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Electrospray Ionization in the Strong Magnetic Field of a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer Sir: As an alternative to particle'+ and laser desorption'-" sourcea for Fourier transform ion cyclotron resonance (FI'ICR) mass spectrometry detection of large molecules, Henry and co-workers recently coupled electrospray ionization (ESI) to FTICR.12-15 This instrument was based upon an external source design with electrospray ions formed outside the magnetic field and guided with quadrupole lenses to the trapped ion cell. One rationalization for employing an ESI source is that FTICR limitations at high mass16 are circumvented by generating multiply charged eledrospray ions for detection in the mass-bcharge (mlz)range between 500 and 2000.'7-30 For example, Henry acquired a spectrum for bovine albumin with a molecular weight 66 266 Da, the highest m w ion detected to date by FTICR.16 Another motivation for coupling ESI to FTICR is the superior mass resolution and mass accuracy of the mass analyzer in the mlz region where ESI spectra are generated. At pressures in the lo+' Torr regime, Henry, Quinn, and McLafferty achieved a mass resolution of about 63 OOO at mlz 942 for the +16 charge state of cytochrome c; the calculated mass value for cytochrome c from this spectrum was 12288.71 f 0.07.14 The principle limitation of their external source configuration was the inefficient transfer of ions from the source to the trapped ion cell. Typical ion currents of only a few picoamperes were measured at the cell and lengthy data acquisition periods were necessary to achieve acceptable During the past few years we have constructed a variety of probe-mounted sources for FTICR that are positioned in the magnet bore to confine radial ion motion and increase ion transfer efficiency to the ~ e l l . ~Extension ~ 9 ~ of this concept to construction of a high magnetic field electrospray source is especially attractive given the difficulties experienced by Henry and others in delivering adequate ion current to the cell from external source^.^'^^^^^ There are no fundamental reatrictions to operation of an ESI source in the magnetic field, but constraints imposed on vacuum system design are considerable since the magnet bore must simultaneously accommodate both the gas load of the ESI source at atmospheric pressure and the ultralow pressures required for FTICR detection. Previous efforts to obtain pressure differentials between source and analyzer regions within the magnet bore, notably by Nicolet Analytical Instruments with their differentially pumped dual trapped ion cell,%achieved a pressure differential of only 103. An additional 6 orders of magnitude pressure differential would be necessary to facilitate the practical coupling of a high magnetic field ESI source with FTICR detection. We present here preliminary results from construction of a new electrospray ionization source. The source operates in the magnetic field within 25 cm of an FTICR analyzer trapped ion cell that is maintained at mid-10-7 Torr pressures. The electrospray ion current of 375 pA that is detected at the trapped ion cell confirms the strong confining nature of the magnetic field. Among several ESI spectra to be presented is one for the largest molecule analyzed to date by FTICR, bovine albumin dimer with a molecular weight of 132532 Da. EXPERIMENTAL SECTION The vacuum system for the probe-mounted electrospray interface consisted of five concentric vacuum chambers of increasing diameter which extended into the high-field region of the magnet as shown in Figure 1. The innermost chamber was a hollow 3/rin.
0.d X S i n . length stainless steel probe which allowed both sample delivery through 22-gauge Teflon tubing and electrical connectians and mechanical support for the ESI interface. This interface was baed on a design by Katta and co-workers." A solids probe inlet allowed the interface to be inserted and removed from the instrument without venting the vacuum chamber. A 12-cm length Delrin plug inserted in the hollow probe and fitted with O-rings to provide the vacuum seal was machined to accommodate a 1Wpm id. bluntended stainlesssteel syringe needle and a 5OO-pm i.d. X 20-cm stainless steel capillary. The needle and capillary were separated by a distance of 6.5 mm, and the capillary extended 6 cm beyond the end of the probe. Delrin material surrounding the ends of the syringe needle and capillary was machined away to create a spray cavity between the needle and the capillary. The capillary was resistively heated to -150 "C by a current of -2.2 A at a bias of 330 V. The syringe needle was maintained at high potential to obtain the spray, with 3.5 kV generating the optimum current. A 1.375-in. i.d. vacuum chamber surrounding the probe terminated at a blunt-ended 500-pm copper skimmer cone/lens assembly. The pressure in this region was maintained at 1.5 Torr by a 13 L/s mechanical rough pump. The skimmer cone was mounted in a Delrin guide assembly to ensure proper alignment at a critical capillary to skimmer distance of 4.5 mm. A cylindrical lene 0.750-in. i.d. X 1.5 cm was attached to the base of the skimmer cone and biased at 15 V. The 1.375-in.sleeve was inserted into a 2.187-in. i.d. vacuum chamber that terminated with a 6-mmgrounded conductance limit that was pumped by a 1100 L/s cryopump to pressures in the mid-10-4 Torr range. The distance between the end of the cylindrical lens and the grounded conductance limit was 13 mm. Finally, this entire assembly was inserted into the analyzer side of the standard 4.375-in. i.d. vacuum chamber of the Extrel FTMS-2000. A 2-cm distance separated the 6-mm conductance limit from the first trap plate of a commercial dual cell assembly which consisted of adjacent cubic cells of 5-cm dimension separated by a common trap plate that also served as a conductance liiit with 2-mm orifice. Twin 700 L/s diffusion pumps provided pumping on either side of the dual cell conductance limit. However, in its present design, pressures were limited to l0w-l0-~ Torr and mid-lO-' Torr at the two cells, respectively. With future designs, the addition of cold fingers, replacement of the analyzer cell pumping stage with a higher speed cryopump, and reduction of the conductance limit orifice on the 2.187-in. sleeve should substantially reduce analyzer cell pressures. Despite the leas than desirable pressures for FTICR detection, the primary goal in constructing the interface, electrospray ion formation in the strong magnetic field, was accomplished. Specifically, the external source configuration described above allowed ionization to occur at atmosphericpressure in a 1.5 T field, while differential pumping through the concentric tube network provided a 9 order of magnitude pressure reduction within a 25-cm length of the vacuum chamber. Sample Preparation. Solutions were fed to the syringe needle through a 22-gauge Teflon tube that was connected to an Isco-500 micro-flow syringe pump delivering a flow of 1-4 pL/min. Solutions were prepared by dissolving the sample in an aqueous solution of acetic acid and then diluting with methanol to achieve a final solution concentration of 5-10 pmol/pL in a solution consisting of 30682 H20-MeOH-HOAc. Horse myoglobin (molecular weight 16951 Da), turkey conalbumin (molecular weight 77 773 Da), and bovine albumin dimer (molecular weight 132532 Da) were used as obtained from Sigma. FTICR Detection. The spectrometer used for the experiment was assembled from componenta that constitute the FTMS-2000 from Extrel. A 3.0-T magnetic, dual trapped ion cells with cubic geometry, standard analog electronics, and Nicolet 1280 data station are included. Extrel software version 6.0 was used to acquire the data. Spectra to be shown were obtained with a standard FTICR pulse sequence. Source trap plates were continuously maintained at static 5-10-V potentials while electrospray
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ANALYTICAL CMMISTRY, VOL. 64. NO. 5. MARCH 1. 1992
Table I. Spectral Figures of Merit for Protein Samples Analyzed By High Mametic Field ESI/FTICR
sample
Mw
M w (ob)
% error
re1 std deviation
chaw
SIN
m/Am
horae myoglobin conalbumin bovine albumin dimer
16950.7 77 773 132 532
16950.5 71672 132628
0.001 0.13 0.07
0.06% 0.04% 0.03%
21-13 54-35 63-52
200 (26 seana)
65. 69 91
34 (100 scans) 71 (Zoo0 scnns)
“Resolution is data-point limited.
I
noun
1. me high magnetlc neld ESI~FTICR instnrment used IO acquhe ttm spectra In Figand IO=’ Ton. respechrely. analyzer ~011sunder full electrospray conditions were
Flpxa 2. ESllFTlCR apechum of horse myoglobin (8.3 pmol carsunad)owlhedhom25coaddsdbansisms. A36-ld-kb~dwWJ711nd 2000 W @sweep rata were used lor exciiaIbn. and 16K data Wts were acquired over a 2.66MHz banmidm lor dete&n.
2-4. Opra~lnppfe8suea In
ttm IIOUCI)and
Fbum 4. ESIlFTICR spsctMn of bwlne albumin d l m (600 pmol consumed) obtalned fmm 2000 coaddsd lransients. A 27kM bandwldm and 500 &/@ sweep raw were used fa ex&Ilon. and 4096 data polnts were acqulred over a l5okHz band* fadalecM.
ions accumulated in the cell for a period of about 0.5 8. Trap potentials were then loweredto 1.0 V for detection. Initial efforts to effciently transfer ions to the analyzer cell were not sucoessful presumably due to alignment problem with the dual cell, 80 the high-pressure trapped ion cell was used instead for detection. Parameters for the linear excitation sweep and the broadband detection eventa were seleeted to maximize aensitivityand mass resolution for each protein and are found in the f w e captions. Typically several spectra were coadded to yield a transient to which an equivalent number of zero8 was added, followed by sinebell apodizationand magnitudemode Fourier transformation to yield the mass spectrum. Broadband calibration tables for maas assignment were generated from ESI spectra of cytochrome c or bovine albumin.
ngun 3. ESIlFTICR speclrum 01 m i b u m l n (30 pmol consumed) obtained han 100 c=aaM lransients. A 404d-k bandwldthand 1000 Hzl@ sweep rate were used fa exdtstion. and 32K data odnta were acqulred over a 2.85MHz bandwm fw datectlon.
RESULTS AND DISCUSSION
Ofprimary concern in the construction of the electrospray interface waa an increase in ion current entering the cell. It
ANALYTICAL CHEMISTRY, VOL. 64, NO. 5, MARCH 1, 1992
was hoped that by positioning the electrospray skimmer cone in the magnetic field several complications associated with ion formation outside the magnetic field would be eliminated. First, although the number of analyte molecules ionized by electrospray is very high compared to other sources, the fraction introduced to any mass analyzer is less than 0.001% because of the wide spatial and kinetic energy dispersion of the spray. Efforts to focus the spray with a jacketed gas flow have not been completely satisfactory." In contrast, the intense magnetic field should reduce the large solid angle of the spray by c o n f i i the radial trajectories of ions, even with large kinetic energies, to small cyclotron orbits. A second advantage derived from positioning the source in the magnetic field is that ion loss associated with transfer over several meters from an external source to the trapped ion cell is minimized. Evidence that the new source performs as deaired in delivering increased numbers of electrospray ions to the trapped ion cell comes from comparative current measurements performed in vacuum chambers both inside and outside the magnetic field. For the identical interface in a vacuum chamber without an applied magnetic field, a peak current of 60 pA was measured on a collector positioned immediately beyond the skimmer cone. This current decreased rapidly without focussing as the collector was withdrawn from the skimmer. In contrast, in the magnetic field of the FTICR vacuum chamber a 375-pA current was measured at the collector, and even as the collector was displaced in the magnetic field up to 40 cm from the source, current readings were within 10% of the initial value. Evidence that the increased current at the trapped ion cell translates into increased FTICR performance is demonstrated with the ESI mass spectra presented in Figures 2-4 and the corresponding figures of merit for each spectrum which are compiled in Table I. Consideringfirst the sensitivity, it was found that a precise matching of ion kinetic energy out of the skimmer cone with trapping potential was necessary to trap ions that could be detected. Under these conditions the ion population in the cell was found to accumulate with time until the space charge limit was reached, t y p i d l y in about 0.5 s. Spectra shown in Figures 2-4 yielded S / N values of 34-200, and in general it was found that for proteins in the molecular weight range up to 20000 Da, spectral S/N exceeded 100 within 25 scans. An increased number of scans were required to achieve an equivalent S/N for larger proteins due to broadening in the charge envelope and reduced solubility. Attempts to transfer ions to the low-pressureanalyzer cell were unsuccessful, requiring that FTICR data acquisition occur in the high-pressure source trapped ion cell. It is believed that the inability to transfer ions is due to alignment difficulties with the dual cell; support of this theory comes from the fact that during ion injection a large amount of ion current is collected on the front source trap plate and on the dual cell conductance limit but no ion current is observed on the rear analyzer trap plate or beyond. It is also possible that the ion beam is aligned with respect to the dual cell assembly, but due to the relatively large cyclotron or magnetron radius of the ion beam, efficient transfer through the 2-mm conductance limit is not facilitated. Studies are currently in progress to map the spatial characteristics of the ion beam, and a self-centering guide assembly is under construction to insure proper alignment when the modified source is reinstalled. Consequently for these data, mass resolution at low-lo6 Torr pressures was pressure limited to about 100 at m/z 1100. This resolution value was similar for all proteins examined, independent of mass, and contrasts with Henry's observation of deteriorating resolution a t the same mlz for larger proteins.15 It should be pointed out that his work was performed at lo4 Torr with different relaxation processes in effect.
571
One impetus for developing an ESI/FTICR interface is the high mass resolution of FTICR which should allow mass assignment from a single charge state, as was shown with the external source in~trument.'~Given the present pressure limitations in our high-field source, this approach was not possible and instead molecular weight determinations from spectra in Figures 2-4 were based upon m / z differences between adjacent charge states.lg Constraintson mass resolution are much leas severe with this algorithm, and as the data in Figures 2-4 indicate, at preseures of lo6 Torr the charge states are adequately resolved to permit molecular weight determinations even with proteins beyond 100OOO Da. Mass calculations presented in Table I indicate that if at least a partial overlap occurs between charge envelopes in external calibrant and analyte spectra, then errors of less than 0.1% are achieved. ACKNOWLEDGMENT This work is supported by the Welch Foundation (F-1138), the Arnold and Mabel Beckman Foundation,and the National Science Foundation (CHE9013384 and CH9057097). REFERENCES (1) McIver, R. T., Jr.; Hunter, R. L.; Bowers, W. D. Int. J . Mess Spec. Ion Roc. 1985, 64, 67-77. (2) Lebrllla, C. B.; Amster, I. J.; McIver, R. T., Jr. Znt. J . Mass Spec. Zon R O C . 1989, 8 7 , R7-Rl3. (3) Hunt, D. F.; Shabanowltz, J.; McIver, R. T., Jr.; Hunter, R. L.; Syka, J. E. P. Anal. Chem. 1985, 57, 765-768. (4) Hunt, D. F.; Shabanowtiz, J.; Yates, J. R., 111; McIver, R. T.. Jr.; Hunter, R. L. Anal. Chem. 1985. 57, 2728-2733. (5) Wiikms, E. R.; McLafferty, F. W. J . Am. Soc.Mess Specfrom. 1990, 7 , 427-430. (6) Watson, C. H.; Kruppa, G.; Wronka, J.; Laukien, F. H. RapM Comm. MeSS SpeCtrom. 1991, 5 , 249-251. (7) Nuwaysir, L. M.; Wiikins. C. L. In Laser and Spectromefry (Oxford S a rles On Optical Sciences); Lubman. D. M., Ed.; Oxford Unhrersky Press: New York, 1980 Chapter 13. (8) Shomo, R. E.; Marshall, A. 0.; Weisengerger, C. R. Anal. Chem. 1985, 5 7 , 2740-2744. (9) WllklnS, V. L.; Weil, D. A.; Yang, C. L. C.; Ijames. C. F. Anal. Chem. 1985, 57, 520-524. 10) Ijames, C. F.; Wilkins, C. L. J . Am. Chem. SOC. 1988, 770, 2887-2688. 11) HetIich, R. L.; Buchanan, M. V. J . Am. Soc. Mess. Spectrom. 1991, 2 , 22-28. 12) Henry, K. D.; Mclafferty. F. W. Org. Mass Spectrom. 1990, 25, 490-492. 13) Henry. K. D.; Wliiiams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. W. Roc. M t l . Acad. Sci. U . S . A . 1989, 86, 9075-9078. 14) Henry, K. D.; Qulnn, J. P.; McLafferty, F. W. J . Am. Chem. Soc. 1991, 773, 5447-5449. 15) Henry, K. D. Electrospray Ionization Fourier Transform Mass Spectrometry for Characterization of Large Biornoiecuies. Ph.D. Dissertation, Comeii Unhrerslty, 1991. (16) Henson, C. D.;Castro, M. E.; Russell, D. H.; Hunt, D. F.; Shanbanowk,J. ACS Symp. Series "FTMS of Large (m/z>5000) Biomolecuies," 1987, 359(FTMS), 100-115. (17) Loo, J. A.; Edmonds, C. 0.; Udseth, H. R.; SmAh, R. D. Anal. Chem. 1990, 62, 893-898. (18) Smith, R. D.; Loo, J. A.; Edmonds, C. 0.;Barinaga. C. J.; Udseth, H. R. J . ChrometOgr. 1990, 516, 157-165. (19) Mann, M.; Meng, C. H.; Fenn, J. B. Anal. Chem. 1989, 67, 1702. . - - 1708. . . - -. (20) Katta, V.; Chowdhury, S. K.; Chait, B. T. J . Am. Chem. SOC. 1990, 772. 5348-5349. (21) Men& C. K.; Fenn. J. B. Ofg. Mess Spectrom. 1991, 26, 542-549. (22) Brulns, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987, 59, 2642-2648. (23) Mann, M.; Meng, C. K.; Fenn, J. B. Anal. Chem. 1989, 67, 1702-1708. (24) McLuckey, S. A.; Van Berkei, G. J.; Giish. 0. L. J . Am. Chem. Soc. 1990, 772, 5668-5670. (25) Calaycay, J.; Rusnak. M.; Shiveley, J. E. Anal. Eiochem. 1991, 792, 23-3 1. (28) Loo, J. A.; Udseth, H. R.; Smith, R. D. Eiomed. Env. Mass Spectrom. 1988, 77, 411-414. (27) Loo. J. A.; Edmonds, C. G.; Smith, R. D.; Lacey, M. P.; Keough, T. Eiomed. Env. Mass Spectrom. 1990. 79, 286-294.
Anal. Chem. 1992, 64, 572-575
Witkowska, H. E.; Oleen, E. N.; Smith, S. J . E&/.
Chem. 1990, 265, 58624665. LOO, J. A.; Udseth, H. R.; Smith, R. D. Anal. Blochem. 1989, 179, 404-4 12. Fenn, J. E.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mess Spectrom. Revs. 1990, 9 , 37-70. Henry, K. D. Personal communlcatlon. Hogan, J. D.; Beu, S. V.; Majldl, V.; La&, D. A., Jr. Anal. Chem. 1991, 63,1452-1457. Hofstadler. S. A.: Lade, D. A., Jr. Anal. Chem. W9l. 63, 2001-2007. Ijems, C. F.; Markey, S. P. Proceedings of 39th ASMS Conference on MaSa Specbometry and AWM Topics, May 19-24, 1991, Nashville,
TN. Kerley, E. L.;
Buchanan, M. V.; Cook, K. D.; Shahgholi, M.: Proceed-
(36)
ings of 39th ASMS Conference on Mass Spectrometry and Allied Topics, May 19-24, 1991, Nashvllie, TN. Nlcolet Anaiytlcal Instruments. U.S. Patent Number 4581533, 1986.
Steven A. Hofstadler David A. Laude, Jr.* Department of Chemistry and Biochemistry University of Texas at Austin Austin, Texas 78712
RECEIVED for review September 4,1991. Accepted November 27, 1991.
TECHNICAL NOTES Speciation of Iron( II)and Iron( III)Using a Dual Electrode Modlfied with Electrocatalytic Polymers Andrew P. Doherty, Robert J. Forster, Malcolm R. Smyth, and Johannes G. Vos* School of Chemical Sciences, Dublin City University, Dublin 9, Ireland Speciation of metal ions has received considerable attention in diverse areas such as ecotoxicology,' geology,2 process analysis,3and aquatic studies: Several approaches have been adopted for the speciation of Fe(I1) and Fe(II1). Electrochemical methods predominate in this type of but chromatographid' and spedrometrii?~' methods have also been described. For the chromatographic and the spectrometric methods, interconversion from one redox state to another? the use of different chelating agents? or the use of more than one detection system6is required to differentiate between the two redox states. Polarographic methods appear almost universal in the electrochemical approach, but as the Fe(II)/Fe(III) couple is reversible,'O the use of reagents to separate the half-wave potentials (Ell2) for the oxidation and reduction processes is necessary. Several reports concerning the determination of Fe(II) and Fe(III) at solid electrodes have also appeared."J2 However, the redox chemistry of the Fe(II)/Fe(III) couple is poor at glassy carbon,ll and at platinum, electrode fouling is encountered.12 In this laboratory, the synthesis and characterization of polymer-bound e1e~trocatalysta'~J~ and their application as sensors in flow analy~is'~7'~ have received considerable attention. In this technical note we demonstrate a novel amperometric sensor system for the flow injection speciation analysis of unbound Fe(II) and Fe(II1) using a dual-electrode assembly using glassyarbon electrodes modified with the electrocatalysta [M(bpy),(PVP),,Cl]Cl (M = Os, Ru; bpy = 2,2'-bipyridyl; PVP = poly(4-vinylpyridine)) to provide simultaneous detection of both Fe(I1) and Fe(II1) in a singlesample aliquot. The merits of this approach are discussed in relation to existing methods.
EXPERIMENTAL SECTION Construction and Operation of the Dual Sensor. The detection system used consisted of an EG&G Princeton Applied Research (PAR) Model 400 electrochemical detector with dualpotentiostat function. The two working electrodes were glassy carbon (3 mm diameter) shrouded in a single teflon block. One and the other electrode was modified with [R~(bpy)~(PvP)~~C1]C1, with [Os(bpy)2(PVP)loC1]C1 by droplet evaporation. These ma-
* To whom correspondence should be addressed. 0003-2700/92/0364-0572$03.00/0
terials were synthesized as previously described.'~'' The structure of these polymers is shown in Figure 1. The working electrodes were placed in parallel in the thin-layer electrochemical flow cell. This parallel arrangement allows the sample plug to reach and be detected at each sensor simultaneously. This is represented in Figure 2. Detection of Fe(I1) was achieved at the ruthenium polymer-modified electrode using an applied potential of 0.85 V vs SCE. The osmium polymer-modified electrode was used for the detection of Fe(1II) at an applied potential of 0.12 V vs SCE. The simultaneous responses were recorded on a Philips PM8252 dual-pen recorder. The flow injection system was that previously described,18connected to the detection system described above. The carrier electrolyte was 0.2 M Na804adjusted to pH 1.0 and a flow rate of 1.0 mL min-' was used. (NH,)z[Fe(S04)2].6H20 and NI&[Fe(SO&z]-12Hz0 were used to prepare solutions of Fe(II) and Fe(III), respectively. Deionized water obtained by passing distilled water through a Milli-Q water purification system was used. The recoveries of Fe(I1) and Fe(II1) from an acidified drinking water sample were determined from samples 'spiked" with various ratios of [Fe(III)]/[Fe(II)].
RESULTS AND DISCUSSION Principle of Operation. The dual sensor is based on the use of two polymer-bound electrocatalysts for electrode modification with half-wave potentials which envelop the formal potential of the Fe(II)/Fe(III) redox couple. The EIl2 of the ruthenium and osmium centers in the electrolyte used here are 0.75 and 0.25 V vs SCE, respectively, and the formal potential of the Fe(II)/Fe(III) couple is 0.46 V vs SCE.l9 This produces electrochemical driving forces of 0.21 V for the reduction of Fe(II1) and 0.29 V for the oxidation of Fe(I1). The ruthenium electrocatalyst with an Ell* more positive than the formal potential of the analytes is capable of the electrocatalytic oxidation of Fe(II) via the mediated cross reaction given in reaction 1. The osmium electrocatalyst with an Ellzless Ru(II1) + Fe(I1) Ru(I1) + Fe(II1) (1) positive than the analyte formal potential can mediate the reduction of Fe(III) via reaction 2. It has been observed that Os(I1) Fe(II1) Os(II1) + Fe(I1) (2) the ruthenium polymer can mediate the reduction of Fe(III)lg as a result of negative shifting of the wave toward the formal potential of the Fe(II)/Fe(III) couple. However, the high
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