Anal. Chem. 1990. 62. 2639-2643
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Week after b i r t h Figure 3. Change of fluoride ion concentration in rat bone wtth age: (0)male; (0)female. I. Fluoride ion in each sample was precisely quantified irrespective of the sample size, showing that the assay data correspond to the whole fluoride present in the bone as the ion. Recovery of Fluoride Ion from Bone. The fluoride ion content in dried rat bone recovered at the specified reaction times (Figure 2) remained constant after more than 2 h at 40 "C with no increment of fluoride ion found after 24 h. This indicated that all of the fluoride in the bone could be recovered by 2 h of reaction time at 40 "C. Bone sample was ignited at 500 "C for 2 h for ashing. The fluoride in the ashes was assayed by the microdiffusion method. The assayed values were compared with those obtained by the text method without incineration. Fluoride in the incinerated femur sample of 23-week-rat (male) was determined to be 80.0 f 0.81 pg/lOO mg (n = 3). The fluoride measured by the text method was 80.9 f 1.00 pg/lOO mg (n = 3). The fluoride values obtained by the two methods showed good agreement. Thus, the fluoride was fully recovered from the bone sample by our microdiffusion analysis method. Fluoride Levels of Various Bones in Rat. Various bones were removed from 11-week-old rats and divided into the
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femur, tibia (including fibula), humerus, skull and the part of lower jaw in skull (including teeth and fangs), and vertebra (from atlas to sixth lumbar vertebra). The first three bones were separated into those from the right and left sides of the body. Table I1 shows that fluoride ion in the bones of five rats were present in the range of 41-54 pg/lOO mg. The amount was slightly lower in the skull. Little or no difference was observed between bones of the right and left sides. Change of Fluoride Ion Content with Rat Age. Femur bone samples were collected from rats of 3 to 33 weeks that had been bred under the same conditions. Figure 3 shows that the fluoride ion content in the femur differed greatly between the young and adult animals, increasing with age. This indicates that fluoride from food intake accumulates in the bone.
ACKNOWLEDGMENT The authors thank Mr. Yuichi Ushioda for his advice and helpful discussions. Registry No. F,16984-48-8. LITERATURE CITED (1) Ikenishi, R.; Kitagawa, T.; Nishiuchi, M.; Takehara, K.; Yamada, H.; Nishino, I.; Umeda, T.; Iwatani, K.; Nakagawa, Y.; Sawai, M.; Yamashita, T. Chem. Pharm. Bull. 1988, 36, 662-669. (2) Ikenishi, R.; Kitagawa, T. Chem. pharm. Bull. 1988, 36, 810-814. (3) Weathereli, J. A. Handbook of Experimental Pharmacology; SpringerVerlag: Berlin, 1966; Vol. 20, Part I,pp 141-172. (4) Smith, G. E. Sci. TofalEnviron. 1985, 43, 41-61. (5) Taves, D. R. Talanfa 1968, 15, 969-974. (6) Sara, R.; Wanninen, E. Talsnta 1975, 22, 1033-1036. (7) Yoshda, M.; Makihara, Y.; Katswa, T. Nihon Kagaku Kaishi 1978, 10, 1375-1379. (8) Charen, J.; Taves, D. R.; Stamm, J. W.; Parkins, F. M. Calcif. Tissue Int. 1979, 27, 95-99. (9) Hattab, F.; Frosteil, G. Acta Odontol. S a n d . 1980, 38, 385-395. (10) Kobayashi, S. Kdtoebeigaku-zasshi 1982, 32, 51-69. (11) Hailsworth, A. S.;Weathereli, J. A. Dtsch. D . Anal. Chem. 1976, 48, 1660-1664. (12) Reddy, J.; Grobler, S. R. J . Clin. Periodontol. 1988, 15, 217-221.
RECEIVED for review April 24,1990. Accepted August 23,1990.
CORRESPONDENCE Photodissociation-Photoionization Mass Spectrometry of n -0ctene Isomers Sir: One long-standing problem in analytical mass spectrometry is distinguishing isomeric structures. Often times, isomeric cations have low energy barriers for rearrangement that cause them to lose memory of the initial ion structure prior to fragmentation. One approach toward overcoming this problem is to probe neutral rather than ionic fragmentation, since the barrier for rearrangement of the neutral precursor is frequently much higher than that of its ionic counterpart (I). We propose a new method for studying neutral decomposition by mass spectrometry, photodissociation-photoionization (PDPI). Neutral molecules are photodissociated in the source region of a time-of-flight mass spectrometer with a pulsed excimer laser beam. After an appropriate time delay, a vacuum ultraviolet beam traverses the same region and softly ionizes the dissociation products and any remaining undissociated parent molecules. The overall process is summarized in Scheme I. As in conventional mass spectrometry, the mass 0003-2700/90/0362-2639$02.50/0
Scheme I
M
u2
>M
+
spectrum contains both the parent ion and various fragment ions. However, unlike conventional mass spectrometry, fragment ions in PDPI result predominantly from neutral rather than ionic decomposition. PDPI should exhibit high sensitivity,since both dissociation 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62,
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Xe/Ar CELL
1 118 AND 193
n
FOCAL POINTS
FLIGHT TUBE
LASER
Y D E T E C T O R DIGITAL OSCILLOSCOPE
I
Flgure 1. PDPI experimental setup.
and ionization occur in the source region and ions are analyzed by a high-transmission time-of-flight mass spectrometer. It should also be widely applicable. Many organic compounds absorb 193-nm radiation, and virtually all absorb at 157 nm. In each case, the photon energy is more than sufficient for bond cleavage. Recent technological advances have allowed reasonable pulse energies of coherent vacuum ultraviolet radiation to be generated from high-power laser systems (2). The most convenient wavelength to use is perhaps 118 nm (10.5 eV), since it can be easily produced by frequency-tripling the third harmonic of a Nd:YAG laser (355 nm) in a phasematched mixture of xenon and argon (3). The photon energy of 118-nm radiation is above yet close to the ionization potentials of most organic compounds and their neutral dissociation products, so fairly soft ionization should be achieved. The advent of coherent vacuum ultraviolet radiation is significant, since the alternative laser-based detection method, multiphoton ionization, is neither as universal nor as soft as single-photon ionization ( 4 ) . Finally, the use of pulsed lasers for PDPI makes it possible to study the kinetics of neutral decomposition by varying the time delay between the two lasers. Other methods have been developed to probe neutral decomposition. Neutralization-reionization mass spectrometry (NRMS) probes neutral decomposition by a multistep process ( I , 5,6). A sample is ionized, and the appropriate parent ion is mass-selected. A neutral species is formed, usually by charge-exchange neutralization, and subsequently characterized by reionization and mass analysis in a second mass analyzer. Another method, charge-remote fragmentation of a closed-shell ion (7, B), approximates neutral decomposition chemistry, since bond cleavage is directed at locations in the ion that are remote from the charge site. Each of these methods can provide enhanced structural information over normal ion fragmentation, but each has its drawback. NRMS is widely applicable but suffers from poor overall efficiency, since so many steps are involved. Charge-remote decomposition is applicable only to closed-shell ions, which have relatively high activation energies for charge-initiated fragmentation. Both methods are able to handle complex mixtures or impure samples, since they are based upon tandem mass spectrometry. In this correspondence, we present a preliminary investigation of the PDPI spectra of 1-,2-, and 4-octene. Olefinic
cations are well-known examples of ions that exhibit facile rearrangement prior to decomposition. This process usually compromises the analytical utility of conventional electron impact or collisionally induced dissociation spectra. For example, the 70-eV electron impact spectra of 2- and 4-octene are virtually indistinguishable, while the spectrum of 1-octene exhibits only minor differences. We show that the PDPI spectra are sufficiently different to allow these compounds to be distinguished. These results demonstrate the feasibility of PDPI as an analytical technique and reveal the potential shortcomings of this method, which must be addressed in the future.
EXPERIMENTAL SECTION Figure 1 shows the experimental setup for PDPI. The sample is introduced into a time-of-flight mass spectrometer by a roomtemperature molecular leak. In the source region, the sample is photodissociated with 193-nmradiation from a Questek (Billerica, MA) Model 2110 excimer laser. After a delay of 1-3 M, the neutral photodissociation products plus any remaining undissociated molecules are photoionized with 118-nm coherent radiation. This radiation is produced by frequency-tripling the third harmonic (355 nm) of a Spectra Physics (Mountain View, CA) DCR-2A Nd:YAG laser in a phase-matched mixture of xenon and argon. The Xe/Ar frequency-tripling cell and the time-of-flight mass spectrometer have been described in detail elsewhere (9). In order to observe only ions produced by 118-nm photoionization, it is necessary to remove those ions produced by 193-nm multiphoton ionization. Normally, the backing plate of the mass spectrometer is held at +3000 V, the first grid is at +2WV, and the flight tube is at ground. For PDPI, the backing plate is held at ground during the photodissociation laser pulse so that the ions formed by multiphoton ionization are accelerated into the plate. After these ions have been removed, the backing plate is pulsed to +3750 V just prior to the photoionization laser pulse. The high-voltage pulse is generated by a Velonex (Santa Clara, CA) Model 350 high-voltage pulse generator with a 1088 plug-in that gives an adjustable pulse from 0.1 to 3 ws and 0 to +5000 V. With the backing plate at +3750 V, ions formed by the photoionization beam are accelerated into the flight tube. These ions are detected by a dual microchannel plate detector (R. M. Jordan Co., Grass Valley, CA). The resulting transient signal is monitored by a Nicolet (Madison, WI) Model 4094 digital oscilloscope with a Model 4180, 200-MHz plug-in. The timing of the high voltage and the laser pulses is controlled by a California Avionics Laboratory, Inc. (Campbell, CA), Model 123 digital delay generator. The flashlamp out-pulse of the
ANALYTICAL CHEMISTRY, VOL. A
Chart I
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Ill
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B x2
2-octene
C%=CH
-C%
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spectra of (A) 1-octene, (9) 2-octene, and (C)
4-octene. A
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2-0ctene-
x2
C x6
4-Octene
I 20
1
I
1
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A
I
L
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I
,
I 80
I
I
I
100
10.5-eV photoionization mass spectra of (A) lsctene, (9) 2-octene, and (C) 4-octene.
Flgure 3.
NdYAG laser, which runs at 10 Hz, triggers the delay generator. After 225.6 ps, the delay generator triggers the excimer laser. The high-voltage pulse is applied to the backing plate 0.500 ps after the excimer laser fires. Finally, the Nd:YAG laser Q-switch is triggered so that the 118-nm pulse occurs ca. 1.850 ps (adjustable) after the excimer laser pulse. Under these conditions, all of the ions produced by the excimer laser are rejected, and the 118-nm pulse occurs after the high voltage has stabilized. 1-Octene (97%), 2-octene (98%), and 4-octene (99%) were obtained from Aldrich (Milwaukee, WI) and used as received.
RESULTS Dissociation Products. Figure 2 shows the PDPI mass spectra of I-, 2-, and 4-octene. Figure 3 shows the 10.5-eV photoionization spectra taken by blocking the excimer laser. It is clear from the 10.5-eV spectra that ionization of the parent
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CH2--CH2-CH2-CH2-CH3
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CH,-CH,+H,-CHICH-CH&CH,-
Flgure 2. PDPI mass
DECEMBER 1,
C H ~
molecules yields relatively weak fragmentation and that the fragment ion current increases substantially when the excimer laser is turned on. The three PDPI spectra can be readily distinguished by the relative abundances of the fragment ions, most notably m / z 41, 54, and 55. m f z 41 is enhanced in 1-octene, m / z 54 is enhanced in 4-octene, and m / z 55 is enhanced in 2-octene. This result is noteworthy because these ions correspond to @-cleavagefragments of the three parent molecules, as shown in Chart I. Photochemical studies of small linear alkenes show that @-cleavageis by far the most common fragmentation path (10). 4-Octene is particularly interesting because it has two potential @-cleavagesites. A single @-cleavagegives C6Hll* ( n / z 83) and CzH5. (m/z 29). (Mass-to-charge ratios are for the corresponding ion.) Very little ion current is seen at m / z 83. However, a strong signal is observed at m/z 54, which corresponds to @-cleavageat bath ends of the molecule, as shown in Chart I. In the 1-octene and 2-octene PDPI spectra, only the resonantly stabilized, unsaturated, @-cleavagefragments have high relative abundances. The saturated alkyl radicals apparently undergo secondary fragmentation. Secondary fragmentation is evident in the PDPI spectra. Very few high-mass ions are seen, except for those produced by direct 118-nm photoionization of the undissociated parent molecule. 1-Octene undergoes @-cleavageto form C3H5*(mlz 41) and C5H11. ( m / z 71). C3H5' is resonantly stabilized and has a high relative abundance. CsHIi' is not stabilized and is not observed in the PDPI spectrum. The ion current observed in the m/z 70-100 region can be attributed to 10.5-eV photoionization of the parent molecule, since the peak shapes and relative abundances are identical with those in Figure 3A. Apparently C a l l . undergoes secondary fragmentation, either by neutral or ionic pathways. The nonstabilized @-cleavage fragment from 2-octene, C4H9*( m / z 57), is also missing from the corresponding PDPI spectrum and must undergo secondary fragmentation. Another indication of secondary fragmentation is the high abundance of low-mass fragment ions. In all three PDPI spectra, strong peaks are seen for CzH3' ( m / z 271, CzH4*(mlz 281, and CzH5*( m / z 29), all of which correspond to likely radical decomposition products from the nonstabilized @-cleavagefragment. It is also interesting that C3H5'( m / z 41) and its probable decomposition product, C3H3' (m/z 39),are seen in all three PDPI spectra. Only 1-octene can directly form C3H5*. 2-Octene and 4-octene both must rearrange and/or undergo secondary fragmentation to form these ions. Finally, it should be noted that none of the PDPI fragment ions appear to be metastably broadened, which indicates that secondary fragmentation caused by the photoionization step must be occurring on a time scale of less than 1 ps.
ANALYTICAL CHEMISTRY, VOL. 62, NO.
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11
delay
h
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Figure 4. PDPI mass spectra of l-octene with delay time of (A) 1.650 and (B) 1.850 ps.
ps
Dissociation Kinetics. By varying the delay time between the photodissociation and photoionization pulses, it is possible to determine how the relative abundances of different fragments vary with time. Unfortunately, MPI ions produced by the photodissociation beam must be removed, so measurements are currently limited to the microsecond time scale. Neutral dissociation processes that occur much faster than this will be complete by the time the photoionization pulse arrives. To illustrate this capability, PDPI spectra of 1-octene,taken with different time delays between the photodissociation and photoionization laser pulses, are shown in Figure 4. Relative to mlz 41, the abundance of mlz 39, which corresponds to 6-cleavage followed by loss of H,,changes dramatically with delay time. With a delay of 1.650 ps, m / z 39 is 36% as abundant as mlz 41. When the delay is increased to 1.850 ps, the relative abundance of m / z 39 increases to 46% of mlz 41. Assuming first-order kinetics, these spectra indicate a rate constant on the order of 7 x lo5 s-l for formation of mlz 39 relative to mlz 41. Of course a precise determination of this value would require additional data points. DISCUSSION Secondary Fragmentation and Ion Formation. The fragment ions observed in PDPI mass spectra can in principle be formed by several pathways. Since our goal is to exclusively probe primary fragmentation of the neutral molecule, secondary processes must be understood and controlled. In these initial experiments, secondary fragmentation is possible by spontaneous decomposition after absorption of a single 193-nm photon, by absorption of additional 193-nm photons, by the 118-nm photoionization step, or by absorption of 355-nm photons either before or after photoionization. A single 193-nm (6.5 eV) photon has more than enough energy to break a C-C bond (3.6 eV typical), so spontaneous secondary fragmentation is possible. In principle, spontaneous secondary fragmentation can be reduced by using a less energetic photon for dissociation or by performing the dissociation step in a high-pressure region so that the primary products are collisionally stabilized. These approaches will be pursued in future work. Absorption of a second 193-nm photon is an unlikely cause of secondary fragmentation in these experiments. When the power of the excimer laser is varied, the abundances of the PDPI fragments change with respect to the molecular ion but not with respect to each other. If some of the fragments were caused by absorption of a second 193-nm photon, their relative abundances would be expected to exhibit a strong power dependence.
It is also unlikely that 355-nm radiation causes much fragmentation. The refractive index of the MgFz lens, which is used to focus the 118-nm radiation, changes significantly between 118 and 355 nm. As a result, the 355-nm beam is not focused until 162 mm past the 118-nm focal point. This, in addition to the doughnut profile of the 355-nm beam, means that the 355-nm flux is relatively low at the ll&nm focal point. Previous work using 118-nm photoionization did, however, find some 355-nm effects (9). In future experiments, the two beams will be physically separated, eliminating this possibility. Secondary fragmentation can arise from the photoionization step. Furthermore, ion current in the PDPI spectrum can be masked by ionic fragmentation of the parent ion created by 118-nm photoionization. This latter problem is particularly difficult in the 1-octene spectrum and is expected to be a problem with other aliphatic compounds as well (9). By varying the wavelength of the ionizing radiation, it should be possible to reduce ionic decomposition of the parent ion and determine if some of the fragmentation observed in PDPI is caused by excess energy during ionization. A variety of mixing schemes makes it possible to generate coherent radiation throughout the vacuum ultraviolet region (2). Future work will incorporate tunable vacuum ultraviolet radiation to reduce the extent of ionic decomposition. Finally, one must consider the possibility that adsorption of a 193-nm photon merely activates the parent molecule so that more extensive ionic decomposition accompanies the photoionization step. Although this process cannot be eliminated a priori, it is an unlikely contributor in these experiments given the near-unity total quantum yield for dissociation and the short excited molecule lifetime (
