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Anal. Chem. 1993, 65, 2776-2783
Molecular Beam Spectroscopy of Molecules with Low Volatility via Laser Desorption from Thin Films Containing Particulate Silver R. Tim0 T. Karaiste,t*sIan M.Atkinson,tJ Jeffrey A. Shorterfsl Alan E. W.Knight:)$ and
F.Richard Keene'J Department of Molecular Sciences, James Cook University of North Queensland, Townsville, Queensland 4811, Australia, and Molecular Dynamics Laboratory, Faculty of Science and Technology, Griffith University, Brisbane, Queensland 4111, Australia
A new polymer matrix containing particulate silver has been developed for the introduction of analytes with low or negligible volatility into molecular beams via laser desorption. These silver-containing film matrices (SCFM) permit stable desorption to proceed for extended periods (ca. 10 h) and have been applied successfully to the nondestructive volatilization of a number of amino acids and a transition-metal compound. Analytes can be extracted directly from solution onto the SCFM surface by simply dipping the film into the solution and then air-drying the film. The laser desorption apparatus, when coupled to a supersonic molecular beam/lascr ionization timeof-flight mass spectrometer, permits the detection of the analytes at femtogram levels. Furthermore, we have developed a reel-to-reel tape transport device that allows extended usage of the analyteloaded SFCM, thereby permitting the measurement of wavelength-scanned mass-selected resonance-enhanced multiphoton ionization spectra of analytes with low or negligible volatility. We demonstrate this application with the aromatic amino acids L-tryptophan and L-phenylalanine. The degree of cooling achieved in the molecular expansion is shown to be comparable with that achieved using conventional beam sources, and the desorption yield is sufficiently stable for highquality spectra to be measured.
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INTRODUCTION Supersonic jet methodologies are now the techniques of choice for probing electronic spectroscopy for both analytical and fundamental photophysical studies. The rarefaction and cooling afforded by molecular beams result in spectra which are dramatically simplified and are therefore significantly easier to interpret.' However, most spectroscopic applications of jet expansions have been directed toward relatively volatile molecules that may be introduced conveniently into gaseous flows. Molecules with low or negligible volatility present a greater challenge. While there have been numerous techniques developed for introducing nonvolatile analytes into Authors to whom correspondence should be addressed. +James Cook University of North Queensland. Griffith University. 1 Deceased, April 1991. I Visiting Scholar from Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139. (1)Levy, D.H.Ann. Rev. Phys. Chem. 1980,31,197-225. 0003-2700/93/0365-2776$04.00/0
molecular beams for mass spectrometric analysis, there has yet to be a routine application of jet methods for recording their electronicspectra, especiallyfor thermally fragilespecies of appreciable molecular weight. There is significant need for the development of a general purpose method for generating stable, cold jet expansions of such species in order to permit the measurement of their electronic spectra. The analytical utility of such a technique in biochemistry and pharmacology is obvious, where the extreme sensitivity and selectivity (optical and mass) of the combined mass spectroscopic and laser ionization techniques may be exploited.2J Great potential also exists in inorganic chemistry, where gasphase methodologies have made little impact in photophysical or photochemical studies due to the involatilityof the majority of the compounds of interest. However, high-resolution spectroscopic methods would be of great value in elucidating the photophysics of many potentially important transitionmetal coordination and organometallic species.4~5 Techniques that exist for the introduction of nonvolatile molecules into the gas phase for mass spectral analysis include plasma desorption," fast atom bombardment,7,8electrospray? and supercritical fluid expansions.8 Of these methods, only supercritical fluid expansions appear to have been coupled successfullyto molecular beams for the purpose of measuring jet-cooled spectra.lb12 However, various technical problems, including solvent clustering, limited range of solvents,vacuum pump loading, and signal instability, have restricted the widespread application of the supercritical expansion methods to spectroscopic experiments.8JlJZ Laser ablation methods, wherein a single laser pulse induces evaporation, fragmentation, and ionization of a solid analyte, are used in the study of refractory materials.13J4 More recently, laser desorption (LD)methods have emerged, where the evaporation and ionization steps are accomplished (2)Lubman, D.M. Anal. Chem. 1987,59,31A-40A. (3)Hahn,J. H.;Zenobi, R.; Zare, R. N. J. Am. Chem. SOC.1987,109, 2842-2843. (4)Geoffroy, G. L.; Wrighton, M. S. Organometallic Photochemistry; Academic Press: New York, 1979. (5)Juris, A.;Barigelletti, S.; Campagna, S.; Balzani, V.; Beleer, P.; von Zelewsky, A. Coord. Chem. Rev. 1988,84,85-277. (6)Cotter, R. J. Anal. Chem. 1980,60,781A-793A. (7)Ashcroft, A. E.; Chapman, J. R.; Cottrell,J. S.J. Chromatogr. 1987, 394,15-20. (8) Vestal, M. L. Mass Spectrom. Rev. 1983,2,447-480. (9)Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989.246.64-71. (10)Brennecke, J. F.;Tomasko, D. L.; Peshkin, J.; Eckert, C. A. 2nd. Eng. Chem. Res. 1990,29,1682-1690. (11)Sin, C. H.;Pang, H.M.; Lubman, D. M.; Zorn, J. Anal. Chem. 1986.58.487-491. (12)Sin, C. H:; Linford, M. R.; Goates, S. R. Anal. Chem. 1992,64, 233-236. ~ . .
(13)Vertes, A.; DeWolf, M.; Juhasz, P.; Gijbels, R. Anal. Chem. 1989, 61, 1029-1036.
(14)Gray, A. L. Analyst 1985,110,551-556. 0 1993 American Chemlcai Society
ANALYTICAL CHEMISTRY, VOL. 85, NO. 20, OCTOBER 15, 1993
separately using different laser sources.15 The advantage of the latter methodology is that desorption can be effected “softly”,without significant fragmentation or ionization.Many reports have demonstrated the potential for analytical sensitivity and selectivity of laser desorption, particularly with fragile biomolecules.16 Recently matrix materials have been employed to reduce fragmentation of molecules in the desorption step.17-19 The combination of laser desorption with supersonic jet/ resonance-enhanced multiphoton ionization (REMPI)/timeof-flightmass spectrometry (TOF-MS)to record high-quality wauelength-scanned spectra as opposed to simple mass spectra has been explored in a few a~counts.20-~5 However, much of this work has dealt with molecules which have low rather than negligible vapor pressures. Indeed, often the molecules could be volatilized into a molecular beam by sufficient heating, the desorption process being simply a means of flash heating. Two limiting mechanisms are involved in these experiments: heating of the sample via direct absorption of the desorption laser (e.g., p-aminobenzoic acid desorbed with 248 nm excimer radiation22) and absorption of the laser radiation by a substrate which mediates the heating of the analyte.26 The advantage of the latter approach is that the desorbing laser need not be in electronic resonance with the analyte, therefore potentially reducing photofragmentation or ionization of sample molecules. However, substratemediated heating has been restricted in application due to the difficulties in maintaining a sufficiently stable yield of desorbing molecules for periods long enough to allow reproducible wavelength-scanned spectral measurements to be taken. Several approaches have been applied to the problem of signal reproducibility in desorption experiments, mostly involving sample preparation. Examples include the preparation of thin films on metal blocks and polymers,26 slurry matrices,’Q and materials such as glycero1.n Movement of the sample is advantageous, allowing a clean surface to be exposed to each laser shot, thus reducing sample variability with a commensurate improvement in stability, reproducibility, and experimental duration.24,a Unfortunately difficulties exist with LD in the desorption of metal-containing compounds. Fragmentation of the desorbing species is facile as ligand binding energies are usually quite small. Such fragmentation has been observed for many organometallic compounds in the gas phase.w1 However, the use of an Ag/poly(ethylene oxide) matrix has been found (15) Grotemeyer, J.; Boesl, U.; Walter, K.; Schlag, E. W. Org. Mass Spectrom. 1986,21, 645-653. (16) Grotemeyer,J.; Schlag, E. W. Org. Mass Spectrom. 1988,23,38& 396. (17) Hillenkamp, F.; Karas,M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991,63,1193A-l202A. (18) Beavis, R. C.; Lindner, J.; Grotemeyer, J.; Schlag, E. W. Chem. Phys. Lett. 1988,146,310-314. (19) Beavis, R. C.;Lindner, J.; Grotemeyer,J.; Atkinson, I. M.; Keene, F. R. ; Knight, A. E. W. J. Am. Chem. SOC.1988,110,7534-7535. (20) Imasaka,T.;Tashiro, K.; Ishibashi, N. Anal. Chem. 1989,61,15301533. (21) Meijer,G.;deVries,M.S.;Hunziker,H. E.; Wendt, H. R. J.Chem. Phys. 1990,92, 7625-7635. 122) Meiier. G.:deVries. M. S.:Hunziker. H. E.: Wendt. H. R. J. Phvs. Chem: 1996,94,4394-4396. (23) Cable, J. R.;Tubergen, M. J.; Levy, D. H. J. Am. Chem. SOC.1989, 111,9032-9039. (24) Cable, J. R.; Tubergen, M. J.; Levy, D. H. J . Am. Chem. SOC.1987, 109,6198-6199. (25) Li, L.; Lubman, D. M. Appl. Spectrosc. 1989,43, 543-549. (26) Cable, J. R.; Tubergen, M. J.; Levy, D. H. Faraday D k c w s . Chem. SOC.1988,86, 143-152. (27) Li, L.; Lubman, D. M. Appl. Spectrosc. 1988, 42, 418-424. (28) Cheshnovsky, 0.;Yang, S. H.; Pettiette, C. L.; Craycraft, M. J.; Liu, Y.; Smalley, R. E. Chem. Phys. Lett. 1987,138, 119-124. (29) Leutwyler, S.;Evan,U.;Jortner, J. J . Chem.Phys. 1981,&5,30263029. ’
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to reduce fragmentation of a t least one coordination compound, viz, tris(2,2’-bipyridine)ruthenium(II)([Run(bpy)312+), to the point that large parent mass peaks of the parent complex are present in the LD/REMPI mass spectrum of the molecule.19 We present in this paper a simple methodology involving photographic emulsions containing particulate silver which can be used as matrices for laser desorption/MS, which overcome many of the problems associated with LD experiments, and which may facilitate the routine recording of the jet-cooledspectra of molecules with low or negligible volatility. An underlying principle of this method is that the desorption laser is absorbed predominantly by the optically dense silver particles, which in turn heat the remainder of the matrix (including the analyte). Hence, the desorption of the analyte is decoupled from the primary absorption step, and reduced fragmentation of the analyte results. The methodology we describe also has significant potential for analytical use in that it offers a reliable sampling procedure for introduction of nonvolatile analytes into molecular beams for subsequent spectral or mass spectral quantitation. The aromatic amino acids L-phenylalanine, L-tyrosine,and L-tryptophan were chosen as the target substrates for this study. When conventional thermally-heated molecular beam sources have been used to measure the mass spectra of these species, significant fragmentation has been observed.nJm Nevertheless, despite the thermal lability and low vapor pressure of these amino acids, their jet-cooled electronic spectra have been measured previously using a variety of desorption methods.27~3- The present paper compares mass and electronic spectral results obtained using desorption from the silver-containing film matrices (SCFM) with previous studies. In addition, the desorption of [Ru(bpy)#+, a substance with negligible vapor pressure, is also reported.
EXPERIMENTAL SECTION Preparation of the Desorption Matrix. The flexible silvercontaining film matrix (SCFM) for desorption studies was prepared from a diffusion-transfer film receiver (DTFR)material that is available from several manufacturers (e.g., AGFA copyproof film A4 0,Ol). The DFTR sheetsdo not themselves contain any silver or compounds of silver, our intention being to controllably introduce particulate silver into the film emulsion ourselves. Initially, the sheets of DTFR were conditioned by washing in a running water bath to remove any contamination of sulfur-containingimage dye. Under photographic safetylight, silver bromide crystals were then grown within the film by cycling the sheetsalternatively through baths containing aqueousAgNOs solution (0.1-0.15 M) and aqueous KBr solution (0.5 M). Crystal growth (asmeasured by opticaldensity)was gradual and reached a saturation plateau after -8 cycles. The resultant sheets were exposed to a strong white light and reduced to metallic silver using conventional photographic development (AGFA Rodinal) and air-dried. Water-soluble analytes were introduced into the SCFM by soaking the sheets in a solution containing about 5% by weight analyte, removing the film from the analyte bath, squeezing off the residual solution, and blow-dryingthe film. Experimentally, it was found that the DTFR emulsion became fully swollen on immersion in water for about 1min, the measured weight gain being greatest at pH 12 and dropping to -40% of this value at pH 2. It is estimated that up to about 0.5 g m-2 of analyte was introduced into the SCFM structure. (30) Hossenlopp, J. M.;Rooney, D.;Samoriski, B.; Bowen, G.;Chaiken, J. Chem. Phys. Lett. 1986,116, 380-386. (31) Prinslow, D. A.; Vaida, V. J. Am. Chem. SOC.1987, 109, 50975100. (32) Rizzo, T. R.; Park, Y. D.; Peteanu, L. A.; Levy, D. H. J. Chem. Phys. 1986,84, 2534-2541. (33) Chiarelli, M. P.; Gross, M. L. Anal. Chem. 1989,61, 1895-1900. (34) Martinez, S. J.; Alfano, J. C.; Levy, D. H. J. Mol. Spectrosc. 1992, 156,421-430.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993
Silver Crystals Source chamber Nd:YAG beam
nT ih,
Silver Free Layer
J-
Emuision Film Base
i Acceleration ; grids
Main chamber
detectors Deflection
Flgure 2. Cross section of silver-containing film matrix (SCFM) (not drawn to scale).
TOF-MS
I
Excimer/
dye laser beam
Figure 1. Schematic diagram of experimental configuration.
Each A4 SCFM sheet was cut into 3-mm-widestrips and spliced seamlessly (with motion picture splicing tape) together to form the final desired length of tape ( 10 m). Scanning Electron Microscopy. Scanning electron micrographs were recorded using a JEOL JXA-840 scanning electron microscope. The SCFM samples were Au-sputtered to allow contrasting of the polymer matrix in the SEM image. Electron energies of approximately15kV were used, and no surfacedamage was induced at these voltages. Laser Desorption/TOF-MSApparatus. Figure 1shows a schematic diagram of the experimental arrangement, part of which has been described previo~sly.~~ The tape player unit directed the DTFR film parallel to and 1-3 mm below the axis of a pulsed molecular beam valve (GeneralValve Series 9; 150ps duration, 0.8-mm-diameter orifice, Ar carrier gas at -400 kPa) in the source region of the molecular beam apparatus (workingpressure of 3 X 1VTorr). Laser desorptionwas effected by the 532-nm output of a frequency-doubled Nd:YAG laser operating in Q-switch mode (-2-6 mJ/pulse, 15 ns FWHM) weakly focused onto the SCFM through an 80-mm cylindrical lens. Power densities at the laser focus were on the order of -3 X lo8 W cm-2. The tape was advanced at a rate such that the overlap between bleach spots on the film was between 10 and
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selected subsequently and displayed. Either a wavelengthscanned spectrum for a selected mass range or a mass spectrum for a selectedlaser ionizationwavelength range could be extracted. This collection strategy meant that all mass channels were measured simultaneously in a given wavelength scan, a feature that has considerable advantages in noise reduction through background subtraction. Analogue spectra were taken using a PAR 162/165boxcar and gated integrator with output to a chart recorder. All timing and triggering for the experiment, which was run at 10 Hz, were provided by a custom-built digital delay generator, with 16 independently programmable outputs, pre. ~optimal ~ temporal delay between the cision 5100 p ~ The desorption and ionizing lasers was typically 400-500 ps.
RESULTS AND DISCUSSION
The free jet expansion containingthe desorbed moleculeswas skimmed (2-mm-diameter skimmer orifice; nozzle-skimmer separation 80 mm) and directed through the DC extraction region of a Wiley-McLaren configurationTOF-MS (drift length 850 mm, working pressure -2 X 10-6 Torr). Ionizing radiation was generated by a Lambda Physik excimer laser (EMG 101 MSC, 308 nm) pumping a Lambda Physik FL3002 dye laser. For all experiments described herein, the laser dye was C540A. Output light was frequency-doubled by an angle-tuned KDP crystaland weakly focused (bya 1000-mmlens),and the unfiltered beam was directed anticollinearwith the molecular beam between the extraction plates in the source region of the TOF-MS. The laser output was attenuated using a rotating Glan-Taylor prism polarizer and/or apertures to reduce saturation of optical transitions. Cations produced by (1 + 1) ionization of the sample were extracted (-100 V) and accelerated (-1000 V) in the source region of the TOF and directed down the flight tube toward tandem microchannel plate (MCP) detectors. Any correction for non-normal ion trajectories was achieved by adjustable lowvoltage biases (510 V) appliedto horizontaland vertical deflector plates. Output signal from the dual MCP detectorswas buffered and impedance matched by a 400-MHz video amplifier (NS LH0063) before display on a digital storage oscilloscope (Tektronix 2432). The oscilloscope was interfaced with a microcomputer (MacintoshSE/30)via an IEEE-488 connection. Discrete spectra were recorded by collecting an averaged mass spectrum (16 or 32 shots) for each laser step (typically 0.0005 nm in the UV region). Custom-written graphics interface software%permitted individual mass/wavelength regions of interest to be
Film Structure. The intent of the design of the desorption matrix was to produce a film in which the analyte was dispersed evenly in a layer in which finely divided metal particles (silver)were also present. Water-swellablematerials such as conventional black-and-white photographic film were investigated but were rejected because of instability of the gelatine emulsion and a substantial gelatine-related background present in the LD-generated mass spectra of such materials. The final choice for the matrix host was a commercially available DTFR material which contains a synthetic polymer emulsion layer. (The precise composition of the emulsion layer was considered proprietary information by the manufacturers.) Particulate silver was introduced into the emulsion layer as described above. The exact quantity of silver crystals introduced was difficult to determine due to changes in the equilibrium moisture content of the emulsion layer. However, we estimate that the silver content was in the range of 3 g cm-2. From SEM studies, the silver crystal size range was evaluated to be in the range of 0.1-0.35 pm, never exceeding 0.5 pm. The effective silver crystal size in the emulsion was estimated at about 0.08 pm. (Silver particle size was calculated using the Nutting equation,%which relates the optical density of a silver emulsion with the number of silver particles in an observed area, and estimating that the 2.0-pm-thick emulsion layer is unlikely to contain more than 3 g/m2of silver.) Figure 2 shows schematically the dimensions of the SCFM. If potassium chloride, rather than potassium bromide, is used in the preparation of the matrices, the silver particle size is reduced, never exceeding -0.03 pm. Volatilization of Amino Acids. Mass Spectra. Figure 3 displays the mass spectra of the three aromatic amino acids L-tryptophan, L-phenylalanine, and L-tyrosine, taken using the SCFM/LD technique. All of the spectra were measured using ionization wavelengths coincident with the SI So electronic origin of the molecules concerned, with ionization laser energies of 50 pJ/pulse. No contamination from the DTFR polymer matrix was ever observed in the mass spectra. This indicates that laser powers in the range 510 mJ, while
(35) Bieske, E.J.; Rainbird, M. W.; Atkinson, I.M.; Knight, A. E. W. J . Chem. Phys. 1989,91, 752-61. (36) Butz, K. W. unpublished software. Rock, A. B., Ph.D Thesis, Griffith University, 1993.
(37) Beames, S.,Facultyof Scienceand Technology,Griffith University; information available on request. (38) James, T. H. The Theory of the Photographic Process; Macmillan: New York, 1977.
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50%.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993
a
Ag
(107, 109)
Ag, (214, 216, 218)
4
b
(214, 216,218)
Mass (mlr) Flguro 3. Mass spectrum of amino acMs laserdesorbedfrom SCFM
matrix. (a) L-Tryptophan (desorptbn X = 532 nm, ionization X = 286.755 nm); (b) L-phenylalanine (desorption X = 532 nm, ionization X = 266.084 nm); and (c) L-tyroshe (desorption X = 532 nm, ionization X = 281.350 nm).
still sufficientto induce substantial desorption of the analyte, are insufficient to produce any fragmentation of the DTFR polymer matrix. (No evidence for any DTFR composition products could be found, even when higher ionization laser powers were used (220 &pulse). However, the possibility also exists that DTFR decomposition products were not ionized in the spectral region scanned.) Evident in all of the spectra are the silver masses Ag (107, 109) and A g z (214,216,218). Some variation in the yield of Ag, Agz relative to that of the amino acid is apparent. Partial explanation of the differing AgJamino acid ratios in these spectra lies with the varying absorption cross sections of the amino acid and the different loadings into the SCFM that could be achieved with each analyte. However, the primary explanation lies with the nature of the silver desorption/ ionization. A g z has a rich vibronic spectrum in the wavelength regions spanned in these studies?@vaand as each spectrum is taken at a different ionization wavelength, the magnitude of the Agz signal will correlate with the proximity of the ionizing wavelength to features in the A g z absorption spectrum. (39) Beutel, V.; Bhale, G.L.; Kuhn, M.;Demtrtider, W. Chem. Phys. Lett. 1991,186, 313-318. (40) Willey, K. F.; Cheng, P. Y.; Yeh, C. S.; Robbm, D. L.; Duncan, M. A. J. Chem. Phys.1991,95,6249-6256.
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Laser ablation of Ag/Agz from metallic or crystalline silver has been studied previously414 and may be compared with the laser desorption/molecular beam data reported here. Although there is no a priori reason to exclude the formation of Ag,+ clusters for n > 2 in a laser ablation experiment, only the monomer and dimer species have so far been reported. In accordance with the ablation data,a Agz was the largest silver mass observed in our experiment, the dimer/monomer ratio decreasing with increasing desorption laser power. With appropriate adjustment of backing pressures, nozzle pulse width, and desorption laser intensity, Agz(Ar), clusters of up to n = 20 could be observed. We note in passing that Willey et d.M have measured electronic spectra of AgZAr using one-color REMPI spectroscopy. The spectra reported by these authors match those collected in our own work using SCFM, although the SCFM spectra indicate that the vibrational temperatures for AgzAr are higher than those obtained by Willey et al." The power dependence of the desorption signal for Ag/Agz is not as sharp using the SCFM method as reported for other desorption matrices44 and displays a fairly wide plateau (- 2 mJ/pulse). The observed power dependence profile is relatively flat compared to most other desorption methods, and therefore the desorption yield of SCFM is less sensitive to laser power fluctuations than these other methods.21122 This single feature is a significant advantage of the SCFM approach to desorption for spectroscopicmeasurements, since it is the poor stability of the desorption yield that is the major limiting factor in applying most other desorption methods to measuring wavelength-resolved spectra. There is indication that the kinetic energy distributions of Ag and Agz may be different from those for the amino acids. The temporal profile of the TOF-MS signal for the organic molecules is fairly sharp, corresponding to a packet width of -20 ps in the jet. However, Ag and Agz display a temporal profile -2-3 times as long. Wavelength-Scanned Spectra. A sensitive test of the efficacy of the SCFM method in producing a steady and significant concentration of the desorbed analyte in a molecular beam is the recording of wavelength-scanned spectra. Figure 4a shows the REMPI spectrum of the origin region of tryptophan measured using the SCFM technology. The quality of this spectrum can be contrasted with Figure 4b, which presents the REMPI spectrum of the same molecule recorded by Levy et al.32 using a continuous jet thermal desorption source. (In their original experiments with tryptophan, Levy et al.6 used a thermospray source, but it was later realized that the thermospray simply coated the walls of the channel nozzle from which tryptophan was thermally desorbed.46~47 Some slight instability may exist in such a source because of uneven or discontinuous desorption from the channel walls.) Tryptophan is sufficiently stable to permit the use of a relatively stable thermal source in its volatilization. Comparison of the spectra in Figure 4 establishesthat there is no reduction in spectral quality using our pulsed-laser desorption source compared with the intrinsically stable ~~
(41) Herminghaus, S.; Leiderer, P. Appl. Phys.Lett. 1991,58, 352354. (42) Helvajian, H.; Welle, R.J. Chem. Phys. 1989,91, 2616-2626. (43) Helvajian, H.; Welle, R. P. Mater. Res. SOC.Symp.Proc. 1989, 129,359-364. (44) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Moss. Spectrom. Ion Processes 1987, 78, 63-68. (45) Rizzo, T. R.;Park, Y. D.; Levy, D. H.J. Am. Chem. SOC.1985,107, 277-278. (46) Rizzo, T. R.;Park, Y. D.; Peteanu, L. A.; Levy, D. H. J. Chem. Phys. 1985,83,4819-4820. (47) Park, Y. D.; Rizzo, T. R.;Petaanu, L. A.; Levy, D. H. J . Chem. Phys.1986,84,6539-6549.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15, 1993
1
a
1
of the latter,49 there is indication that some conformers of L-phenylalanine may be generated in the molecular beam with higher vibrational temperatures, perhaps up to 50 K. The position of the tape with respect to the nozzle was found to be important (but not critical) to the cooling achieved in the molecular beam, although the quantity of material entrained varied significantly. In general, the best cooling and beam flux density were obtained when the tape edge was flush with the exit face of the nozzle and located 0.5-1 mm below the axis of the nozzle orifice. With distances less than 0.5mm, beam flux dropped as the beam quality was degraded (presumably due to physical obstruction of the Mach bottle by the tape). Moving the tape to a position further than -3 mm from the nozzle resulted in only 10% of the potential maximum signal obtainable. It was found that moving the tape further from the nozzle along the axis of the molecular beam was very destructive to the molecular beam profile, distances of >2 mm causing drastic signal reductions, presumably because the tape edge interfered with the sheath of the Mach bottle associated with the expansion.* Desorption Signal Strength and Detection Limits. It is an important requirement that the SCFM/LD method to be able to yield a sufficient density of neutral molecules so that reasonableREMPI ion currents can be generated without the need to resort to high-power ionizing lasers. Low ionizing laser powers are advantageous in that saturation of spectral transitions can be minimized in one-color REMPI spectra, although the use of LIF or two-color REMPI would reduce the problem of spectral saturation. Quantification of the desorption flux in the present experiments is difficult because of the many variables involved, and no attempt was made to experimentallycalibrate the necessary instrument-dependent parameters. However, some reasonable approximations may be made. Peak ion signals for L-phenylalaninewere 150 mV with a 20-ns FWHM peak width ( 5 0 4 oscilloscope input, electron multiplication -2 X 1@),which corresponds to -100 ions detected per laser pulse. The ionization efficiency is probably 1% ,Hwhile the fraction of neutral species entrained in the molecular beam passing through the 2-mm skimmer is about 0.176.60 Several factors are not accounted for in this estimation (e.g., losses in the TOF drift region), but the orderof-magnitude estimate of about lo7 neutral molecules being desorbed per pulse is probably close to the real value. Although approximate, this figure reflects the high analyte loadings that can be achieved with SCFM and correlates to a minimum amount of phenylalanine required to produce detectable signalof 1.5 fg. Phenylalanine has an extinction coefficient of about one-sixth that of tyrosine and about onetwentyfifth that of tryptophan, so the detection limits for these analytes are correspondingly lower. Desorption Signal Stability. Several conditions must be satisfied for the successful measuring of wavelengthscanned spectra with accurate, reproducible intensities and band contours. Smallfluctuations of the signal intensity can be tolerated and compensated for with smoothing; however, this increases the time required for a spectral scan and therefore places more emphasis on longer term (Le., several hours) desorption stability. Workers who have previously applied LD to spectral collection have reported pulse-to-pulse variations in the desorption yield of 130% and total scan times limited to 0.5 h or less.21922*% Lubman et al.27have reported the LD of tyrosine analogues from a glycerol matrix
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b
34850
34900
34950
Energy
35000
(c")
Figure 4. (a) REMPI spectrum of L-tryptophan vla laser desorption from SCFM. (b) AEMPI spectrum of L-tryptophan (Levy et aL3* Reproduced wlth permlsslon from the Journal of chemical physics).
continuous thermal desorption source. Temperature and/or different conformer populations may be partly responsible for the difference in some of the relative band intensities between the spectra in Figure 4. Differences in spectral saturation levels may also contribute to the differing intensities.32 The pulsed-LD-derived spectrum has slightly more noise, but also more structure, than the thermal desorptionanalogue. If the LD data are smoothed with a sliding 5-point average, the spectrum is found to display resolution and noise almost identical to that found in the spectrum of L-tryptophan in reference 32. A scan time of -30 min was required to measure the LD spectrum, which means that a considerable amount of data can be collected from a 10-m strip of SCFM tape (translated at -1-1.5 m/h). Molecular Beam Character and Cooling. The extent to which the desorbed molecules are cooled in the supersonic expansion is reflected by the rotational bandwidths of the vibronic transitions in the REMPI spectra. The FWHM peak widths of the bands in the LD-derived spectra of L-tryptophan are -1.3 cm-1, compared with those of the previously reported spectra of 2.5 cm-l.32 This indicates that our SCFM/LD method permits cooling in the expansion at least as dramatic as that observed previously for amino acids such as tryptophan. The rotational temperature estimated from rotational contour analysis48of the origin band in Figure 4a indicates that the beam temperature is no higher than -5 K. From the absence of hot bands in the REMPI spectra of L-tryptophan and phenylal alanine," the vibrational temperature is estimated to be 115 K. However, in the spectrum
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(48) Atkinson, I. M. Ph.D. Thesis, James Cook University of North Queensland, 1992.
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(49) Atkineon, I. M.; Shorter, J. A.; Karaiste, R. T. T.;Keene, F. R.; Knight, A. E. W., manuscript in preparation. (50) Arrowsmith, P.; deVries, M. S.; Hunziker, H. E.; Wendt, H. R. Appl. Phys. B 1988,46, 165-173. (51) Li, L.; Lubman, D. M. Anal. Chem. 1989,61, 1911-1915.
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h
c .v)
c 3
@
5 % c .v)
a
c
0
10
5
15
20
Time (minutes)
Flgure 5. Stability of neutral desorptionyield of L-phenylalanine laser desorbed from SCFM (desorption X = 532 nm, ionizationX = 266.084 nm). J
set on a Macor rod, but, once again, the instability of the desorption yield compromises the quality of the spectrum. Computer averaging may be used to average spectra from several different scans, Le., where the wavelength range for the spectrum is scanned repetitively, rather than taking a single, long scan. This procedure has the advantage of suppressing any noise due to random surface variations of the SCFM and pulse-to-pulse instabilities of the desorption
orption source since the stability of the SCFM technology was quite sufficient for high-quality spectral scans. Pulseto-pulse desorption stability was usually 10%. An additional advantage of the SCFM is that a 532-nm Nd:YAG laser (which is usually considerably more stable than excimer or COasources) can be used for the desorption light source, and the desorption yield of SCFM is less sensitive to laser power fluctuations than direct desorption without matrix mediation (vide supra). Experiments were conducted in which the laser wavelength was tuned to the SI SOorigin transition of phenylalanine and the laser adjusted so that the REMPI signal was saturated, and the neutral desorption yield was monitored under these conditions. Figure 5 illustrates a typical trace of the signal stability observed. These data were taken using a boxcar gated to the mass of L-phenylalanine, and time constants were set to be much more sensitive than would be the case in a wavelength-scannedexperiment, so that smoothing did not unduly influence the trace presented. Figure 6 illustrates the SI SOorigin band of L-phenylalanine scanned successively and repetitively over the sample spectral range. Desorption and ionizing laser powers differed by no more than 5% between any of these runs, which took a total of 90 min to accumulate. No smoothing or averaging has been applied to these data, so as to give a realistic impression of the reproducibility of the SCFM technology. As may be seen, all of the profiles have the same shape, and their integrated intensities vary by no more than &lo%. Although the signal stability from SCFM technology is reasonable, it could be improved. We currently believe that attention should be focused toward removing any surface inhomogeneities that are present in the film structure. Physical Damage to the SCFM. The physical effect of the laser on the SCFM was investigated by subsequent SEM analysis using a series of samples that had endured a single laser pulse. At low laser powers, very little effect was observed on the SCFM surface. Using a 100-mm lens to focus the desorption radiation onto the SCFM and a laser power of 1 X 107 W cm-2, the surface appeared physically intact to the naked eye, with the exception that a brown discoloration was apparent: SEM studies revealed the surface to be peppered with small round holes (-0.2-1.0 pm in diameter) together
I
I
c
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Figure 7. Single shot laser damage spot of SCFM (AgBrderived Ag, desorption laser power -4 X lo7 W cm-*).
with some significantly larger holes (- 10pm) that resembled burst bubbles. The overall impression was that a considerable gas or vapor pressure had developed underneath the surface layer of the SCFM. At higher laser powers (about 1.7 X 107 W cm-2),surface damage was obvious to the naked eye. The higher power damage suggested that a thin layer of the emulsion polymer had expanded as a bubble and then burst, leaving pieces of the surface layer around the damage site (Figure 7). In this case, a large amount of gas or vapor may have been generated underneath the cool transparent surface layer of the SCFM, causing it to stretch and expand until it finally burst, releasing the analyte as a vapor into the stream of supersonic gas sweeping across the SCFM. It was apparent from desorption studies of a stationary SCFM that all of the analyte was desorbed in 5-10 laser pulses. Moreover, the desorption yield from the first laser pulse was not as high as the succeedingfew pulses. This indicated that disruption of the surface layer from the SCFM enhanced the desorption process and explainswhy stronger signals were observed when the tape player was run with some overlap between successive laser shots. Further increases in laser power increased the surface damage. This led to an eventual loss of signal due to fragmentation/ionization of the sample molecules when the
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 20, OCTOBER 15. 1993
The SCFM possesses several attributes that invite further development. Firstly, the emulsion polymer, whilst not completely characterized, is amenable to modification. The degree of ionic cross-linking which binds the emulsion structure may be adjusted over a wide range by simple treatment of the emulsion during the preparation of SCFM.
The moisture content in the emulsion lowers the melting temperature of the polymer, and experiment has shown that this enhances anal@ release. Any moisture inside the SCFM will be rapidly vaporized during the laser pulse, and this expandingvapor may assist in “flushing”the adsorbed analyte into the molecular beam. The analyte is uniformly distributed throughout the silver-containing matrix structure, and it is possible that any analyte that is in intimate contact with the silver crystals becomes thermally fragmented during the desorption. It is probable that several desorptionmechanisms are operative in SCFM-based desorption. It is conceivable that SCFM could be produced that is swellable in solvents other than water, further extending the utility of the matrix. In principle, it is possible to manufacture matrix structures from colloidal silver powder mixed with various polymers. The main advantage envisaged of these materials would be an increased analyte loading and more control over the analyte introduction into the matrix. While these experiments are incomplete,we provide in the following paragraphs a summary of some initial results that are relevant in the present context. One of the aims of our work was to volatilize transitonmetal compounds in preparation for their spectroscopic interrogation via molecular beam techniques. We had demonstrated previously that tris(2,2’-bipyridine)ruthenium was introduced into a molecular beam from a slurry of poly(ethylene oxide) and colloidal Ag or Au using a pulsed C02 laser for desorption.lg However, the yield of parent ion was relatively low ( 4 5% of the total ion signal) due to substantial fragmentation. Our current approach produces greater than 50% parent ion signal. Tris(2,2’-bipyridine)ruthenium(II)acetate was mixed with an aqueous Ag colloid supported with 10% poly(viny1alcohol) (PVA) and applied with a home-built film applicator to a Mylar backing sheet. Inspection of the resulting air-dried matrix by optical and scanning electron microscopy showed that the analyte was bound to the PVA binder, which in turn surrounded the Ag colloid granules to a depth of -100 nm. The matrix had a void volume of 64% and provides a high surface area medium that can be heavily loaded with analyte ( 1% by weight) while simultaneouslyserving as an optically dense medium for laser absorption. The material is flexible, as required for its use on the tape drive (described above). Laser desorption of the coordination compound was achieved using 532-nm irradiation, with subsequent laser ionization at 290 nm, which is sufficient to two-photon-ionize the 2,2’-bipyridine ligand.53 The desorption mass spectrum was dominated by a single broad peak centered at m/z = 570, corresponding to that expected for the singly charged monocation, [Ru(bpy)J+. The spectrum contained no evidence for the fragments [Ru(bpy)~]+(m/z = 414) or [Ru(bpy)]+ (m/z = 258), corresponding to loss of one or two bipyridine fragments from the parent cation. In our previous work,19 substantial fragmentation of the [Ru(bpy)3]”+moiety was observed. When 270-nm irradiation was used as the ionization wavelength, the mass spectrum remained unchanged. No evidence for production of laser ablated ions (desorption and ionization within the desorbing laser pulse) could be found within the sensitivity of our apparatus, indicating that neutral [Ru(bpy)31was the dominant desorbed species. In our previous report,l9 we suggested that fragmentation of the parent complex may be the result of ionization-induced fragmentation of [Ru(bpy)S]. However, we have used similar laser powers for ionization in both sets of experiments, so that the reduced fragmentation reported here is presumably
(52) Ready, J. F. Effects of High Power Laser Radiation; Academic Press: New York,1971.
(53) Barone, V.; Cauletti, C.; Piancastell, M. N.;Ghedini, M.; Toscano, M. J . Phys. Chem. 1991,95,7217-7220.
Flgure 8. Single shot laser damage shot of SCFM (desorption laser power = 1.7 X 1O7 W cm-*). (Left) AgBrderived silver (larger particles), and (right) AgCIderived silver (smaller particles).
onset of ionic desorption/ablation was reached, typically at desorption laser fluences of (5-6) X 107 W cm-2. The exposed layer of silver crystals showed no evidence of melting or fusion, but the crystals were covered with a layer of emulsion polymer adhering to the surface. In between the silver crystals, there were deep crevices and channels leading into the silver-emulsion polymer matrix structure. Surface temperatures of the laser heated film were estimated by assuming that the silver particles were the primary photon absorber, approximating the laser pulse profile as triangular, and applying the model proposed by Read~:5~
where I ( t ) is the incident power as a function of time (W cm-2),Ris the surface reflectivity, k is the thermal conductivity (W cm-l K-l), and D is the thermal diffusivity (cm2s-l) {D= k/pC,, where p is the density of the surface, and C , the heat capacity at constant pressure). Using this equation, calculations of the rise in surface temperature of the SCFM suggest peak values around the Ag particles of -1250 K, with the temperature in the region between Ag granules somewhat less than this value. (Calculation based upon: D A =~ 1.688 cm2 s-l; kAg = 4.154 W cm-l K-l; t o = 16 ns; t, = 18 ns; IO = 1 X 107 W cm2. As the intersilver spaces are not directly heated by the desorption laser, the temperature in these regions may not reach the predicted maximum.) This figure is regarded as only a rough guide to the true peak temperature because of the difficulty in determining the exact thermal parameters of the SCFM material. Smaller silver particles (ca. 300 nm) could be introduced into the DTFR by using AgC1-based precipitations as well as the AgBr process described previously. As illustrated in Figure 8, the laser couples more efficiently with the smaller particles, yielding a larger damage spot. However, better signal stability was observed from the AgBr-based SCFM. Nonetheless, the chloride-based film may have some advantages for specific analytical applications.
FURTHER DEVELOPMENTS
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ANALYTICAL CHEMISTRY, VOL. 65,
a consequence of either the different sample medium or the differences in the characteristics of the desorption laser. The CO2 laser used for desorption in our previous work19 had a relatively long pulse width (- 1-ps spike, 10-ps tail) and a power density of -104 W cm-2. In our current experiments, the temporal width of the desorption signal is -20-40 ps. Hence it is likely that the longer pulse width of the C02 laser could have caused secondary laser-induced fragmentation of [Ru(bpy)~Ifollowing the initial desorption step. This preliminary study demonstrates that the use of polymer-coated colloidal metals, in conjunction with shortpulse laser desorption, is a potentially useful technique for mass spectral study of transition-metal coordination and organometallic compounds. We believe that by appropriate choice of the binding polymer, the technique could be expanded for use in other solvents if required by different solubility characteristics of the analyte. At this stage, while the mass spectral results are encouraging in terms of the
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NO. 20, OCTOBER 15, 1993 2783
nondestructive volatilization of the coordinationcompound, the signal was not sufficiently stable for the measurement of wavelength-scanned spectra.
ACKNOWLEDGMENT The assistance of the staff of the mechanical and electronic workshops within the Faculty of Science and Technology, Griffith University, is gratefully acknowledged. The US.Australian Cooperative Research Program [DITAC (Australia) and the NSF International Division (U.S.A.)] supported the visit of J.A.S. to G.U. Financial assistance from the Australian Research Council is also acknowledged.
RECEIVEDfor review April
12, 1993. Accepted July 7,
1993.'
Abitract published in Advance ACS Abstracts, September 1,1993.
