Electrospray Ionization Ion MoMlity Spectrometry

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1 Anl. Chem , Electrospry Ioniztion Ion MoMlity Spectrometry Dou(lw er Milllpore Corportion, Wters Chrmtogrphy Division, 34 Mpie Street, Milford, Msschusetts Yong Hong Chen, Wn K. LuckenMII, nd Herbert H. Hill, Jr. Depltmnt of Chemisby, Wshington Stte Unhws&, Pullmn, Wshington An improved electrospry ioniztion source for ion d i t y spectrometry ws developed by mking three slient modifictions to the electrospry needle. First,the needle ws removed from the ion drift region, reducing neutrliztion of chrged drops nd im on the wlls of the io mobility drift tube. secod, the tip of the spry needle ws electriclly insulted, exteding the voltge rnge with which stble electrospry could be chieved. Third, the needle ws cooled, eliminting problems ssocited with solvent voltiliztion in tbe spry needle. Ion mobility spectr of cytochrome c nd Triton X-100 were compred with those obtined by electrospry ioniztion mess spectrometry. While direct mss/chrge comprison with ion mobility ws not possible, similr electrospry ion ptterns between the IMS nd MS dt suggest tht the electrospry ioniztion source for IMS behved in er consistet with thpt of other electrspry ioniztion surces. In ddition, mss ssignments of IMS ion peks, mde by mtching ptterns from mss spectr, fit well with those expected from IMS theory. The inherent high resolution of cpillry electrophoresis (CE) nd microbore liquid chromtogrphy (MLC) long with ese of smple preprtion hs mde high-performnce liquid-phse seprtion (HPLPS) the method of choice for mny nlyticl pplictions. For the most prt, however, detection of smples in the liquid phse hs been limited to those nlytes which bsorb or fluoresce in the UV nd visible regions of the spectrum. For compounds which bsorb poorly, dditionl smple clenup or derivtiztion procedures re required. Thus, development of simple nd dependble detection method for non-w-bsorbing compounds in solution would significntly expnd the nlyticl pplictions for HPLPS. In gs chromtogrphy, ioniztion methods hve provided bsis for universl detection. Most of these methods, however, rely on the selective ioniztion of nlytes in the presence of the mobile phse nd, with the exception of mss spectrometry, hve not been successfully interfced to HPLPS methods becuse of solvent interferences. Ion mobility spectrometry (IMS) is technique for detecting ions in the gs phse which re seprted on the bsis of differences in cross sectionl re. Since most nlytes in HPLPS give rise to ions of lrger cross section thn those of the solvent, IMS would seem logicl choice s n ltemtivedetection method. Furthermore, the fesibility of IMS-bsed detection for HPLPS hs lredy been demonstrted1 using n electrospry nebulizer s n ioniztion source Anlyticel ChemWy, Vol. 66. No. 14, JI& 15, 1994 Electrospry ioniztion ion mobility spectrometry (ESI- IMS) ws first described in 1972 by Gieniec et l.,2 who modified Plsm Chromtogrph mnufctured by Frnklin GNO Corp. (West Plm Bech, FL). In their design, the norml smple entrnce nd the 63Ni ion source were removed from the commercil instrument nd replced with n electrospry chmber. Liquid smples were introduced into the enclosed glss spry chmber by syringe pump vi Teflon tube connected to 27-guge hypodermic needle. The spry chmber ws seprted from the ion drift tube by brss plte contining lrge orifice for the bth gs nd ions to enter the drift region. Electrospry ws developed by pplying potentil between the needle tip nd the brss plte. The bth gs ws introduced into the spry chmber t the rer of the needle. According to the uthors, this heted gs flow served to help evporte the droplets nd to sweep the ions downstrem through the orifice in the brss plte. Unfortuntely, ll of the solvent ws swept towrd the ion drift region nd pprently contminted this region, decresing both sensitivity nd resolution in the spectrometer. Smith nd co-workers hve reported the ESI-IMS spectr for cytochrome c3 nd ly~ozyme.~ These spectr, obtined t low tempertures (25-90 C), weresimilr in resolution, shpe, nd drift time to those first reported by Gieniec et le2 The peks drifted t reltively long drift times (>50 ms), nd only one brod pek (pek width t hlf-height of bout 25 ms) ws observed for ech compound. An obvious concern with respect to these spectr ws presented by Fernndez de l Mor.5 *... ESI mss spectrum (of cytochrome c) generlly contins between 15 nd 20 chrges. Should not then the mobility spectrum contin lso six peks t lest for cytochrome C? Improvement in both sensitivity nd resolution ws reported by Shumte nd Hill by incorporting n electrospry needle directly into unidirectionl flow ion mobility spectrometer.6 While this design worked well for low moleculr weight compounds, high moleculr weight, nonvoltile compounds were not detected. In ddition, enclosure of the needle in the stinless steel drift tube mde it difficult to chieve electrospry conditions, nd the most stble opertion of the detector (1) Hill, H. H., Jr.; Siems, W. F.; St. Louis, R. H.; McMinn, D. G. Ad. Chem. 1990, 21(5), A362. (2) Gieniec, J.; Cox, H. L., Jr.; Ttcr, D.; Dole, M. Abstrcts, 20th Annul Conference on MS nd Allied Topics, Dlls, TX, 1972, 216. (3) Smith, R. D.; Loo, J. A.; Loo, R. R. 0.; Busmn, M.; Udscth, H. R. Mss Spectrom. Rev. 1991, 10, (4) Smith, R. D.; Loo, J. A.; Loo. R. R. 0.; Udscth, H. R. Mss Specrrom. RN , (5) Fernndz de l Mor, J. Mss Spcrrom. RN. 1992, 11, (6) Shumte. C. B.; Hill, H. H.. Jr. Anl. Chem. 1989, 61(6) /94/036&2348$04.50/ Americn Chemlcl Society

2 occurred when coron dischrge ws visible t the tip of the spry needle. Nevertheless, with this coronspry ioniztion ion mobility spectrometer (CSI-IMS), wide vriety of compounds could be detected from solution with good sensitivity. Subsequently, potentil pplictions of IMS s detector for liquid chromtogrphy? s detector for cpillry electrophoretic methods: nd s n on-line liquid-strem process sensor were reported.9 Although initil investigtions of the IMS s detection method for liquid smples were encourging, lck of response for high moleculr weight compounds indicted tht the spry source ws not behving in mnner nlogous to tht observed when electrospry sources were used with mss spectrometry. Our experience, coupled with tht reported by others, led us to further investigtions of ESI-IMS for the purpose of identifying the cuse of these differences between dt observed with ESI-IMS nd those expected from ESI-MS. The overll objective ws to design n IMS with predictble response chrcteristics nd which could be used for detection fter HPLPS. EXPERIMENTAL SECTION Two ion mobility spectrometers were used in these investigtions: The first ws identified s coronspry ion mobility spectrometer (CSI-IMS) since it ws primrily operted in tht mode. The second ws identified s the electrospry ioniztion ion mobility spectrometer (ESI-IMS) nd ws the design developed s result of informtion gined from experiments with the CSI-IMS instrument. The drift tubes of both spectrometers were bsed on the stndrd stcked ring configurtion used by vriety of commercil instruments. A series of stinless steel rings lternting with series of insultor rings were ssembled to produce the tmospheric pressure ion drift tube. The insulting rings served to isolte the stinless steel rings from one nother while the stinless steel rings, clled gurd rings, were connected by series of mtched megohm resistors. Applying high voltge to the first ring of the tube generted uniform electric field in the center of the tube, directing the coronspryed or electrospryed ions down the tube to terminl Frdy plte where current ws collected nd mesured. The CSI-IMS. This spectrometer ws designed by Shumtelo nd constructed under consignment by ScienTech, Inc. (Pullmn, WA). A schemtic digrm of the spectrometer is shown in Figure 1. Gurd rings in this design hd n internl dimeter of 2.5 cm nd were 0.8 cm in width. Teflon insultors, fitted round ech ring, served to isolte the gurd rings from the luminum housing s well s from ech other. The oven ws constructed from 10 in. long temperture-controlled luminum cylinder. Figure l provides view of the entire drift tube ssembly which ws locted inside the luminum oven, nd Figure lb shows cut-wy schemtic of the (7) McMinn, D. G.; Kinzer, J. A.; Shumte, C. B.; Siems, W. F.; Hi11, H. H., Jr. J. Microcolumn Sep. 1990, 2, (8) Hllen, R. W.; Shumte, C. B.; Siems, W. F.; Tsud, T.; Hill, H. H., Jr. J. Chromtogr. 19%9,480, (9) Shumte, C. B.; Hill, H. H., Jr. Electrospry Zon MobilitySpectrometry--lts Potentil s Liquid-Strem Process Sensor In Pollution Prevention in Industril Processes-The Role of Process Anlyticl Chemistry; Breen, J. J., Dellrco, M. J., Eds.; Americn Chemicl Society: Wshington, DC, (10) Shumte, C. B. An Electrospry Nebuliztion/Ioniztion Interfce for Liquid Introduction into n Ion Mobility Spectrometer. Ph.D. Thesis, Wshington Stte University, IONIZATION REGION,A, SPACER & +, Gurd rirg - region DRIFT REGION Figure 1. Cut-wy design of the ion mobility spectrometer showing the gurd ring nd ion gte ssembly (, top) nd the luminum housing which served s the IMS oven (b, bottom). luminum oven with only three gurd rings. In opertion, ll of the gurd rings shown in Figure l were inserted into the luminum oven. Electronic components of the spectrometer included n IMS controller, current mplifier, dt cquisition/control system, n interfce box, nd temperture controller. The IMS controller ws custom-built device contining electrospry high-voltge supplies (Model P, Bertn Assocites, Inc., Hicksville, NY), drift field high-voltge supplies, nd gte driver. The electrospry high-voltge supply provided both positive nd negtive voltge which could be vried up to V. The drift field supply provided positive nd negtive voltges which were vrible up to 5000 V. The coronspry needle ws modified from the design given in refs 6 nd 7 nd is shown in Figure 2. It consisted of 3 16 stinless steel union with ferrules nd compression screws (Wters Division of Millipore, Milford, MA). A commercilly vilble 25-guge polished needle (P/N 80726, Hmilton, Reno, NV) ws cut to length of pproximtely 1.3 in. The cut end of the needle ws inserted into Teflon sleeve (l/16 in. 0.d in. i.d.) nd pushed through loosened compression screw nd ferrule in the steel union. The compression screw ws tightened nd the entire ssembly inserted into Teflon housing which ws clmped to ring stnd. The polished needle tip protruded 0.05 in. inside the first of two stinless steel drift tube rings. The spry ssembly ws fed by prototype dul syringe micropump vi 3-ft length of PEEK tubing (l/16 in. o.d., in. i.d.). For solvent spry experiments, the first gurd ring ws bised t +250 V nd the second gurd ring ws t ground s shown in Figure 2. Solvents evluted were wter, methnol, cetonitrile, tetrhydrofurn, isopropyl lcohol, ethyl cette, methylene chloride, toluene, hexne, nd ism- AnlyticlChemistry, Vol. 66, No. 14, July 15,

3 "1 I I v, = I f I44 1Cm U Teflon Block \ Reducing Eledrorpry / Voltge solvent Row + solvent Rn + Plte I 'Drifi Dritt / I Figure 2. Schemtic digrms of spry ioniztion sources. (, top) Design used in solvent spry experiments. Needle: 25-guge polished SS, noninsulted. Solvent flow rte: 10 pl/min. Voltges: 5-6 kv on needle; 250 V on first gurd ring; second gurd ring t ground. Temperture: mbient. (b, middle) Design used in coronspry experiments. Needle: uninsulted 25-guge polished stinless steel inserted into IMS tube. Solvent flow rte: 10 pl/min. IMS nitrogen drift gs flow rte: 800 ml/min. Voltges: 5-6 kv on needle; 2-3 kv on first drift ring, generting drift field of V/cm in the IMS tube. Temperture: 'C. (c, bottom) Open design of electrospry IMS. In this design the needle is locted in the mbient temperture of lbortory ir nd outside the ion drift field. The focusing screen shown in this design served to direct the electrospryed ions down the center of the ion drift region. tne in ddition to severl mixtures typicl of HPLC mobile phses. In ech cse, flow rtes were djusted to 10 pl/min. For coronspry experiments, the IMS ws interfced to the Teflon spry ssembly s shown in Figure 2b. The spry ssembly ws identicl to tht shown in Figure 2 to which gurd ring stck, shutter grid, nd collector ssembly were dded to form typicl IMS drift tube s shown in Figure l. Four l/g-in. holes were drilled in the Teflon block which housed the needle ssembly, llowing the drift gs to exit the tube. An injector (Rheodyne 7000, Rheodyne, Cotti, CA) with 20-pL loop ws instlled between the pump nd the needle, leving 3 ft of PEEK tubing between the needle nd injector. The drift tube field strength ws set t V/cm. The drift gs ws nitrogen with flow rte of 800 ml/min. The drift tube temperture ws set t 60 or 80 OC. Needlevoltge ws estblished for ech set of drift tube conditions by the following procedure: The IMS ws turned on, nd spectrl scns were monitored with no solvent flow to the needle. Needle voltge ws initilly set to equl tht of the first gurd ring nd ws successively incresed until rectnt ion spectr were observed, indicting coron dischrge t the needle tip.6 Solvent flow rte ws then set t 8 pl/min, nd smples were injected. The solvent used for this study ws 50% methnol/ wter. With the sme drift tube described bove, the coronspry source ws modified s shown in Figure 2c to produce stble electrospry. The needle ssembly described bove ws 1 1 Region "'.*...,,,,.,,. I...( / Focusing Screen Desolvtion Figure 3. Closed design of the electrospry ion mobility spectrometer showing cooled electrospry needle, insulted electrospry needle, nd spry focusing screen. removed from the Teflon housing nd directly ttched to ring stnd with n insulted clmp. A grid consisting of nickel mesh ws fused to the first gurd ring of the drift tube to direct ionizd spry into the tube. Finlly, double-lyer Teflon sleeve, fbricted from shrink-tubing, ws slipped onto the needle tip. In opertion, the insulted tip ws plced outside of the drift tube, */&/4 in. from the focusing grid. Conditions for experiments, unless otherwise specified, were s follows: drift mode, positive; drift gs, nitrogen t 800 ml/min; pressure, brometric; desolvtion region, 4 cm; drift region, 10 cm; field strength, 310 V/cm; drift temperture, 167 OC; pulse width, 0.5 ms; gin, lo9 V/mp; scn time, 50 ms; scns verged, 50 or 100. The ESI-IMS. A schemtic digrm of this instrument is shown in Figure 3. As with the IMS described previously, the ion drift tube ws constructed from stcked stinless steel rings, seprted by Teflon insultors, nd electriclly connected by series of megohm resistors. This configurtion provided uniform electric field inside the drift region. The inside dimeter of the tube ws 4.22 cm, nd the outside dimeter ws 5.71 cm. All of the chrcteristics required for stble electrospry described for the previous design were incorported into the closed design. First, the spry needle ws removed from the drift tube of the spectrometer, minimizing the electricl field strength between the needle nd the drift tube wll. Second, focusing screen ws dded to the first gurd ring of the drift tube, directing the spryed ions into the desolvtion region of the ion drift tube. And third, the 2350 AnlfliclChemistry, Vol. 66, No. 14, July 15, 1994

4 27-guge SS spry needle ws insulted with 0.4 mm i.d. Teflon tubing with wll thickness of 0.2 mm. The slient difference between this design nd the one described bove ws tht the spry needle ws completely enclosed in the spectrometer, eliminting contmintion from lbortory ir nd stbilizing the temperture inside the drift tube. As result of enclosing the needle in the spectrometer, the spry needle hd to be externlly cooled in order to void voltiliztion of the liquid strem in the needle. When the liquid strem vporized before it reched the tip of the needle, the spectr becme unstble. As shown in the figure, externl cooling ws pplied to the needlevi wter-cooled/ir-cooled jcket. The entire ssembly consisted of wter chmber surrounding n ir chmber which housed the insulted spry needle. The electronics of the electrospry IMS included highvoltge supply for the spry needle, high-voltge supply for the drift field, temperture controller for the IMS oven, gte driver under softwre control, nd current mplifier to mplify the ion signl. The spry voltge ws supplied by two high-voltge supplies, one for positive nd one for negtive. Both were Model 602B-200P (Bertn Assocites, Inc. Hicksville, NY) with mximum potentil of V. Typiclly, V ws pplied to the needle for generting the electrospry. The drift field potentil ws provided by single 5000-V high-voltge supply (Model PMT-50 A, Bertn Assoc. Inc., Hicksville, NY). The current limit of the power supply ws 0.5 ma. At the totl drift tube resistnce of bout 16 M, only bout 0.31 ma ws flowing through the resistor chin connecting the gurd rings. The gte driver ws controlled by CACTUS PC/XT computer (Cctus Computer Co., Moscow, ID). The computer ws equipped with Burr-Brown PCI dt cquisition/control system (Burr-Brown Corp., Tucson, AZ) which included high-performnce crrier (PCI-20041C crrier with DMA cpbility, plugged into the 1/0 chnnel of the computer), high-speed A/D module (PCI-20019M), counter timer (PCI-20007M), nd PCI s high-speed nd DMA softwre. The high-speed A/D moduk (12-bit dynmic rnge, 54-kHz mximum smpling rte) ws used to mesure 0-1 voltge of the electrometer output nd convert this nlog signl to digitl one. The counter-timer module of the Burr-Brown bord, controlled entirely by lb-designed softwre, provided timing for digitiztion nd gtes. The solvent used throughout this portion of the investigtion ws mixture of wter, methnol, nd cetic cid in volume rtio of 47.5/47.5/5. The liquid ws pumped to the electrospry needle by dul piston syringe pump (Microgrdient System, BrownLee Lbs, Applied Biosystems, Snt Clr, CA). The syringe pump provided pulseless flow t low flow rtes (l-lopl/min.). Theinjector (C6W, VlcoInstruments Co., Houston, TX) ws six-port vlve with 6-pL externl injection volume. A 100 pm i.d. nd 200 pm 0.d. dectivted vitreous silic tubing (J& W Scientific, Folsom, CA) of proximtely 1 m in length ws used s trnsfer line. One end of the tubing ws connected to the pump through '/IS '/s*-in. zero ded volume internl reducer (lzrlst, Vlco Instru. Co. Inc.). Theother endofthetrnsfer linewsinserted through the 27-guge stinless steel electrospry needle. A l/1&/32-in. zero ded volume internl reducer (lzrl ST, Vlco Instr. Co. Inc.) ws used to hold the trnsfer line, so tht the exit of the fused silic trnsfer line ws recessed bout 1 mm from the end of the electrospry needle. Electrospry Ioniz~9. MssSpectrometry. A previously chrcterized lb-mde mss spectrometer with n electrospry interfce ws used to obtin mss spectr.ll For these studies, however, there ws no grid between the needle nd the inlet nozzle, nd the needle ws mintined t constnt positive voltge rther thn pulsed s ws described in ref 1 1. Mss spectr were cquired using Qudstr (Blzers) dt system. Resulting dt files were converted to ASCII formt nd further reduced using Microsoft Excel (Microsoft). Finished spectr were generted using Deltgrph (Deltpoint). Computer Simultion. Computer modeling of the electric fields generted by the ppliction of bis voltges ws ccomplished using the McSimion (Montech PTY, Ltd., Clyto, Victori, Austrli) ion trjectory simultion progrm performed on n Apple Mcintosh personl computer (Apple Computer, Cupertino, CA). Although the progrm ws designed to clculte nd disply ion trjectories in vcuum, the progrm module lso clculted the electricl fields nd displyed equipotentil contours imposed by the IMS geometry. With the electric fields ccurtely represented, the electrosttic forces experienced by the ions were described. Ion trjectories under tmospheric conditions re orthogonl to equipotentil contour lines of the electric fields. RESULTS AND DISCUSSION As stted in the introduction, the objective of these investigtions ws to identify problems ssocited with CSI- IMS with the finl gol of constructing stble electrospry ioniztion source for IMS which behved in mnner consistent with electrospry ioniztion sources commonly used in mss spectrometry. Initil experiments served to stndrdize hrdwre nd operting conditions, compring results with those chieved previously. The first experiments were performed without the drift tube in order to observe the spry nd evlute the spry of severl solvents. Through the use of the configurtion shown in Figure 2, severl solvents were spryed. These experiments permitted visul inspection of the needle tip, s well s thespry plumegenerted under field conditions similr to tht of stcked ring IMS drift tube. For ech solvent, needle voltge ws successively incresed until typicl plume ws observed, chrcterized by the ppernce of the "Tylor cone" t the needle tip. When voltge ws incresed beyond tht of the Tytor cone formtion, this single cone disppered nd severl smller jets could be observed. Most of the spry generted in this experimentl configurtion collided with one or both of the rings. At 5-6 kv, coron electricl dischrge ws clerly visible t the needle tip. Solvents investigted in this study included wter, methnol, cetonitrile, tetrhydrofurn, isopropyl lcohol, ethyl cette, methylene chloride, toluene, hexne, nd isooctne. Results of the test indicted tht dry hexne nd isooctne were not menble to electrospry. A spry for dry toluene ws ~ ~ ~ ~~ ~~~~ ~ ~ ~ ~ (1 1) Tomny, M.; Wittmcr, D.; Gbelcr, S.; Jrrell, A. Design nd Perfo"nce Chrcteristics of Prticle Bem Interfce nd n Electrospry Interfce: Procecding of the 40th ASMS Conference on Mss Spectrometry nd Allied Topics; Wshington, DC, My 31-June 5, AMmIC%e&W, Vd. 66, NO. 14, Ju& 15,

5 Tble 1. 8p.drrl D.1.otkn unlb of CSI-1m tor sekdod Compoundr th. StoRll Mod. "pd clss MW PdPL pmol/rl methiocrb pesticide tributylmine industril streptomycin sulfte ntibiotic phenyllnine minocid observed but only t voltges well into the coron regime. However, when solvent of higher dielectric such s methnol, cetonitrile, or THF ws dded in proportion of 5% (v/v) to ny of these, the mixture redily electrospryed t voltges somewht higher (0.5-1 kv) thn tht required for the pure modifier. All other solvents produced electrospry within the rnge of 2-3 kv. Stbility of the electrospry plume vried from solvent to solvent t givenvoltge. It generlly ppered tht plume stbility incresed with voltility nd polrity of the solvent, nd decresed with viscosity. A stble spry could be obtined by djusting the needle voltge. Initil investigtions sprying into the ion mobility spectrometer were conducted using the configurtion shown in Figure 2b. In this configurtion, IMS spectr for severl compounds were obtined nd good sensitivities observed. Exmples of detection limits for severl compounds re given in Tble 1. With this method, detection of mines I6oked prticulrly promising. Thus series of mines were evluted, nd ll provided good IMS response lthough KO vlues did not mtch those in the literture. Other problems ssooited with this design were lso encountered. At low tempertures, stble spectr were obtined for low moleculr weight compounds. When homologous series of orgnic cids successively were observed, the signl decresed drsticlly for hexnoic cid, nd no signl ws observed for heptnoic cid. A signl for heptnoic cid ws observed t higher (80 "C) temperturend ppered to be the result of well-resolved clusters. At tempertures greter thn 80 OC, the rectnt ion pek becme unstble, presumbly s result of outgssing or boiling of the solvent in the needle. Severl smll chnges were mde to the hrdwre, ech with n increse in performnce, but the problems inherent t low drift tube tempertures proved too troublesome to continue. Results of computer-simulted studies of the CSI-IMS design re shown in Figure 4. Equipotentil lines t 250-V intervls were drwn inside the drift tube. The collecting electrode, locted below the perture grid t the bottom of the figure, ws t ground potentil. The perture grid served to shield the collection from inductive effects of the ion cloud s it migrted down the drift tube towrd the collector. Just bove the perture grid ws reltively steep electricl grdient which focused the ions to the collecting electrode. In the min portion of the drift tube, the field grdient ws found to be reltively smooth with simulted field of 250 V/cm. Above the ion entrncegte, equipotentil lines indicted tht ions were directed towrd the wlls of the detector. This simultion, coupled with results found using the spry configurtion in Figure 2, indicted tht when the needle ws locted inside the drift tube, sensitivity ws lost due to neutrliztion of ions on the gurd rings of the spectrometer. In order to focus ions into the center of the ion drift region where the field ws uniform nd to void problems ssocited Sorry - Neele Aluminum, Oven In,ultor, I rte 4 om I Reglon 0.0 cm 4prnture Qdb - OlleCIor- Gwd ~IQ 1.d. Qmrror 2.5 cm m--, 4-4. cnpner-tknofowresprey~moblltyspectrometer showhg the contour profk of the equipotentll Held lines. Ech equipotentll Held line in the flp6 potentiel drop of 260 V. Note the okwkr nture of the eqqulpotentll Ih.les ner the spry needle^. Slnceb~trevd~tofbklHnee, moetweredlrected rdilly to gurd rings insted of bnghudlnlly to the center of the drlft tube. with heting the needle, the needle ssembly ws removed from the drift tube s shown in Figure 3c. A gfid consisting of nickel mesh ws fused to the first gurd ring to direct the spry into the drift tube. With the needle removed from the drift tube, temperture of the drift gs could be incresed to bout 170 OC, permitting efficient desolvtion of the spry drops prior to reching the ion entrnce gte. In ddition, the needle remined wol, voiding buildup of nonvoltile mteril t the needle tip. Becuse the needle ws locted outside of the drift tube, the ion spry could be observed. Under these conditions coron dischrge could be seen similr to tht observed in the experiments described for Figure 3. Insulting the tip of the spry needle with Teflon tubing eliminted visul observtion of the coron dischrge. With the insulted needle, there ws less tendency for rcing from the needle tip to the ring or grid, llowing higher field strength to be generted t the origin of the plume, either by moving the insulted spry tip closer to the grid or by incresing needle voltge. With incresing needle voltge, it ws found tht the mximum flow rte t which stble plume nd spectr were obtined hd incresed from typicl 6-8 pl/min to typicl 16 pl/min. Minimum flow remined 1 pl/min, the limit of the pump. Under this configurtion, electric current from both noninsulted nd n insulted needle ws monitored nd recorded s function of voltge with no solvent flow pssing through the needles. Results of this experiment re shown in Figure S. With the noninsulted steel needle, onset of the coron dischrge could be clerly seen s shrp increse in 2952 Am~IcIChemis~, Vd. 66, No. 14, Ju& 15, 1904

6 . steel m Insulted needle needle 5 1 elmktednccdlr ledu&w Flgure 5. Electrospry needle current. (, top) Comprison of totl ion current from Insulted nd noninsulted needles. (b, bottom) Comprison of totl ion current from n insulted needle with nd wlthout solvent flow. These dt illustrte the fct tht the insulted needle increses the potentil rnge In which stble electrospry cn be obtined. Tble 2. Reduced Ion Mobl#u.r (&) of Olgnlc Amlm from ESI-IMS Compred with NI Source Literture Vlues mine MW Ko (ESI) KO (NOR) KO (63Ni) mmoni methylmine n-but ylmine triethylmine lutidine isoquinoline dibutylmine tributylmine tetrbutylmmonium tetrpentylmmonium tetrheptylmmonium tetroctdecylmmonium Moleculr Weight of Prent Compound Figure 6. Log MW vs Ion mobility constnts (KO) for quternry nd tertirymlnes. Dt wereobtined with ESI-IMS configurtion shown in Figure 2c. Dt plotted re shown in Tble 2, demonstrting tht tetrlkylmmonium Ions pprently do not dissocite during soft electrospry ioniztion ~~ r I / *KO = (&/Vt)(P/760)(273/r) where d is the drift distnce, Vis the potentil cross the drift distnce, t is the ion drift time, Pis the pressure in Torr, nd Tis the temperture of the drift region in degrees Kelvin. needle current round 8-10 kv. While smll current ws observed for the insulted needle, no shrp increse in current ws noted in this region, indicting tht bckground ioniztion from the coron ws significntly reduced with the insulted needle. Figure 5b compred the needle current observed for 8 pl/min of solvent (50% methnol/wter) flowing through the insulted needle with tht observed for the no solvent flow condition. In similr experiments reported in ref 6, it ws found tht, with noninsulted needle locted inside the drift tube, the region in which solvent flow enhnced current over the no flow condition ws quite limited. Thus the primry dvntge of the insulted needle ws to provide n incresed rnge of needle voltge nd flow rte over which true electrospry ioniztion spectr could be obtined. To investigte the extent of desolvtion with this design, series of mines were electrospryed into the ion mobility spectrometer nd their KO vlues clculted. Indequte desolvtion resulted in ions of lower thn expected mobilities. As shown in Tble 2, most KO vlues compred well with those obtined by KrpsI2 using conventionl 63Ni ioniztion source. This ws especilly true when KO vlues were Drift Time (mr) Flgure 7. Electrospry ioniztion ion mobility spectrometry of cytochrome c. (, top) Open electrospry design nd (b, bottom) closed electrospry design. By comprison with ESI-MS spectr In Figure 9, this figure Illustrtes the first exmple of multiply chrged ions seprted by IMS. normlized to 1.95, the KO vlue for lutidine. The exceptions were those of low moleculr weight, which ppered in the region of the bckground solvent (rectnt ion) pek. With regrd to orgnic mines, electrospry ioniztion did hve t lest one distinct dvntge over the 63Ni source. The KO vlues of quternry mines hve been reported s difficult to mesure, presumbly due to dissocition in the IMS cell. This ppered not to be the cse with this electrospry configurtion. Figure 6 shows reltively smooth curve when log MW ws plotted s function of KO for quternry nd tertiry mines together. Figure 7 nd Figure 8 re ion mobility spectr of high moleculr weight compounds. Detection of these compounds (12) Krpes, 2. Anl. Chem. 1989, Anlyticl Chemistty, Vol. 66, No. 14, July 15,

7 h /IB tb ws not possible with CSI-IMS. Figure 7 is n ESI-IMS spectrum of protein, cytochrome c, nd Figure 8 is the ESI-IMS spectrum of polymer, Triton X Both spectr produced response for ions drifting from bout 20 ms to bout 35 ms. In ech cse, some resolution of individul ion peks cn be observed. Figures 7b nd 8b show ESI-IMS for the sme compounds with the closed design shown in Figure 3. This instrument differed from the prototype in tht the electrospry needle ssembly ws seled from the lbortory tmosphere nd tbe internl dimeters of the gurd rings were incresed from 2.5 to 4.22 cm. To prevent voltiliztion of the solvent in the spry needle, the needle ssembly ws wter cooled. Advntges of the seled configurtion were tht the temperture in the drift region of the spectrometer remined uniform throughout the desolvtion nd drift region nd contminnts from the lbortory ir were excluded from the ioniztion region. Spectr obtined with the seled source ppered to exhibit both higher sensitivity nd higher IMS resolution. Figure 9,b shows ESI-MS spectr of cytochrome c nd Triton X For cytochrome c, seven ions were observed, corresponding to multiple chrged ions contining +12 to +18 chrges. Muitiplechrgedionsrechrcteristicof ESI- MS of proteins which hve number of bsic sites. Compring the MS spectrum with the IMS spectr, it is possible to speculte tht themultiplepeksokrvedintheimsspectflun were lso due to multiple chrged ion species. If this is true, it is the first time tht multiple chrged peks hve been reported in IMS. The ESI-MS spectrum of Triton X-100 ws of singly chrged ions corresponding to 1 1 different oligomers rnging in degree of polymeriztion (n) from 5 to 15. In the IMS spectrum using the open needle design, totl of men ion peks were detected nd with the closed design 14 peks were observed. In both cses the ptterns of IMS spectr were similr to those obtined by ESI-MS. CONCLUSIONS True electrospry ion mobility spectrometry hs been chieved. To chievo elotrospry colrdith, three importnt problems which occurred in previous designs OP ion spry ion mobility spectrometers were identified d comctd. The problems were the following: (1) bokgrctund contribution nd dissocitive ioniztion from corm diwhrge, (2) reduced response due to ion collection on the wb d the drift tube, nd (3) prcspry precipittion of nonvoltiio mpounds in the needle due to solvent voltiliztion. In ddition, voltiliztion of the solvent in the & prior to sprying led to unstble spry due to bubbk of vpor Sppttins from the tip of the needle. Thus, successful opertion of IMS in the electrospry ioniztion regime requires three specific design modifictionsofthespryneedlefromtht nportedin previous studies: First, ion contribution of coron dischrge to the overll current of IMS cn be eliminted by enclsing the n d e in nonconductive (e.& Teflon) sleeve. Under thecoditions, ions re only produced s result of dectrospmying solvent. When no solvent flow is present through the needle, no ions in the IMS will be observed A w l chemlsby, Vd. 66, No. 14, JurL 75, lssl

8 Second, when spry needle is inserted into the drift region of the IMS, electric field lines re produced such tht trjectories of the electrospryed ions re towrd the wlls of the drift tube, reducing the current directed down the tube. Creting the spry outside of the dirft region nd dding focusing screen which is positioned cross the entrnce to the ion drift region nd few centimeters in front of the tip of the spry needle directs the electrospryed ions into the center region of the drift tube. With this modifiction, more of the ions rech the ion drift region nd the sensitivity of the instrument is substntilly enhnced. Finlly, desolvtion of the spryed ions requires elevted tempertures of the drift gs to evporte solvent from the ionized spry drops s they migrte down the drift tube nd before they rech the first ion gte of the spectrometer. When the electrospry source is incorported s n integrl prt of the instrument in closed design, het trnsfer from the drift gs to the needle is sufficient to voltilize the solvent in the needle prior to electrospry. To prevent solvent voltiliztion in the spry needle, the needle must be cooled. With cooled nd electriclly insulted ion spry needle, nd with the electrospryed drops focused into the desolvtion region of the IMS, stble nd well-resolved response ions for nonvoltile high moleculr weight compounds cn be chieved. Moreover, the ion ptterns produced with this electrospry IMS were similr to those observed in electrospry mss spectrometry, indicting tht the response nd behvior of n electrospry ioniztion source for IMS cn be predicted from mss spectrl dt. Although optimized sensitivity studies were not conducted in this investigtion, estimtes mde from these studies predict tht the detection limit for IMS response will be similr to or less thn tht chieved for most opticl detection methods. Thus, the electrospry IMS ppers to offer vible complementry detection method for liquid chromtogrphy nd cpillry electrophoresis. Further investigtions of this design with respect to liquid-phse detection re currently being conducted. ACKNOWLEDGMENT The uthors express their grtitude to Andy Jrrell, Steve Gbeler, nd Mike Tomny for their ssistnce in these studies. Initil dt included in this pper were first reported by D. P. Wittmer t the 1992 Workshop on Ion Mobility Spectrometry held in Mesclro, NM, June Received for review December 21, Accepted April 11, 1994.' Abstrct published in Advunce ACS Absrrucrs, My 15, An&fklChemistry, Vol. 66, No. 14, July 15,

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