Anal. Chem. 2005, 77, 4378-4384
Preparative Separation of Mixtures by Mass Spectrometry Philip S. Mayer,† Frantisˇek Turecˇek,*,† Hak-No Lee,†,§ Adi A. Scheidemann,†,⊥ Terry N. Olney,†,| Frank Schumacher,† Petr S ˇ trop,‡ Martin Smrcˇina,‡ Marcel Pa´tek,‡ and Daniel Schirlin‡
Department of Chemistry, University of Washington, BG-10, Seattle, Washington 98195, and Selectide-Aventis, Tucson, Arizona 85737
A specially designed mass spectrometer which allows for preparative separation of mixtures is described. This mass spectrometer allows for large ion currents, on the order of nanoamperes, to be produced by electrospray and transmitted into a high vacuum. Accumulation of nanomole quantities of collected and recovered material in several hours is demonstrated. The use of high-velocity ions reduces space charge effects at high ion currents. Separation of mass occurs simultaneously for all ions, providing a 100% duty cycle. The use of a linear dispersion magnet avoids compression at higher m/z ratios. A deceleration lens slows the ions to allow for soft landing at low kinetic energy. The ions are neutralized by ion pairing on an oxidized metal surface. Retractable landing plates allow for easy removal of the separated components. Mass spectrometers, in addition to their analytic uses, have also been used in preparative separation of components of mixtures. Large-scale magnetic sector mass spectrometers, called Calutrons, were used in the Manhattan Project to separate the 235U isotope from mixtures of naturally occurring uranium isotopes containing primarily the 238U isotope.1 Since the Calutron separated atomic ions which cannot fragment, the ions were landed and discharged at high (keV) kinetic energies. Such an approach is unsuitable for separation and isolation of organic ions, which will fragment if landed on a solid target at high kinetic energies. There have been several recent efforts that used ion soft landing on various solid or liquid substrates. Soft landing2 refers to the nondestructive landing of a gas-phase ion on a target in a way that it can be retrieved and reanalyzed or used otherwise. Soft landing is not required for identification of an ion in a conventional mass spectrometer, but it is essential for recovery of an intact molecule and is essential when dealing with compounds of biological interest. Busch and co-workers used massselected organic sulfonium ions that were generated by fast-atom †
University of Washington. Selectide-Aventis. § Current address: Pacific Northwest National Laboratory, Richland, WA. ⊥ Current address: Intelligent Ion, Inc., Seattle, WA. | Current address: Thermo Electron, San Jose, CA. (1) (a) Smith, L. P.; Parkins, W. E.; Forrester, A. T. Phys. Rev. 1947, 72, 9891002. (b) Yergey, A. L.; Yergey, A. K. J. Am. Soc. Mass Spectrom. 1997, 8, 943-953. (2) Franchetti, V.; Solka, B. H.; Baitinger, W. E.; Amy, J. W.; Cooks, R. G. Int. J. Mass Spectrom. Ion Phys. 1977, 23, 29-35. ‡
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bombardment and, following deceleration, successfully landed on a metal plate.3 Siuzdak and co-workers used electrospray ionization to produce ionized virus particles in the gas phase that were transported through a quadrupole mass spectrometer that was operated in the radio frequency-only mode, and the ions were nondestructively landed.4 Smith and co-workers isolated a negatively charged DNA fragment in an ion-cyclotron resonance cell and soft-landed it on a nitrocellulose membrane.5 The identity of the soft-landed DNA was established after PCR amplification. Recently, Cooks and co-workers used quadrupole mass filter6 and linear quadrupole ion trap mass spectrometers7 for isolation of multiply charged protein ions that were soft-landed in a liquid matrix consisting of fructose and glycerol.8 An exciting feature of these experiments is that even relatively labile proteins, such as trypsin, retained most of their enzymatic activity after ionization, mass selection, and soft landing. Concurrently, soft landing of large polysaccharide9 and protein10 ions on plasma-treated dry metal surfaces has been shown to yield intact biomolecules that retained their biological activity after having been washed into solution and tested by standard assays. Perhaps the most exciting finding in these studies was that by adjusting the ion kinetic energy, large biomolecules (hyaluronic acid of 420 kDA and trypsin of 16 kDa) can be immobilized on the plasma-treated metal surface while retaining biological activity.9 The previous pioneering work has established the feasibility of ion soft landing for isolation of organic molecules. However, practical applications of soft-landing mass spectrometry as a separation method face several problems. First, the amount of the soft-landed material is limited due to inefficiencies in the ionization of the starting material, ion transport into and through the vacuum system of the mass spectrometer, ion discharge upon landing in the solid or liquid phase, and isolation of the soft-landed (3) Geiger, R. J.; Melnyk, M. C.; Busch, K. L.; Barlett, M. G. Int. J. Mass Spectrom. 1999, 182/183, 415-422. (4) Siuzdak, G.; Bothner, B.; Yeager, M.; Brugidou, C.; Fauquet, C. M.; Hoey, K.; Chang, C. Chem. Biol. 1996, 5, 45. (5) Feng, B.; Wunschel, D. S.; Masselon, C. D.; Pasa-Tolic, L.; Smith, R. D. J. Am. Chem. Soc. 1999, 121, 8961-8962. (6) Ouyang, Z.; Takats, Z.; Blake, T. A.; Gologan, B.; Guymon, A. J.; Wiseman, J. M.; Oliver, J. C.; Davisson, V. J.; Cooks, R. G. Science 2003, 301, 13511354. (7) Gologan, B.; Takats, Z.; Alvarez, J.; Wiseman, J. M.; Tallaty, N.; Ouyang, Z.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2004, 15, 1874-1884. (8) Morozov, V. N.; Morozova, T. Ya. Anal. Chem. 1999, 71, 3110-3117. (9) Kitching, K. J.; Lee, H.-N.; Elam, W. T.; Turecˇek, F.; Ratner, B. D.; Johnston, E. E.; MacGregor, H.; Miller, R. J. Rev. Sci. Instrum. 2003, 74, 4832-4839. (10) Volny, M.; Elam, W. T.; Branca, A.; Ratner, B. D.; Turecek, F. Proc. 52nd Conf. Mass Spectrom. Allied Top., Nashville, TN, May 2004. 10.1021/ac050444j CCC: $30.25
© 2005 American Chemical Society Published on Web 05/18/2005
material. Furthermore, the mass spectrometers used so far for soft-landing experiments were single-channel mass filters or traps that allowed only one component at the time to be isolated and deposited. Thus, the duty cycle of soft landing was low, and depositing more than one component required extended time and moving the position of the target. Our approach to preparative mass spectrometry addresses some of these problems. We focus on the separation of medium-sized organic molecules in the molecular mass range of m/z 200-600. This encompasses the majority of synthetic drugs and drug candidates that are routinely screened for biological activity. The screening assays often encounter the problem of false positives, whereby an unknown impurity simulates biological activity that does not belong to the desired compound. Another, perhaps even more serious, problem arises when the target compound contains an unknown component that is cytotoxic and invalidates the whole assay. Purification by chromatography of a small amount of synthetic product can be problematic because of contamination and limited separation ability of the column. For example, the components of a mixture produced from combinatorial libraries typically have very similar polarities, since they are derived from the same scaffold with the same functional groups. However, the components of the mixture would have different masses, since they would have different alkyl groups or other nonpolar substituents added onto the carbon skeleton. A second goal of this study was to achieve high yields in ionization, ion transmission through the mass spectrometer, and multiplex separation by mass of several, ideally many, components that are soft-landed simultaneously on a solid support from which they can be retrieved by simple washing with solvent. These goals required a new approach to the design and construction of a special mass spectrometer that is described in this article. We chose confocal imaging ion optics of a modified Mattauch-Herzog geometry that disperses ions by their mass-to-charge ratios such that they exit the magnetic field along a focal plane. In this way, all ions within the mass range of the analyzer are separated and collected simultaneously, providing a multichannel advantage over scanning mass analyzers based on magnetic sectors, quadrupole filters, or ion traps. EXPERIMENTAL SECTION Instrument Design. A specially built mass spectrometer11 was used (Figures 1 and 2). The instrument consists of an electrospray ionizer,12 an electrodynamic ion funnel lens,13 a radio frequency octopole ion guide, an electrostatic acceleration lens, an electrostatic sector, an inhomogeneous-field magnet analyzer, an ion bender, a multichannel deceleration lens, and a multichannel array collector. The vacuum system consists of the main housing, the magnet flight tube, and the collector housing. The main housing is made of a welded aluminum box that is separated by two (11) Turecek F.; Scheidemann, A. A.; Olney, T. N.; Schumacher, F. J.; Smrcina, M.; Strop, P.; Patek, M.; Schirlin, D. Preparative Separation of Mixtures by Mass Spectrometry; U.S. Patent 6,750,448 B2, June 15, 2004. (12) Seymour, J. L.; Syrstad, E. A.; Langley, C. C.; Turecˇek, F. Int. J. Mass Spectrom. 2003, 228, 687-702. (13) (a) Shaffer, S. A.; Tang, K.; Anderson, G. A.; Prior, D. C.; Udseth, H. R.; Smith, R. D. Rapid Commun. Mass Spectrom. 1997, 11, 1813-1817. (b) Shaffer, S. A.; Prior, D. C.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1998, 70, 4111-4119. (c) Shaffer, S. A.; Tolmachev, A.; Prior, D. C.; Anderson, G. A.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1999, 71, 2957-2964.
Figure 1. Layout diagram of the preparative mass spectrometer. A, syringe pump; B, electrospray needle; C, glass-lined transfer capillary; D, funnel lens; E, octopole; F, acceleration lens; G, electrostatic sector with shunts; H, movable slit mounted on a linear motion feedthrough; I, Faraday cup ion collector mounted on a linear motion feedthrough; and J, HV-floated Faraday cages.
bulkheads with conductance limits into three differentially pumped chambers. Another stage of differential pumping is applied in the collector housing. Ion Formation and Transport. Ions are produced at atmospheric pressure by standard electrospray ionization14 from a stainless steel capillary that was tapered to 20° at the tip.12 A high positive potential (3-4 kV from a Bertan HV power supply) is applied to the needle. The ions are transported to the first differentially pumped vacuum chamber by a glass-lined stainless steel capillary (0.8 mm o.d., Scientific Instrument Services) that is floated at 300-450 V. The capillary is embedded in an aluminum block that is heated by an immersion cartridge to 150-190 °C and is electrically insulated from the vacuum housing. The first vacuum chamber is pumped to 0.5-1 Torr by a roots blower (Leybold Ruvac WAU251, 85 L/s). The ions exiting the transfer capillary enter the electrodynamic ion funnel lens,13 where they are radially focused by an applied rf field and axially transported by a dc gradient. The typical settings on the funnel lens are as follows: rf frequency, 800-850 kHz; rf amplitude, 135-150 Vp-p; dc bias, 250-400 V. The rf is provided by a function generator (Tenma Universal Test System) and amplified by a broad-band amplifier (ENI 310L, 250 kHz-110 MHz). The ions enter a 2-mm aperture that serves as a vacuum conductance limit. The aperture is mounted on a bulkhead separating the first and second vacuum chambers and is either grounded or floated at a low dc potential (-5 to +5 V). The ions passing the conductance limit enter the second chamber, which is differentially pumped to 5 × 10-4 Torr by a turbomolecular pump (Leybold TW250). There, the ions are transmitted by an octopole ion guide (12-cm length, 4.5-mm inscribed diameter), which is powered by an rf from another Tenma frequency synthesizer (1.35 MHz), and amplified by an ENI 240L (20 kHz-10 MHz) to 350 Vpp. The octopole can be floated on a 0 ( 50 V dc potential to finely tune ion transmission. The octopole is placed in a cradle, and its axial position can be adjusted within 5 mm. Since the first and second conductance limits are fixed on the vacuum chamber bulkheads, changing the octopole position changes the acceptance and emittance angles and affects ion transmission. The ions are extracted from the downbeam end of the octopole by an electrostatic lens (dc voltage) (14) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451.
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Figure 2. Top view of the main vacuum chamber showing (from right) the ion funnel, octopole, extraction/acceleration lens assembly inside a Faraday cage, ESA, Faraday cages, and entrance into the magnet.
and pass through a 2-mm orifice into the third differentially pumped vacuum chamber, which is maintained at 1-5 × 10-6 Torr by a Leybold TW250 turbo pump. The ions are accelerated to 2 keV by a special seven-element electrostatic lens and refocused to produce an object for the electrostatic analyzer (ESA). Mass Analyzer Ion Optics. This consists of a confocal Mattauch-Herzog arrangement of electrostatic and magnetic analyzers.15 The ESA is a 31.82° angle sector (ion beam radius R ) 160.3 mm, sector pole gap 15.2 mm) where the center voltage is floated at -2 kV and the elements are symmetrically biased by ( 379 V about the float potential. Four-element shunts precede and follow the ESA both to terminate the field of the ESA and to provide a small amount of horizontal steering of the ion beam. The ion beam is steered by applying a voltage difference on the shunt plates relative to the -2 kV master voltage. The ions exiting the ESA form a 2.8-mm-wide (full width at half-maximum) paraxial beam. The ion beam width was measured by moving a 0.4-mmwide retractable slit across the beam and monitoring the current on a Faraday cup collector behind the slit (Figure S1, Supporting Information). The ion beam can be intercepted by a collector plate or collimated by the slit. The collector plate and slit, which are mounted on separate linear motion feedthroughs, can be floated at the desired potential (0 to -2 kV). The collector plate serves as a Faraday cup ion detector for tuning the ion transmission, or, if floated close to -2 kV, as a soft-landing plate for ions passing the ESA. The ion beam outside the shunts and ESA is surrounded by a Faraday cage made of reinforced stainless steel mesh, which is floated at the acceleration voltage. Next, the ion beam enters the copper flight tube of the mass analyzer, which is electrically insulated from the main vacuum (15) Nier, A. O.; Schlutter, D. J. Rev. Sci. Instrum. 1985, 56, 214.
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chamber by a massive Delron washer. The magnetic analyzer is a specially designed 1.6-T permanent magnet with an inhomogeneous field that provides linear dispersion along the 38-cm-long focal plane and separates the ions by momentum.16 The inhomogeneous magnet causes the ions to be dispersed equidistantly as a linear function of ion m/z valuessrather than being compressed as a square root of masssso that beam mass separation is not decreased at higher m/z ratios. The entire magnet and the flight tube are floated at the acceleration potential (-2 kV). The magnet is set on Thompson rails and can be slid in and out. It is shown in the “out” position in Figure 1. The rails are set on a fiberglass insulating platform, and the magnet is protected by a safety Plexiglass shield. Ion Deceleration and Soft Landing. The mass-separated ions exit the magnet at large angles (60-67°, depending on the m/z value) to the focal plane normal. Both the magnitude and the divergence of the exit angles make deceleration of the ions from 2 keV to the soft-landing energy (5-15 eV) difficult. Therefore, a deflector lens made of two layers of a fine copper mesh (90% transmission) is placed between the magnet exit and the deceleration lens assembly. The first copper mesh is mounted plane-parallel to focal plane of the magnet and is maintained at the flight tube potential (usually -2 kV). The second mesh (also plane-parallel and ∼4.5 mm from the first mesh) is mounted on an insulating plastic washer and is floated at an adjustable potential, usually -6.5 to -7 kV. This not only reduces the exit angles to ∼30° by deflecting the ion paths, but also virtually eliminates their divergence (i.e., ion paths exiting the second mesh are parallel to each other; see Figure 3). (16) Scheidemann, A.; Robinson, K. E.; Jones, P. L.; Gottschalk, S. C. Magnetic Separator for Linear Dispersion and Method for Producing the Same; U.S. Patent 6,182,831, February 6, 2001.
Figure 3. Deceleration lens assembly with SIMION simulated trajectories for eight ion beams.
Scheme 1. Electric Potential (volts) Diagram for the Selectide Instrument
The dc potentials applied to the ion optics components are summarized in Scheme 1. A deceleration lens lowers the ions’ kinetic energy in a stepwise manner to allow for refocusing and nondestructive soft-landing on the ion collector array (Figure 3). The deceleration lens assembly consists of 16 equidistant channels of a rectangular cross section (10.6-mm height, 5.3-mm width) that are arranged in six lenses of various voltages and terminate in a 16-bin collector array. Figure 4 shows the ion entrance into the deceleration lens with the 16-bin collector array. The first five lenses are connected in series by a resistor chain. The potentials of the first five lenses
Figure 4. Deceleration lens and landing plate array.
can be adjusted proportionately to the voltage placed on the second copper mesh, which is equal to the voltage placed on the first deceleration lens. For example, if the potential on the second mesh is -7 kV, then the potentials on the first five deceleration lenses will be -7, -3.5, -1.75, -3.5, and -1.75 kV, respectively. Notice that the potential on the fourth lens increases (in absolute value). This is necessary to refocus the ions as they enter the lower potential regions and is analogous to a convex lens refocusing a diverging light beam. Figure 3 shows an ion trajectory simulation17 of ions through an eight-channel deceleration lens. The distances separating the ions of different masses as they exit the magnet are arbitrary in this simulation, which is intended to demonstrate the operation of the deflector lens and the decelerator lens assembly. The actual channel separations are about an order of magnitude larger. The ions can be seen to be diverging through the first three lenses and are then refocused by the fourth lens. The last lens is adjustable from 0 to -120 V. The ion current is usually at its maximum when the potential on the last lens is set at -120 V. The ion collector (Figure 4) is a retractable array of 4-mm bins, each containing a collector electrode and a counter electrode. The collector array or landing plates have an adjustable potential of from 0 to -50 V (-10 to -15 V is normally used) to allow soft landing. The current, which corresponds to the number of ions discharging on the surface in any given channel, is transduced by a multiplexer to a current-to-voltage converter op-amp circuit. Each landing plate can be removed individually to work up the separated, soft-landed molecules. The 4-mm collecting bins can fit into the neck of a small (1.5-mL) ampule and are rinsed directly into the ampule with a known volume of solvent. The solutions are then analyzed on a Bruker Esquire LC ion trap mass spectrometer. Yields have been calculated by comparison with a calibration curve of known concentrations. Materials. Crystal violet dye and Rhodamine B dye were purchased from Aldrich. (Monoisotopic masses of the cation (17) Dahl, D. A. SIMION 3D Version 6.0 Users Manual Publication No. INEL 95/0403 Lockheed Idaho Technologies Company, Idaho Falls, ID, 1995.
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without the chloride counterion are 372 and 443 u, respectively.) A 100-µm solution of each dye was made using Pyrex-distilled methanol. Two peptides, GlyGlyHis and AlaHis, were purchased from Sigma. A 100-µM solution of each peptide was made using a 90:10 mixture of distilled methanol/water and 1% acetic acid. RESULTS AND DISCUSSION Ion Currents and Transmission. Ion currents were measured at several points along the ion trajectory, for example, at the aperture after the funnel lens, at the aperture after the octopole, at the collector plate after the ESA, and at the final collector after the deceleration lens. Thus, ion transmission through the ion optics components could be established and optimized. The ion currents were tested by electrospraying 100 µM crystal violet dye in methanol. The ion current intercepted in the first vacuum chamber at the entrance of the funnel lens was as high as 130 nA, depending on the dye concentration and the inner diameter of the transfer capillary used (0.4-0.8 mm). However, only ∼16 nA maximum current was transmitted by the funnel lens, as measured at a collector plate mounted over the first conductance limit. The ion current measured at various settings of the funnel lens (background pressure, radio frequency, and amplitude) is shown in Figures S2-S4 (Supporting Information). The current of the accelerated ion beam that was measured right after the electrostatic analyzer but before the magnet was typically 10-13 nA. This indicates that a substantial fraction of ions passing the first conductance limit were transmitted to the high-vacuum region of the instrument. Since optimizing the octopole axial position resulted in g90% ion transmission through the guide, the ∼80% overall ion transmission (100 × 13/16 ) 81%) implies that the ions are also transmitted efficiently (∼90%) through the electrostatic lenses and apertures. The ion currents after the magnet, 2-2.7 nA, have been achieved when a -3 kV bias was applied to the collecting plate. A -3 kV bias would not allow for nondestructive soft-landing of ions. At a -10-V bias, suitable for collection of intact parent ions, the currents were 2-3 nA before the magnet and 0.2-0.3 nA after the magnet using the deceleration lens. In terms of material collection, landing 1 nA for 10 h represents deposition of ∼0.5 nmol of a singly charged molecule. The ion currents before and after the magnet indicate that ∼90% of the ions are lost as they undergo mass separation and deceleration. Since there are two copper meshes, each with a 90% transmittance efficiency, we expect a 20% loss in ions hitting the mesh. However, it is difficult to pinpoint where the remaining 70% of the ions are lost. Some improvement was achieved by refocusing the 2 keV ion beam along the vertizal (z) axis before entering the magnet. Since the cylindrical electrostatic analyzer does not focus along the z axis, without refocusing, the height of the divergent ion beam from the acceleration lens can exceed the 6-mm clearance in the magnet flight tube and be collimated by it. However, the main difficulty with achieving more efficient ion transmission is in characterizing the ion trajectories past the focal plane of the inhomogeneous magnet. According to ion trajectory simulations, both the trajectories and the focal plane position depend on the ballistic entrance angle into the magnet, which we can change only within narrow limits. Since the deceleration lens assembly is mounted at an ∼30° angle with respect to the magnet 4382
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focal plane, the mass-resolved ion beams enter the deceleration lens at different distances from the magnet. This means that the ion beams passed their focal points and became divergent before deceleration, which is, of course, undesirable. An improved ion transmission could be achieved by changing the magnet design to provide steeper exit ion trajectories, or using a larger negative potential for bending the exiting ion beams. Each of these solutions has an obvious limit. Redesigning the magnet would be costly ($100 000), and increasing the deflector voltage above -10 kV meets serious problems with electrical insulation and arcing. Despite less than perfect ion transmission, the currents of mass-resolved and decelerated ions are sufficient for preparative isolation of involatile components from mixtures, as described in the next section. Landing Surface and Preliminary Soft-Landing Experiments. Efficient recovery of soft-landed material critically depends on the charge neutralization of the ions impacting the collector. The mechanism of ion capture, retention, and discharge is not well-understood.18 To study the soft landing of ions and recovery of the neutral molecules, crystal violet dye was collected on a stainless steel plate (at a -10 V bias) placed after the ESA but before the magnet. Mass spectrometric analysis of the collected crystal violet dye showed fragment and rearrangement ions but little or no molecular ion. Mass spectrometric analysis of an electrosprayed stock solution of crystal violet dye showed that the parent ion (the cation without the chloride counterion) was very stable under mass spectrometric conditions. We suspected that the ion was landing intact but that a surface interaction on the stainless steel plate was fragmenting the ion after the soft landing. Cations could be neutralized in two ways at the collecting plate. There could be an acid/base reaction to deprotonate or form an ion pair. Alternatively, the molecular ion could be neutralized by electron transfer, with its subsequent fragmentation and rearrangement. To maximize recovery of the intact neutral parent, electron transfer neutralization followed by subsequent fragmentation and rearrangement must be minimized. Evidently, electrontransfer neutralization predominated when using an untreated stainless steel plate as the landing surface. To maximize acidbase neutralization, a stainless steel plate with an oxidized layer was tried as the landing surface. A layer of iron, chromium, manganese, and nickel oxides (the main ingredients of stainless steel) is basic or amphotheric.19 A basic oxidized layer could deprotonate or form an ion pair with the molecular ion and so suppress neutralization by electron transfer. Three methods of oxidizing the stainless steel surface to prevent this fragmentation were tried. One method was to burn ions onto the plate surface at -3 kV before collecting dye at -10 V. A second method, chemical oxidation, involved treating the surface with a saturated solution of NaOH/water for 1 h and then with hydrogen peroxide for 1 h. The third method was to treat the stainless steel plate with oxygen in a plasma chamber for 10 min at 60 W, as described previously.9 Crystal violet dye was soft-landed on the “burned-in” plate. Analysis of the recovered (18) Godbout, J. T.; Halasinski, T.; Leroi, G. E.; Allison, J. J. Phys. Chem. 1996, 100, 2892-2899. (19) Shriver, D. F.; Atkins, P.; Langford, C. H. Inorganic Chemistry; 2nd ed.; W. H. Freeman: New York, 1994; p 197.
Figure 5. (a) Soft landing of a mixture of crystal violet and Rhodamine B dyes. Graphs from front to back are Rhodamine B dye, crystal violet dye, and mixture of the two. The peaks of the mixture are shifted two bins to the right due to a different setting of the ESA. (b) Yields of soft-landed and recovered Rhodamine B.
mixture showed the parent ion was the second-highest peak (80% of the base peak). After collecting on the chemically oxidized plate or on the plate that was treated with oxygen in a plasma chamber, the molecular ion was the base peak, and only small amounts of fragment ions were observed. A chemically oxidized copper plate was also used, and the results were similar to the chemically oxidized stainless steel plate where the parent ion was the base peak. Figure S5 (Supporting Information) shows a picture of crystal violet dye landed on a copper plate before the magnet. It
was intended that the sample would land inside the well in the plate to make recovery of the sample easier. Instead, evidently, an electrostatic field formed around the well (the plate was at -10 V), and the dye landed along the rim. This occurred even when the plate was repositioned so that the previous landing site was aimed at the center of the well. Two peptides, GlyGlyHis and AlaHis, have also been softlanded onto a chemically oxidized stainless steel plate after the ESA but before the magnet. Both show that the unaltered parent Analytical Chemistry, Vol. 77, No. 14, July 15, 2005
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can be recovered. The recovery of peptide molecules from the surface was found to depend on the surface properties. The highest yields, approaching 0.8% of the electrosprayed material, were achieved when collecting the peptide ions on a plasma-etched collector. The yield from collecting on a wet-oxidized metal plate were lower. Soft Landing with the Deceleration Lens. Figure 5a shows the current on each bin from spraying two dyes, Rhodamine B and crystal violet dyes, and the current from spraying a mixture of the two. Ion current was ∼0.3 to 0.6 nA on the targeted bins. Most of each dye, when sprayed individually, landed in one bin with small shoulders on each side. The graph of the mixture shows a decrease in current when compared to the dyes injected individually. This occurs even when the concentrations in the mixture are the same as the concentrations of the individually sprayed dyes and can be due to mutual suppression of the analyte electrospray ion currents, as reported for other ionic analytes.20 The two dyes have a mass difference of ∼70 amu and land about seven bins apart. Figure 5b shows the collection and analysis of Rhodamine B dye. On bin 6, 0.13 nmol was landed (average current of 0.23 nA for 15 h), and 17% was recovered. Figure 5b also shows a small peak at three bins lower mass from the parent peak. Although this peak may be from loss of CO2 from the parent ion, nothing was recovered from that bin to confirm its identity. The present efficiency of preparative scale mass spectrometry is
