Fission Fragment Ionization Mass Spectrometry of Chlorophyll a

The title study has been made by using a time-of-flight mass spectrometer, placing primary emphasis on fragmentation reactions and rates. The mass spectrometer was modified so that segmented fields with segments of different magnitudes could be applied in the ion acceleration region and retarding potentials in the region between the flight tube and the ion detector. Methods using these retarding potentials were developed for determining the identities of fragmentations in the flight tube and for measuring the rate constants of fragmentations occurring in the ion acceleration region. Positive and negative spectra were determined with and without retarding potentials at the end of the flight tube. Extensive amounts of fragmentation occur both close to the sample foil (prompt fragmentation) and in the flight tube. For example, 98.9% of the positive molecular ions from chlorophyll a entering the flight tube fragment before reaching the detector. In the spectra with retarding potential applied, M+. and ( M + H)’ ions are the ions in the positive molecule-ion region, and (M H)-, M-., and (M + H)are the ions in the negative molecule-ion region. Networks of fragmentation paths were deduced from retarding potential measurements. Rate constanis for certain fragmentations are deduced from tails on peaks and from measurements utilizing segmented fields in the ion acceleiation region. It is deduced that fragmentations occur with rate constants ranging from >lo9 s-l to lo4 s-’. It is suggested that the fission fragment induced fragmentation processes observed in Chl a involve many of the concepts embodied in the quasi-equilibrium theory of mass spectra, e.g., formation of reactant ions with a wide range of energies, which results in a network of sequential and competing unimolecular reactions with variable and wide-ranging rate constants. This paper is a report of our study of the mass spectra of chlorophyll a (Chl a) obtained with our fission fragment ionization mass spectrometer. Chl a has been resistant to treatment by conventional mass spectrometry (E1 and CI) because of its low volatility and relative chemical fragility, and consequently little is known about the detailed mass spectrometry of this important compound. Recently spectra have been obtained by Hunt, Macfarlane, Katz, and Dougherty’,2 using fission fragment ionization mass spectrometry (referred to by these workers as plasma desorption mass spectrometry) and by Dougherty, Dreifuss, Sphon, and Katz3 using field desorption mass spectrometry. Dougherty has written two pertinent reviews of the mass spectrometry of tetrapyrrole^.^^^ One of the fission fragment mass spectrometry papers’ is concerned mainly with chlorophyll aggregates; the field desorption paper is concerned mainly with chlorophyll hydration. The second fission fragment ionization paper treats some aspects of the ionization and fragmentation of the compound, but it is of limited extent. W e had been using fission fragment mass spectrometry for the analysis of Chl a, and some of the hypotheses advanced in the paper were contrary to our observations. Consequently, we made an extensive study of the fission fragment ionization mass spectrometry of the compound, placing primary emphasis on fragmentation reactions and rates. It is our observation that similarities exist between the spectra produced and the processes involved in the various kinds of impact desorption mass spectrometry (fission fragment ionization, fast-atom bombardment, slow-ion bombardment, and laser desorption) and even conventional mass spectrometry (electron ionization and chemical ionization). Therefore we think that our results will be of interest in types of mass spectrometry other than fission fragment ionization. A preliminary report of our work has been published.6 (1) Hunt, J. E.; Macfarlane, R. D.; Katz, J . J.; Dougherty, R. C. Proc. Nail. Acad. Sci. U.S.A. 1980, 77, 1745. (2) Hunt, J. E.; Macfarlane, R. D.; Katz, J. J.; Dougherty, R. C. J . Am. Chem. SOC. 1981, 103, 6715. (3) Dougherty, R. C.; Dreifuss, P. A,; Sphon, J.; Katz, J. J. J . Am. Chem. SOC. 1980, 102, 416. (4) Dougherty, R. C. In ‘Biochemical Applications of Mass Spectrometry”; Waller, G. R., Ed.; Wiley-Interscience: New York, 1972; pp 591-600. (5) Dougherty, R. C. In ‘Biochemical Applications of Mass Spectrometry”; Waller, G. R., Dermer, 0. C., Eds.; Wiley: New York, 1980; 1st Suuol. Vol., .. pp 693-701. (6) Chait, B. T.; Field, F. H. J . Am. Chem. SOC. 1982, 104, 5519 Experimental Section The mass spectra were obtained with the time-of-flight fission fragment ionization mass spectrometer built in this laboratory and described previously.’ It contains three grids in front of the ion detector, and the purpose of these is to provide a retarding potential for the study of ions produced by fragmentations occurring in the 3-m flight tube of the mass spectrometer.* It also contains three grids in the ion acceleration region. These were used to provide electric fields in the ion accelerating region which were of discontinuously different magnitudes. This in turn enabled us to make measurements of rates of ion fragmentations in the acceleration region. A schematic drawing of the apparatus is given in Figure 1 . The ion acceleration voltage, V,, applied to the sample foil was usually 10 kV. The nominal strength of the 252Cf source was 45 pCi, and this produced a bombarding flux of 3000 fission fragments s-l thru the sample foil. Samples with a surface density of approximately 15 pg/cm2 were prepared by evaporation of Chl a from benzene and CC14 solutions on 1-pm-thick Ni and 2-pm-thick aluminized polyester. Samples of Chl a were obtained from Professor D. C. Mauzerall of The Rockefeller University, the Sigma Chemical Co., St. Louis, MO, and US Biochemical Corp., Cleveland, OH. The spectra from all samples were essentially identical, except for some variation in the pheophytin a (Chl a Mg + 2H) content. The presence of pheophytin a was easily detected from the mass spectra. Solutions were made up in the laboratory atmosphere working as rapidly as possible, and these solution were dropped on the sample foils as rapidly as possible. The solvent was evaporated from the foils by evacuation, and the sample foils thus prepared were rapidly transferred to the mass spectrometer. The fission fragment ionization mass spectra obtained in a time-offlight mass spectrometer exhibit many peaks that are quite broad by conventional mass spectrometric standards, and these peaks often exhibit wings and tails. We pointed out in an earlier works that the broadening of peaks may be ascribed to the Occurrence of fragmentations in the flight tube, and the tails may be ascribed to fragmentations in the ion acceleration region. Fragmentations often entail the conversion of internal energy to kinetic energy of the fragments, and when the fragmentations occur in the flight tube this kinetic energy produces fragment ions with a distribution of velocities and, consequently, of flight times. When the fragmentations occur in the ion acceleration region the conversion of internal energy to kinetic energy is of much less importance than the fact that fragmentations occurring at different places in the strong electric field in the acceleration region will produce ions with differing flight (7) Chait, B. T.; Agosta, W. C.; Field, F. H. In?. J . MassSpectrom. Ion (8) Chait, B. T.; Field, F. H. I n [ . J . Mass Specirom. Ion Phys. 1981, 41 , Phys. 1981, 39, 339.