the inlet aperture to the mass spectrometer, and a positive voltage of 3-4 kV was applied to the needle. For flow injection experiments carried out at low flow rates (l-BpL/min), the Harvard Apparatus syringe pump was used to deliver solvent to the needle at a constant rate, through Teflon tubing, to a Rheodyne (Cotati, CA) Model 7520 injector with a 0.5-pL internal sample chamber and then through a short length (ca. 15 cm) of 100-pm4.d.silica capillary (silylated to prevent peak tailing) to which the ES needle was connected. Flow injection experiments at high flow rates (40 pL/min) made use of the dual-syringe solvent delivery system of our microboreHPLC system (see below). Solvent was pumped at 40 pL/min to a Rheodyne (Cotati, CA) Model 7161 injector with a 1.0-pL internal sample chamber. From the injector the solution traveled through a short length (ca. 15 cm) of 127-pm4.d. PEEK tubing that connected via a 1/16-in. to 1/32-in. zero dead volume bulkhead reducing union (Valco, Houston, TX) and standard fittings to a 120-pm4.d. (500 pm 0.d.) dome-tip needle within a pneumatically-assisted ES source. The dome-tipped needle Austin, TX). passed through a 1/16-in. stainless steel tee (SGE, Nitrogen gas, at a backing pressure of 60 psi, for pneumatically assisting the ES nebulization was introduced through the side port of the tee and traveled between the dome-tip needle and a concentric 20 gauge (584 pm i.d) stainless steel tube. The gas exited at the tip of the inner needle which protruded about 500 pm from the end of the 20 gauge tube. Microbore Liquid Chromatography. The microbore-HPLC system consisted of a Model 140A dual-syringe solvent delivery system with a UV/visible detector both from Applied Biosystems, Inc. (Foster City, CA). The only modification to this system was replacement of the standard 250-pL dynamic mixer incorporated in the plumbing system with a 52-pL guard column to ensure adequate mixing of the solvent components for gradients run at flow rates of 40 pL/min. Sample injection was accomplished with a Rheodyne (Cotati, CA) Model 7161 injector with a 1.0-pL internal sample chamber. The microbore-HPLC columns (Keystone Scientific, Bellefonte, PA), outfitted with an appropriate guard column, were connected to the injector via a 3-cm piece of 178-pm4.d.stainless steel tubing. From the column the effluent could be directed either directly to the pneumatically-assisted ES interface or to the UV/visible detector and then onto the ES interface. These connections were made with 127-pm4.d.PEEK tubing. Three different reverse-phase columns were used in this work. Two of these were C-8columns, viz., Hypersil300 Octyl (300A pore size, 5 pm particles, 100 mm X 1mm)) and Keystone Octyl/b (100 8,pore size, 5 pm particles, 100 mm x 1mm)) while the third, Partisil ODs-3(80 8,pore size, 5 pm particles, 250 mm X 1 mm), was a C-18 column.
414 Energy &Fuels, Vol. 7, No. 3, 1993 Samples. All solvents and reagents used in this study were HPLC-grade unless otherwise specified. For actual HPLC work, solvents and reagents were filtered through 0.2-pm membrane filters (Alltech, Deerfield, IL) prior to use. Octaethylporphyrin (OEP), etioporphyrin-I11 (etio-III), and their various metal chelates, along with mesoporphyrin IX dimethyl ester and chlorophyll a , were obtained from commercial suppliers and used without further purification. For analysis by ES-MS porphyrins were typically dissolved in methylene chloride, toluene, or various mixtures of methylene chloride or toluene and methanol with or without the addition of trifluoroacetic acid or acetic acid. (i) Preparation of OEP/Etio-I11 Standard Mixtures. A Mettler AE160 balance, the calibration of which was tested using 5,20,50, and (5 20 50) g precision weights, was used to weigh out samples. A 53.4 mg (0.1 mmol) sample of OEP was weighed by difference into a 1-L volumetric flask and then dissolved in 1 L of HPLC-grade toluene (Fisher Scientific, Fairlawn, NJ). The flask was covered with aluminum foil and magnetically stirred (3 h) to ensure dissolution and mixing. The procedure was repeated with a separate flask using 47.8 mg (0.1 mmol) of etio111;however, A.C.S. certified 1,4-dioxane (Fisher Scientific) was used in place of toluene as solvent. Visiblespectraof the standard solutions, in a 1 cm path length cell, were measured using a HP8452 diode array spectrophotometer with the corresponding neat solvent as the reference blank. The sample cell was rinsed first three times with the appropriate solvent and then twice with the particular sample solution. Molar extinction coefficients were calculated for each of the four major absorbance bands for both porphyrins assuming a concentration of 0.1 mM and found to compare well with published literature values.49 Therefore, the concentration of each stock solution was taken to be 0.1 mM. The standard mixtures of OEP and etio-I11were prepared from these 0.1 mM stock solutions as follows. The appropriate volume of the OEP stock solution (100,200,300,400,500,600,700,800, 900,1000 pL) was transferred to a brown glass vial using a 1000p L syringe. Each of these solutions was diluted to 1000 pL by addition of appropriate volume of the etio-I11 stock solution. This resulted in standards with OEPietio-I11 ratios (molar) of 9/1, 812, 7/3, 6/4, 515, 416, 317, 218, and 119. For ES-MS determination of the OEPietio-I11 ratios, 1.0 mL of methylene chloride was added to each of the vials containing the standard OEPJetio-I11mixtures, the vials were capped, and then each was sonicated for about 5 min to assist porphyrin dissolution. Into separate brown vials, 0.1 mL of each standard was transferred and then diluted to a total volume of 1.0 mL with a solvent comprised of methylene chloride/methanol/trifluoroacetic acid (90/10/0.1% v/v/v). Total concentrationof porphyrin in each vial was 10 pM. (ii) Isolation of Gilsonite Porphyrins. Gilsonite (100 g, Eocene bitumen, Uinta Basin, UT) was dispersed on sand (1kg) by grinding bitumenlsand aliquots with a mortar and pestle. The gray-brown mixture was placed in a column and a mixture of toluene and methanol (1:2 v/v) was percolated through the mixture. The eluants were monitored by visible spectroscopy. Elution was continued until the absorbance band at ca. 550 nm in the visible spectrum was no longer distinguishable from the background. This crude nickel porphyrin concentrate was chromatographed on silica gel (230 mesh) eluting with hexane, methylene chloridelhexane, and methylene chloride. The porphyrins eluted in 20-30% methylene chloride. The nickel porphyrin extracts were combined and rechromatographed on silica TLC plates (20 cm X 20 cm X 1mm, Whatman plates with a preconcentration zone). Two porphyrin containing bands, A, the most polar, and B, were obtained. Each band was rechromatographed on silica TLC plates ((20 cm X 20 cm X 0.25 mm, Whatman plates with a preconcentration zone). Band B was resolved into five fractions the most polar of which was designated B5. An aliquot of the combined nickel porphyrins from the silica column was demetallated by treatment with a solution of
+ +
(49) Smith, K.M., Ed. Porphyrins and Metalloporphyrins; Elsevier: Amsterdam, 1975.
Van Berkel et al. concentrated H2S04/CF3C02H(1/9 v/v) under nitrogen in the cold. The resulting purple solution was washed with hexane to remove hydrocarbon impurities and then methylene chloride was added. This mixture was then washed in turn withwater,aqueous NaHC03, and water before the methylene chloride layer was isolated and evaporated to dryness to give the Gilsonite total free-base porphyrin mixture.
Results and Discussion Geoporphyrin mixtures or single isomeric species isolated from geological samples are often obtained in microgram quantities or less followinglaboriousseparation procedures. As a result, the samples are precious and each analysis carried out on the samples, especially those analyses that are destructive, such as mass spectrometry, should consume the minimum amount of sample yet yield the desired data. The desired data from mass spectrometry is typically molecular weight, from which carbon number and structural type can be determined, and the relative abundances of the various porphyrins within the mixture. Current mass spectrometric methods do not, however, allow determination of relative abundances of isomeric porphyrins as such compounds have the same molecular weight. Prior isolation of the components or an on-line separation is needed to garner such information. Therefore, the major attributes of amass spectrometric technique for geoporphyrin analysis are low detection limits, the production of a single type of molecular species (e.g., M'+ or (M + HI+) for unambiguous determination of molecular weight, and production of little or no fragmentation. The combination of ES ionization and MS provides these attributes. The ES mass spectrum of free-base etioporphyrin-I11 (etio-111) shown in Figure 2a is typical of that for all freebase alkyl-substituted porphyrins which have been analyzed. The only major peak observed at mlz 479 corresponds to (M + HI+.No fragment ions are observed. In contrast to the free-bases, the major molecular species observed for the nickel and vanadyl chelates of this porphyrin is the molecular radical cation, M*+,rather than the protonated molecule. The major ions in the ES mass spectrum of nickel etio-I11 (Figure 2b) are those attributable to the molecular ion at mlz 534 and its isotope peaks. The much lesser abundant ion at mlz 519 (15% of the base peak) corresponds to loss of a methyl group from the macrocycle via &cleavage. This fragmentation results from collision-induced dissociation (CID) of the porphyrin in the atmospheric sampling interface region. Tuning of the lens voltages in this region and tuning of ion injection conditions can either enhance this fragmentation, so as to gain structural information, or virtually eliminate it.36 The major ions in the ES mass spectrum of vanadyl etio-I11 (Figure 2c) are attributable to the molecular ion at mlz 543 and its isotope peaks. A low abundance fragment ion is also observed in this spectrum at mlz 528. This fragmentation can be either enhanced or diminished through adjustment of instrumental parameters as discussed above. Closer inspection of the vanadyl etio-I11 spectrum reveals, however, that the abundance of the peak at mlz 544, nominally the carbon13 isotope peak of the molecular species, is greater than theoretical expected (38 % abundance expected, 54 % observed). This would indicate that a significant portion of the ions at mlz 544 are (M + H)+. This observation is not unexpected as previous work36 has found that molecular species observed for vanadyl
Energy &Fuels, Vol. 7, No. 3, 1993 415
Geoporphyrin Analysis Using ES-MS
892 M+. -'20H38
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100
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300 400 500 600 mh Figure 2. ES mass spectra of (a) etioporphyrin-I11(MW = 478 u), (b) nickel(I1) etioporphyrin-I11 (MW = 534 u), and (c) vanadyl(V=O(II)) etioporphyrin-I11(MW = 543 u) obtained via continuous infusion at 5 pL/min. Each porphyrin was dissolved in and sprayed from a solvent comprising methylene chloride/ methanolltrifluoroacetic acid (10/90/0.1%v/v/v).
porphyrins can depend on the nature of the solvent system from which the porphyrin is sprayed. The actual mechanism of formation of these different molecular species for vanadyl porphyrins in ES has been the subject of a detailed investigation36750 and will not be discussed here. It suffices to say that the ionization of vanadyl porphyrins by ES can involve the competition of several condensedphase mechanisms, viz., charge transfer, protonation, and sodiation (from adventitious sodium), and the nature of the ES solvent system effects both reaction energetics and reactant concentrations. The difference in the behavior of nickel and vanadyl porphyrins almost certainly stems from the chemistry of the vanadyl moiety, but a detailed understanding is not in hand. At present, therefore, these competing ionization phenomena inhibit the ES-MS analysis of unknown vanadyl geoporphyrins because molecular weight determination may be uncertain and because uncertainties may arise in deconvoluting the (50) Van Berkel, G.J.;McLuckey,S. A.;Glish,G. L. Anal. Chem. 1992, 64,1586-1593.
overlapping isotope patterns of porphyrins of different structural types. An additional word of warning is in order for the analysis of metallogeoporphyrins. That is, care must be exercised in choosing a solvent so that demetallation can be avoided or can be taken into account when interpreting results. For example, in the spectrum of zinc(I1) OEP, dissolved in and sprayed from a solvent system comprised of methylene chloride and methanol, only the molecular ion (mlz 595) is observed. When this porphyrin is dissolved in and sprayed from the same solvent system containing trifluoroacetic acid, the protonated free base (mlz 535)) resulting from demetallation of the zinc porphyrin, Le., (M - Zn + 3H)+, is observed as well as the molecular ion of the zinc complex. With time following sample dissolution the extent of demetallation increases such that only free-base porphyrin is eventually observedm36A similar result is obtained with acid labile magnesium porphyrin complexes. However, the ES mass spectrum of chlorophyll a in Figure 3 demonstrates that magnesium(I1) chlorin and porphyrin complexes can be analyzed intact by ESMS when the appropriate solvent (i.e., no acid added) is used in the analysis. In this spectrum the base peak (mlz 892) corresponds to the radical cation of chlorophyll a. The peak at mlz 871 corresponds to (M - Mg 3H)+.The next most abundant peak (mlz 614) results from loss of the phytyl moiety with hydrogen rearrangement from the radical cation. The remaining peak (mlz593) corresponds to loss of the phytyl chain from the free-base complex. Demetallation of chlorophyll a probably occurs in the solution before the spraying process given the known lability of this metal-chlorin complex and the fact that methylene chloride usually contains traces of acid. Loss of the phytyl chain more than likely results from interface f r a g m e n t a t i ~ nand ~ ~in this case could not be eliminated indicating the fragility of this linkage. Nonetheless, the ES mass spectrum of chlorophyll a is quite simple for such a delicate molecule and indicates that other highly functionalized geoporphyrin precursors might be successfully analyzed using this methodology. In previous work36we investigated the detection limits for free-base and nickel OEP using flow injection analysis at flow rates of 1-5 pL/min. In that work, known quantities of porphyrin were injected into the ES solvent stream with the analyte eluting from the ES needle as a peak. For OEP, detection of 18 fmol injected was demonstrated.
+
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416 Energy &Fuels, Vol. 7, No. 3, 1993
10 pmol 13001
(a)
1
I
x4
