Anal. Chem. 1986, 58,890-894
LITERATURE CITED Martinsen, D. P. Appl. Spectrosc. 1981, 3 5 , 255-266. Rozett, R. W.; Petersen, E. McLaughlin Anal. Chem. 1975, 4 7 , 1301-1306. Justice, J. B.; Isenhour, T. L. Anal. Chem. 1975, 47, 2286-2266. Rozett, R. W.; Petersen, E. McLaughlin Anal. Chem. 1978, 48, 617-625. Ritter, G. L.; Lowry, S. R.; Isenhour, T. L. Anal. Chem. 1978, 48, 591-595. Malinowski, E. R. Anal. Chim. Acta 1982, 134, 129-137. Williams, S. S.; Lam, R. B.; Isenhour, T. L. Anal. Chem. 1983, 55, 11 17-1 121. Hardy, J.; Jardine, I.I n “Advances In Mass Spectrometry”; West, A. R., Ed.; Applied Science Publishers: Barking, England, 1974; Vol. VI, pp 1061-1071. Heller, S. R.; Chang, C. L.; Chu, K. C. Anal. Chem. 1974, 4 6 , 951-952. Ziemer, J. N.; Perone, S. P.; Caprloll, R. M.; Selfert, W. E. Anal. Chem. 1979, 5 1 , 1732-1738. Lowry, S. R.; Isenhour, T. L.; Justice, J. B.; McLafferty, F. W.; Dayringer, H. E.; Venkataraghavan, R. Anal. Chem. 1977, 4 9 , 1720-1722. McGiII, J. R.; Kowalski, 8. R. J . Chem. I n f . Compuf. Sci. 1978, 18, 52-55. Justice, J. B.; Isenhour, T. L. Anal. Chem. 1974, 46, 223-226. Lam, T. F.; Wilklns, C. L.; Brunner, T. R.; Soltzberg, L. J.; Kaberllne, S. L. Anal. Chem. 1978, 4 8 , 1768-1774. Wold, S.; Christie, H. J. Anal. Chlm. Acta 1984, 165, 51-59. Wangen, L. E.; Woodward, W. S.;Isenhour, T. L. Anal. Chem. 1971, 43, 1605-1614. Van Marlen, G.; Dijkstra, A.; van’t Klooster, H. A. Anal. Chem. 1979, 5 1 , 420-423. Van Marlen, G.; Dijkstra, A. Anal. Chem. 1976, 4 8 , 595-598. Van Marlen, G.; Dijkstra, A,; van’t Kiooster, H. A. Anal. Chim. Acta 1979, 112, 233-243. Van Marlen, G.; van den Hende, J. H. Anal. Chim. Acta 1979, 112, 143-150.
(21) Dupuis, P. F.; Dijkstra, A.; van der Maas, J. H. 2.Anal. Chem. 1978, 29 1 ,. -27-33. .
(22) Dupuls, P. F.; Dijkstra, A. Z . Anal. Chem. 1978, 290, 357-368. (23) Bink, J. C. W. G.; van’t Klooster, H. A. Anal. Chim. Acta 1983, 150, 53-59. (24) Dupuis, P. F.; Cleij, P.; van’t Klooster, H. A.; Dijkstra, A. Anal. Chlm. Acta 1979, 112, 63-93. (25) Heite, F. H.; Dupuis, P. F.; van’t Klooster, H. A. Anal. Chim. Acta 1978, 103, 313-321. (26) Dromey, R. G. Anal. Chem. 1978, 48, 1464-1469. (27) Varmuza, K. Anal. Chim. Acta 1980, 122, 227-240. (28) Albano, C.; Dunn, W. J., 111; Edlund, U.; Johansson, E.; Norden, B.; Sjostrom, M.; Wold, S. Anal. Chlm. Acta 1978, 290, 429-443. (29) Wold, S. Pattern Recognit. 1978, 8 , 127-134. (30) Wold, S.; Albano, C.; Dunn, W. J., 111; Edlund, U.; Esbensen, K.; Geladi, P.; Hellberg, S.; Johansson, E.; Lindberg, W.; Sjostrom, M. I n “Chemometrlcs, Mathematics and Statistics in Chemistry”; Kowalskl, B. R., Ed.; D. Reidei Publishlng Co.: Boston, MA, 1964; pp 17-96. (31) Wold, S. Technometrics 1978, 2 0 , 127. (32) Woodruff, H. B.; Lowry, S. R.; Ritter, G. L.; Isenhour, T. L. Anal. Chem. 1975, 47, 2027-2030. (33) Grotch, S. L. Anal. Chem. 1970, 42, 1214-1222. (34) Pesyna, G. M.; McLafferty, F. W.; Venkataraghavan, R.; Dayringer, H. E. Anal. Chem. 1975, 47, 1161-1164.
RECEIVED for review October 7,1985. Accepted December 9, 1985. Although the research described in this article has been funded by the United States Environmental Protection Agency, it has not been subjected to Agency review. It therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred. The mention of trade names or commercial products does not constitute endorsement or recommendation for use.
Photodissociation of Ions Generated by Soft Ionization Techniques Michael J. Welch,* Robert Sams, and Edward White V Center for Analytical Chemistry, National Bureau of Standards, Gaithersburg, Maryland 20899
Photodissociation wlth vlslble light of Ions generated by the
soft Ionization techniques ceslum ion bombardment, fleld desorption, and fleld lonlratlon has been demonstrated. An argon Ion laser was used to irradiate ions In the flrst fleld-free region of a Mattauch-Herrog geometry mass spectrometer. Ions that dlssoclated In this region were detected by means of a linked scan at a constant ratlo of the magnetlc field to the electrlc fleld. Detection of only those ions created by photodlssoclationwas accomplished by use of a lock-in amplifier trlggered by a mechanlcal chopper In the laser beam. A variety of compounds were tested for evldence of photodissociation activity. Only compounds with chromophores capable of absorbing visible llght were found to photodlssoclate. Several peptides tested showed no photodissoclation actlvlty. The photodlssoclationsof (M H)+ Ions from methyl red and bilirubin are used to illustrate the potential of the technlque for providlng structural Information. For methyl red the photodlssoclatlon mass spectrum exhlblts several prominent Ions that are very weak or absent In the normal mass spectrum. Improvements, Including use of shorter wavelengths, wlli be needed before thls technlque can become a useful tool for structure determinations.
+
Photodissociation has frequently been applied as a tool for studying the structure of gaseous ions and the thermody-
namics and kinetics of gaseous ion processes ( I ) . Most of the studies involved ions consisting of only a few atoms. Ion cyclotron resonance instruments (ICR) and sector instruments equipped with lasers have been used to study the fragmentation of somewhat more complex ions generated by electron impact ionization (EI). These studies have been limited to compounds of low molecular weight, generally less than 200, and low polarity, which are amenable to ionization by EI. They have also been directed, for the most part, to determining the structure of the ions generated rather than to the development of a means for the determination of the structures of organic molecules. E1 mass spectra show extensive fragmentation which provides considerable information on structure. Soft ionization techniques such as chemical ionization (CI), field ionization (FI), field desorption (FD), fast atom bombardment (FAB), and secondary ion mass spectrometry (SIMS) often give little fragmentation, and therefore, the spectra from these techniques contain less information on structure. These last three techniques are generally the methods of choice for obtaining mass spectral information on polar molecules for which E1 is not suitable. Fragmentation can be induced by addition of energy to the ions after their formation through collisions with neutral species. Collision induced dissociation (CID), both at low energies in quadrupole mass spectrometers and high energies in sector mass spectrometers, has been extensively used as a tool for structure determinations. An alternative way to add energy to ions thereby inducing fragmentation is through the absorption of
This article not subject to US. Copyright. Published 1986 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
4
V
L2
(),;TE:/
a>
LOCK-IN AtlPLlFlER
i G O N ION:;"'
1
T W O PEN RECORDER
1I ~
B '
MAGNET
EM
51
52
Flgure 1. Schematic of photodissociation instrumentation, illustrating the two laser paths used: A, for CIB; B, for FI and FD. M is a mirror, L, and L, are cylindrical lenses, W is the Brewster window, FFFR is the first field-free region, S, is the source slit, S2 is the first energy resolving slit, ESA is the electrostatic analyzer, and EM is the electron
multiplier.
photons. Photodissociation has potential advantages over CID in that energy will be absorbed only if the ion absorbs at the wavelength chosen for irradiation and that the amount of energy added can be more carefully controlled allowing for greater selectivity in the fragmentations induced. Preliminary reports on the photodissociation of ions generated by soft ionization techniques have recently appeared. These include the following: the work of Bowers et al. (2), who reported the photodissociation of some small peptides ionized by chemical ionization (CI) and dissociated in an ICR instrument by short wavelength UV light from an excimer laser; a study of the photodissociation of n-butylbenzene, which included the use of FI, reported by Welch et al. (3);and a study of the photodissociation of metal chelates and porphyrins, some of which were ionized by FAB, reported by Fukuda and Campana (4). These papers and the present work demonstrate that sufficient energy can be added by photons to induce dissociations even for ions with low average internal energies. We have studied the photodissociation of ions generated by field ionization (FI), field desorption (FD), and cesium ion bombardment (CIB) and dissociated in the first field-free region (FFFR) of a Mattauch-Herzog geometry mass spectrometer. An argon ion laser with continuous output at several wavelengths in the visible and near-ultraviolet (near-UV) was used as the light source. Two light paths were used, one of which is similar to an arrangement described by Harris et al. (5) where the light passed through the ion source and traveled collinearly with the ion beam through the FFFR. The second pathway involved a multipass mirror arrangement installed in the FFFR to allow the laser beam to interact with the ion beam without passing through the source. In order to investigate the potential of the photodissociation of ions as a technique for the determination of the structure of compounds of biological and biochemical interest, a variety of compounds were tested for evidence of photodissociation by visible light after ionization using a soft ionization technique.
EXPERIMENTAL SECTION A schematic of the experimental setup is shown in Figure 1. The mass spectrometer was a Varian MAT 731 double focusing mass spectrometer of Mattauch-Herzog geometry with a FFFR of about 90 cm and equipped with a combination FI, FD, E1 ion source. The laser was an argon ion laser capable of producing 10 wavelengths of visible light with intensities of 11W and several ultraviolet lines centered around 357 nm with a total intensity of up to 1.5 W. For this work the line at 514.5 nm with up to 8 W intensity and a visible range that included the lines from 454.5-514.5 nm with a total intensity of up to 18W output were used. The detection of those processes induced by the laser light
891
was accomplished by use of a mechanical chopper in the laser beam typically operated at 1 kHz, and a lock-in amplifier that extracted the signal in-phase with the light from the ion intensity signal. Ions formed in the FFFR from a given parent ion were selectively detected by using a linked scan at a constant ratio of the magnetic field (B) to the electric field (E). In this mode, B is chosen such that the selected parent ion is the only ion generated in the ion source that is transmitted. When the B/E ratio i s kept constant as the magnetic field is varied, ions formed by decompositions of the selected ion in the FFFR are transmitted. For compounds exhibiting strong photodissociation activity, complete scans were made to determine all of the photon-induced daughters from a given parent ion. When photodissociation appeared to be weak or nonexistent, likely daughter ions were selected and monitored for several minutes using long time constants (signal averaging) in the lock-in amplifier. As a further test for activity, the parent ions were monitored for loss of intensity with laser light. Optics. Two means of overlapping the laser beam with the ion beam were implemented. These are shown in Figure 1. In the first arrangement laser light was introduced through a quartz window in the FD port of a combination FI, FD, E1 ion source and passed collinearly with the ion beam through the ion source, the source slit, and the FFFR. A pair of cylindrical lenses in the laser beam path external to the mass spectrometer changed the laser beam cross section from circular to a narrow vertical elliptical shape. These lenses were positioned such that the beam was focused at the source slit to maximize the light passing through to the FFFR. The laser beam cross section at this point was approximately 5 to 6 mm high and 0.8 to 0.9 mm wide, corresponding to an average power density of about 450 W/cm2 for a laser output of 18 W. However, the intensity across the laser beam was not uniform, with most of the light concentrated in a central region, 0.2-0.3 mm wide. The light and ion beams passing into the FFFR were restricted by slits to a width of approximately 0.3 mm and a height of 4 mm at a resolution of 1000. The divergence of the laser beam in the FFFR and uncertainty about the fraction of the light passing through the slit and about the distribution of detectable photodissociation in the FFFR prevented an accurate determination of the power density in the FFFR. In the second arrangementthe flight tube containing the FFFR was replaced with a stainless steel box into which a multipass mirror device was installed. A brief description of the optics of this device follows: A quartz Brewster angle window in the top of the box allowed the laser light to enter the front of the FFFR. The light then struck a 45' mirror which directed it a low angle across the ion beam to a right angle mirror mounted in a stainless steel mirror mount near the end of the FFFR. The mirrors used were prisms with all surfaces aluminized to prevent charge buildup from the ion beam. The laser beam was reflected up to an identical arrangement which directed it back across the ion beam to the front of the device, where two more mirrors, identically configured, directed the beam back for another figure-eight round trip across the ion beam. The front and back mirrors were 66 cm apart and could be tilted and adjusted up and down; the gap between the rear mirrors was about 6 mm while the front mirrors were about 13 mm apart to allow light from the first mirror to pass. The front mirrors were located about 19 cm from the source slit and laser beam-ion beam interaction volume was calculated to be about 31 cm long, beginning 37 cm from the source slit. For three round trips of the laser beam the interaction length was about 187 cm. In operation three round trips were realizable with a fourth partially completed before the laser beam was too large to have useful interaction. Only the multipass mirror arrangement was used for ions generated by FI or FD, since the FI/FD probe was inserted through the port in which the window for the collinear arrangment was installed. Optimizing this arrangement was difficult since the five mirrors of the multipass assembly had to be adjusted with the FFFR box open. Once the system was under vacuum the only adjustment possible was of the one external mirror. Either mirror arrangement could be used for ions generated by cesium ion bombardment. Initial experiments were with the multipass arrangement, but, with the modifications described
892
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
Table I. Photodissociation of Ions Generated by Field Ionization"
:c s + ......
I ASf K 1 IGHT
.
.
.
:
.
*...
.
compound
................ 0
0
0
...............
.,. ....
disparent sociates ion
n-butylbenzene
+
nitrobenzene 3,5-dinitrobenzoicacid
+
Mt.
product ion(s)
(M - NO,)+
'Linked B/E scans; 514.5-nm light, 8 W. € x trac t ion
Plates
I
focusing Lenses I
('FF-AXI"
[RllBF
Figure 2. Diagram of off-axis probe construction and position in the
ion source. below, the collinear arrangement was found to provide greater interaction and could be adjusted with the system under vacuum, and was therefore used for all further work. The mirrors were adjusted until maximum light was seen on the first energy resolving slit, located at the end of the FFFR. Fine adjustments of the mirrors to obtain the maximum photodissociation signal were made using the photodissociation of the molecular ion of n-butylbenzene, generated by EI, to form the ion a t m / z 91. Field Ionization and Field Desorption. FI and FD were accomplished by using high-temperature carbon emitters and an accelerating voltage of 8 kV. For FI, the source was typically at 150 OC, the counter electrode voltage was -2 to -4 kV, and samples were introduced through the reference inlet. For FD, the counter electrode voltage was 0 to -3 kV and the ion source was generally kept at 90-100 "C. Samples were dissolved in an appropriate solvent and loaded on the emitter wire by dipping. The emitter was heated to provide as steady a total ion current as possible. Cesium ion Bombardment and Off-Axis Probe. The cesium ion gun was mounted in the inlet flange intended for a GC/MS interface and aimed at the direct probe inlet on the opposite side of the ion source. The gun voltage could be set from 0 to +5.5 kV above the source potential and the gun had two focusing lenses for the Cst beam. For this work the gun was operated at 4-5.5 kV, and the ion source temperature was 50-70 "C. The target assembly used is shown in Figure 2. It consisted of three pieces press fitted together: a stainless steel tube, a nylon insulator, and a stainless steel target. The assembly fit through the vacuum lock normally used for the direct probe. One end of the insulator was inserted into the end of the tube. A rubber O-ring provided a vacuum-tight seal. The rest of the insulator extended 27 mm out of the rod and had an 8 mm deep hole drilled in the other end for attachment of the target piece. The target consisted of a 7 mm long, 4 mm 0.d. section, which fit in the insulator, a 7.7 mm long, 6.4 mm 0.d. center section, and a 13.4 mm long, 3.1 mm o.d. section, which passed through an existing entrance in the ionization chamber. The target piece had a hole drilled in from the end inserted in the nylon, which joined a hole in the side of the thick section to allow pumping of the hole in the nylon. The end of the target piece was machined to provide a target surface with a 30" angle of incidence to the cesium ion beam (6). The target piece was maintained at source potential by spring-loaded contact with the ionization chamber. Maximum sensitivity was obtained when the target surface was in the center of the ionization chamber. While the multipass mirror arrangement could be used with this alignment, the collinear arrangement could not because the target blocked the path of the laser beam through the source. In order for the collinear arrangement to be used, the target was moved off-axis,out of the laser path, as is shown in Figure 2. The
end of the target was removed to allow the center of the target surface to be closer to the center of the ionization chamber. The ion beam intensity that could be obtained when operating with the target off-axis was improved by replacing the standard single piece extraction plate which has a 1mm ion aperture with two separately controlled half plates as are found on the EI-only ion source. The gap between the plates was widened to 1.1mm. With this arrangement the target could be moved out of the path of the laser beam and reasonable sensitivity,about 50% of the signal intensity observed at the optimum position, obtained. Samples were dissolved in glycerol, if possible. If the material did not dissolve in glycerol, it was dissolved in an appropriate solvent or an acid or base was added t o convert the material t o an ionic form and then this solution was mixed with glycerol. Best results for bilirubin were obtained when it was dissolved in triethanolamine and applied to the target surface after application of a coating of glacial acetic acid. Collision-Induced Dissociation. A collision-induceddissociation spectrum of methyl red ionized by CIB was generated by leaking air into the FFFR until the intensity of (M + H)+ was reduced by 50% and performing a linked B/E scan. R E S U L T S AND DISCUSSION Field Ionization. Three relatively volatile and low molecule weight compounds were ionized by FI and the molecular ions tested for photodissociation. The ion intensities produced by FI were much weaker and the signal more variable than with EI; thus the detection of photodissociation effects was more difficult. The results are listed in Table I. For n-butylbenzene, the ratio of the intensities of mlz 91 and 92 formed by photodissociation of M+. as a function of photon energy was not significantly different from that observed using E1 ionization and the same instrument (7), despite the expected differences in internal energy of ions generated by the two techniques. In general, the ions observed to form by photodissociation also are observed to form spontaneously, at a lower level, in the absence of light. One ion observed only during irradiation was that formed by the loss of NOz from the molecular ion of 3,5-dinitrobenzoic acid. These results demonstrated that ions formed by soft ionization techniques could be given sufficient energy by absorption of visible photons to dissociate. Field Desorption and Cesium Ion Bombardment. A variety of moderate-sized organic and organometallic compounds were tested for photodissociation using FD and CIB for ionization. The FD results and the CIB results are shown in Tables I1 and 111, respectively. I t is clear from the results that only compounds with chromophores absorbing in the visible range are likely to photodissociate. The peptides and amino acids tested showed no evidence of photodissociating in visible light. Compounds ionized by both FD and CIB generally exhibited the same effects with the exception of a few compounds for which photodissociation was observed when CIB was used but not when FD was used. The differences seen may have been the result of the stronger, more stable, and longer lasting parent ion intensities from CIB, which made the relatively weak photodissociation signal easier to detect. Use of the 18-W visible band (as was done for most of the CIB work) rather than individual wavelengths (as was done for all of the FD work) also improved detection of weak
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986 COOH
Table 11. Photodissociation of Ions Generated by Field Desorption'
methyl red Sudan IV metanil yellow (Na salt) methyl orange (Na salt) ethyl orange (Na salt) neutral red nile blue cresol red crystal violet quinoxalinedithiol adenosine leucine enkephalin silver methanesulfonate silver trifluoromethanesulfonate bilirubin 2,3,5 triphenyl-2Htetrazolium chloride
I
parent ion
product ion
M+. M+. M'., (M + Na)' (M + Na)+
(M - 121)'. (M - 157)'
compound
893
(M + Na)+
C+b C+ M+. C+ M.' M.'
(M- 80)'.
200
150
250
mi2
Figure 3. Photodissociation spectrum of methyl red with linked B/E scan from (M 4- H)' and 18 W of visible light.
(M + H)+ Ag&+, Ag&+ Ag3X2+
of electron-impact ionized n-butylbenzene gave an intensity for the photofragment at m / z 92 of about 0.1% of the parent ion intensity. The strongest photofragments from CIB ionized ions may approach this relative intensity. Nevertheless, much weaker ion intensities have acceptable signal-to-noise ratios with the detection system used here. As an illustrated of information that can be obtained when the photodissociation is sufficiently intense, the photodissociation spectrum of the (M H)' ion of methyl red (2-[[4(dimethylamino)phenyl]azo]benzoic acid), ionized by CIB,is shown in Figure 3 and possible neutral losses are listed in Table IV. These losses are taken from high resolution measurements of fragment ions formed in the ion source. This compound, with a chromophore that has an intense absorption in the visible region, exhibited excellent photodissociation activity, particularly when ionized by CIB. Many of the photon-induced metastables are either absent or very weak in the normal (no light) metastable (B/E)spectrum. The
M+*
C+
'Linked B/E scans; 514.5-nm Light, 8 W. bC' = cation.
+
photodissociation signals. Previous work (3, 7,8) using E1 and FI suggests that, a t least over the visible range available with an argon ion laser, the absorption bands that induce ions to dissociate are broad, although the realtive intensities of fragmentations that occur are known to be wavelength dependent. The intensities of the daughter ions formed by photodissociation are weak and, because of the phase-sensitive detection and amplification, difficult to relate to parent ion intensity. However, measurements of the photodissociation
Table 111. Photodissociation of Ions Generated by Cs+ Bombardment" compound
dissociates
methyl red neutral red crystal violet adenosine adenosine monophosphate L-tryptophan phenylalanine, methyl ester trialanine, methyl ester Phe-Leu-Glu-Ile leucine enkephalin @-casomorphin hexa-L-tyrosine glutathione Mg tetratolylporphyrin Rh tetraphenylporphyrin Hztetraphenylporphine bilirubin cesium iodide riboflavin folic acid cobalamin bisbenzimide rhodamine B benzoxanthene yellow coomassie brilliant blue [Cu(slppy)lNO$ [Ni(slppy)I N03f
+ + +
-
-
-
-
-
+ + + + + + +
+ + + + +
+
parent ion(s)
product ion(s)
(M + H)+, M+., (M - H)+
see Table IV (M - 15)+,(M - 16)' (M - 16)'
(M + H)+ (M - Cl)' (M + H)' (M + H)+ Cs413+, Cs312+,Cs2I+ G+,cCsJ+ (M + H)+ (M + Nax)+d
(M - 91)+, (M - 106)' (M - 77)' (M - 77)' see Figure 4
C+b ' C M'., (M + H)' (M + H)+ (M + H)' (M t H)' (M + H)+ (M + H)+ (M + H)+ (M + H)+ (M + H)+ (M + H)+
' C
(M + H)+
C+ (M + H)+ (M + Na)+
C+ C+
(M - 133)+,(M -119)+ not tested' not tested (M - 70)+, (M -28)+, +others (C-44)+, (C- 30)', +others not tested not tested (C - 134)' + others (C - 121)+,(C - 27)' (C- 160)' + others
'Linked B/E scans; 457.9-514.5 nm Light, 18 W. C ' = cation. G = glycerol. X = 0-5. ONot tested indicates that no daughter ions were tested for formation by photodissociation, but parent ions were found to photodissociate. fCopper(l+), and nickel(l+), [2-[[[3-[(Zpyridinylrnethylene)amino]propyl]imino]methyl]phenolato-N,N',N",O]-, nitrate.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
894
Table IV. C o m p a r i s o n s of Dissociations of Methyl R e d (M H)+Ion from Cs+ B o m b a r d m e n t '
+
m/z
relative intensity of dissociationsb spontaneous collisional photo-
252 236 224 209 196 181 153 148
100 1
3 1 1
22
100 1 2 1 1 2 1 6
100 1 18
37 22
15 11 21
possible losses
from (M + H)+c
HzO HzO, CHI HzO, CO HzO, CHZ-NCH, HZO, CO, Nz HzO, CO, NP, CHSd HzO, CO, Nz, CHZ=NCHB CsH&OzH
'Linked B/E scan; 457.9-514.5 nm light, 18 W. Intensities of the ion at m / z 252 were arbitrarily set to 100. The intensities of the other ions are relative to these. cBased upon high-resolution measurements of ions formed in the source. dIons also observed for losses of HzO and CH2=NCH, with CO or N2
Flgure 4. Bilirubin structure illustrating photon-induced cleavages observed with visible light.
collision-induced dissociation spectrum exhibited increases in all of the metastables over the background spectrum. These two spectra are similar, evidence that the background metastable level is a t least partially a result of collisions (Table IV). Photodissociation was also observed when M+. or (M H)+was used as the parent ion. Because resolution in the B / E scans is not sufficient to clearly distinguish the daugther ions of (M H)+from those of M+. or (M - H )', some of the ions listed in Table IV may come, at least partially, from dissociations of these latter parent ions. Many of the fragment ions are formed by successive losses of small neutrals and would be useful for structure elucidation. While most molecules of biological interest that we have examined do not photodissociate using visible light, those with suitable chromophores such as the porphyrins, some vitamins, and bilirubin do. Cobalamin (vitamin BIZ)is the largest molecule we have photodissociated, thus far. Unfortunately, the cation mass ( m / z 1329) is above the present mass limit of our B / E linked scan unit, preventing the detection of daughter ions at this time. The fragmentations observed for bilirubin are shown in Figure 4. All of the metastables observed in these cases can also be found in normal B / E scans, but in view of the known ability of the photodissociation technique to discriminate between isomers that have identical electron impact spectra ($11) and in some cases also identical collisional activation spectra (12), we believe that further investigation is warranted as a means of distinguishing isomeric structures for compounds requiring the use of soft ionization techniques. We are investigating photodissociation as a means of distinguishing bilirubin isomers. The purpose of this work was to determine if photodissociation of ions with low average internal energy could be observed, to understand what kinds of substances were most likely to photodissociate, and to see if the fragmentations induced would contain useful information for determining the structure of the original molecule. These goals have been achieved, but both theoretical and practical problems must be solved before this technique can become a useful structural
+
tool. The physical and chemical nature of the interaction of photons with large ions must be better understood. If sector instruments are to be used for structural determinations by photodissociation, the interaction between ions and photons must be significantly increased. In ICR cells, the ion-photon interactions are greater because of the long residence times. The difficulty lies in getting large, thermally labile ions into the cell, although progress on this problem has been reported (13-15). The use of excimer lasers, or other light sources that produce short wavelength UV, should considerably extend the range of compounds that can be photodissociated.
ACKNOWLEDGMENT The authors thank John Travis, Stanley Meiselman, Richard Christensen, and Carl Barry for their assistance in planning and constructing the instrumental modifications, and Reinhold Pesch for his advice conerning the off-axis probe. Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Registry No. [Cu(slppy)]NO,,100333-85-5;[Ni(slppy)]NO,, 100333-87-7; n-butylbenzene, 104-51-8;nitrobenzene, 98-95-3; 3,5-dinitrobenzoicacid, 99-34-3; methyl red, 493-52-7;Sudan IV, 85-83-6; metanil yellow, 587-98-4; methyl orange (sodium salt), 547-58-0; ethyl orange (sodium salt), 62758-12-7; neutral red, 553-24-2;nile blue, 2381-85-3;cresol red, 1733-12-6;crystal violet, 548-62-9;quinoxalinedithiol, 1199-03-7;adenosine, 58-61-7;leucine enhephalin, 58822-25-6;silver methanesulfonate,2386-52-9 silver trifluoromethanesulfonate, 2923-28-6;bilirubin, 635-65-4;2,3,5triphenyl-2H-tetrazolium chloride, 298-96-4; adenosine monophosphate, 61-19-8;L-tryptophan,73-22-3;phenylalanine methyl ester, 2577-90-4; trialanine methyl ester, 30802-27-8; Phe-LeuGlu-Ile, 100333-88-8;P-casomorphin, 79805-24-6hexa-L-tyrosine, 6934-38-9;glutathione, 70-18-8; Mg tetratolylporphyrin, 9192846-0; Rh tetraphenylporphyrin, 38856-19-8; H2tetraphenylporphine, 917-23-7;cesium iodide, 7789-17-5;riboflavin, 83-88-5; folic acid, 59-30-3;cobalamin, 13408-78-1;bisbenzimide, 2349145-4; rhodamine B, 81-88-9;benzoxanthene, 76723-61-0;coomassie brilliant blue, 74434-20-1.
LITERATURE CITED Dunbar, R. C. In "Gas Phase Ion Chemistry"; Bowers, M. T., Ed.; Academic Press: New York, 1979; Vol. 2, Chapter 14. Bowers, W. D.; Delbert, S.; Hunter, R. L.; McIver, R. T. J . Am. Chern. SOC. 1984, 106,7288-7289. Welch, M. J.; Hertz, H. S.; Sams, R. L.; White, E., V Presented at the 32nd Annual Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, May 27-June 1, 1984. Fukuda, E. K.; Campana, J. E. Presented at the 1985 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, LA, Feb 25 to Mar 1, 1985. Harris, F. M.; Mukhtar, E. S.; Griffiths, I. W.; Beynon, J. H. Proc. R. SOC. London, A 1981,374, 461-473. Martin, S . A,; Costello, C. E.; Biemann, K. Anal. Chem. 1982,5 4 , 2362-2366. Welch, M. J.; Pereles, D. J.; White, E., V Org. Mass Spectrom. 1985, 2 0 , 425-426. Mukhtar, E. S.; Griffiths, I. W.; Harris, F. M.; Beynon, J. H. I n t . J . Mass Specfrom. Ion Phys. 1981,3 7 , 159-166. Mukhtar, E. S.; Griffiths, I. W.; Harris, F. M.; Beynon, J. H. Org. Mass Spectrom. 1981, 16, 51. Weger, E.; Wagner-Redeker, W.; Levsen, K. Int. J . Mass Spectrom. Ion Phys. 1983,47, 77-80. Collins, G. J.; Kiss, K.; Pereles, D. J.; White, E., V, submltted for publication. Waaner-Redeker. W.; Levsen, K. Org. Mass Spectrom. 1981, 16, 53c-541. Hunt, D. F.; Shabanowitz, J.; McIver, R . T., Jr.; Hunter, R. L.; Syka. J. E. P. Anal. Chem. 1985,5 7 , 765-768. McIver. R. T.. Jr.: Hunter, R. L.; Bowers, W. D. I n t . J . Mass Spectrom. ion Phis. 1985, 6 4 , 67-77. Hunt, D. F.; Shabanowitz, J.; Yates, J. R., 111, Mclver, R. T., Jr.: Hunter, R. L.; Syka, J. E. P.; Amy, J. Anal. Chem. 1985, 5 7 , 2728-2733.
RECEIVED for review September 30,1985. Accepted December 2, 1985.
