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Mass Spectrometry and Chromatography

by: Guiseppe Bednar

Mass Spectrometry and Chromatography CHEM 5181

Marketplace > University of Colorado at Boulder > Chemistry > CHEM 5181 > Mass Spectrometry and Chromatography
Guiseppe Bednar

GPA 3.93


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This 44 page Class Notes was uploaded by Guiseppe Bednar on Friday October 30, 2015. The Class Notes belongs to CHEM 5181 at University of Colorado at Boulder taught by Staff in Fall. Since its upload, it has received 32 views. For similar materials see /class/232186/chem-5181-university-of-colorado-at-boulder in Chemistry at University of Colorado at Boulder.


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Date Created: 10/30/15
Lecture 3 Ionization Techniques Part II CUBouMer CHEM 5181 Mass Spectrometry amp Chromatography J Kimmel Fall 2006 Electrospray Ionization fragmentation Nobel Prize 2002 Liquid elutes through a high voltage tip D l IAtmospheric pressure ionization IEnables MS detection of large nonvolatile V 39quot molecules eg proteins With no z 3 f Coulombic explosions yield a continuous 35 W mist of bare gasphase ions positive or negative Conveniently coupled to liquid separations Characterized by multiply charged ions 391 Needle lip Newobjectivecom Electrospray Mechanism An electrolytic analyte solution is pushed through the conductive end of capillary id 10100 um at very low flow rate 01 10 uLmin held a few mm from the entrance of the MS High potential 24 kV induces a strong electric field 106 107Vm1 For positive field cations will move towards the liquid surface and anions will move towards the conductive tip Repulsions between adjacent cations combined with the pull of the cations towards the grounded MS inlet cause the surface to expand into a socalled Taylor cone IDDD quot 2 Gomez amp Tang Phys Fluids 1994 6404 41 4 ESI Mech con t As this induced electric field overcomes the surface tension of the liquid the tip of the cone elongates into a filament which breaks up and emits a stream of charged droplets towards the inlet of the mass spectrometer Evaporation of solvent from the droplets increases the charge density At the Rayleigh limit repulsion between cations equal surface tension causing Coulombic explosions that produce even finer droplets This process of evaporation and explosion repeats until fully desolvated ions are released The release of ions occurs either by repeated fission events until total evaporation of the solvent Charge Residue Model or by direct ion emission from a charged droplet Ion Evaporation Model EEEDDD V k39 Gomez amp Tang Phys Fluids 1994 6404 41 4 ESI Mass Spectrum IEEDDD 4 g 1 3 2i 1 5 High charge states makemz practicle for most mass analyzer types 2 can be determined by isotope 15 distibution or sequence of peaks see section 181 of De Hoffmann and HW 2 1 5 r 1 Argue ur Jrn lED I I I a I N 5 1 I l q 14 tilt BEIGEquot quot1 1 ll mi ESIMS of Cytochrome C 12360 Da From Fig 1318 Lambert ESI Source Design IEEDDD ESI source must 1 2 3 On1 On 3 Move ions from solution to the gas phase Transfer the gasphase ions from atmospheric pressure to vacuum Yield ion beam with maximum current and minimum kinetic energy distr bution N2 8000 3 to W ions gl H Ii Efzzle Skimme Analyzer Electrospray Metallic caplilrH PD 4quot Stable spray requires user optimization High flow rates may require nebulizing gas to form droplets J 39 Electrospray Sheat gas metallic OW Heated capillary Tube Skimmer capillary lens Heated drying gas capillary encourage Figure 117 Diagram of ESI sources using Skimmers for ion focalization and a curtain of heated nitrogen gas for desolvation top or With a heated capillary for desolvation bottom desolvation and limits solvent analyteadduct formation during expansion Pumping speed places practical limit on size of entrance aperature Transfer of ions between stages of decreasing pressure can result in a total ion loss on the order of four to five orders of magnitude From de Hoffmann For discussion see ESI Source Design and Dynamic Range Considerations A P Bruins in Electrospray Ionization Mass Spectrometry R B Cole 1997 Harnessing expansion Constant Velocity high E distribution Controlled Current Electrolytic Flow Cell D Metal Plate 0 ESI Droplets 10 V T aylur Cone a S pray N eedle 9 gate 275 kV 13quot 3955 Mass Spectrometer 7 a ESI Suluum a a 9 05 39t 9 ee fa39f e e Execs s quot Oxldanon From Cech and Enke Mass Spec Rev 20 362 2001 Spray Current 2 25 kV power supply Eectrica circuit to sustain ESI current Terminal to tip to counter electrode to Terminal Eectroysis at electrodes maintains the charge balance to allow continuous production of charged droplets ln orderto supply demanded current potential at electrodesolution interface has value permitting the oxidation process characterized by lowest oxidation potential in solution This process determines the total of ions that can be produced per unit time ESI Concentration Dependence D tis the excess charge in final droplets that imparts charge to gas phase ions See Fig 124 In De Hoffmann ESI is sensitive to concentration not flow Because limiting current IM is dependent on OXIdation process at tip ESI response can vary significantly among different analytes that have identical concentrations For a system with one analyte spray current for will depend on its concentration and a analytespecific rate constant lA A For system oftwo analytes A and B lr lM A B And currents proportional to relative desorption rates and signal responses are coupled Complicates quantification See section 184 of de Hoffmann For any system dynamic range limited at end 1 mM by Limited amount of excess charge Limited space on droplet surface lon suppression Consider separations prior to ESI to maximize sensitivity Nanospray 10 100 nllmin flow rate with line spray tip 11000 ESI ow Droplets 1001000 times smaller than conventional E Large proportion ofanalyte available for desorption from surface 23 time higherion current than ESI at a given concentration Smaller tip close to ori ce narrow dispersion of droplets yields better transfer in MS 0rders of magnitude 2 improvement in efficiency analyte detected I analyte sprayed At these ow rates ESI becomes mass ux sensitivequot Longer analysis times better SNR andor more options in MS experiment New Objective SillEaTlpS Tip id range mm in an urn Fluvv rate ZEItu iEiEiEi nLmin See VWrnarid Mann A Chem EE irE iBBE phase sample analysis from static matrix El and Cl are methods for molecular analysis of gas APCI and ESI molecular analysis of liquid phase Now desorption techniques that allow molecular Fast Atom Bombardment FAB and Secondary Ion Mass Spectrometry SIMS IDDB primary 5 beam of bombarding particles ion optics for mass analysis of secondary ions analyte dissolved in matrix FIG 91 Bombardment of a sample dissolved in a liquid matrix by a primary beam of atoms or tons to produce secondary ions that are characteristic of the analyte a analyte b bom barding particle m matrix From Watson Desorption techniques Analyze the ions emitted when a surface is irradiated with an energetic beam of neutrals FAB or ions SIMS Producing Primary Beam IEDDD Ar From de Hoffmann Gn ium Surface Ionization ErinIt ten ETD t z o i U gang E jlg j n G lt Ar F g w Electrode jiLlj39lII EE T T rWmi thttrtmntli EE 39 39 a i 5 Figure 112 FTquot Diagram of a FAB gun 1 Ionization of argon the resulting ions are it a I I accelerated and focused by lenses 2 In 3 the argon ions exchange their flquot jnnjut charge with neutral atoms thus becoming rapid neutral atoms As the beam If Emquot path passes between the electrodes 4 all ionic species are de ected Only Ti lamlg PM 39I39 1 rapid neutral atoms reach the sample dissolved in a drop of glycerol 5 all The ions ejected from the drop are accelerated by the pusher 6 and focused by electrodes 7 towards the analyzer 8 SIMS primary ions are Primary Beam of neutrals produced eg as Cs atoms produced by Ionizing and vaporize through a porous accelerating compound into tungsten plug charge exchange collision with neutral eg From SIMS Tutorial Q httpwwweagabscomen Ar rapid Arslow 39 Arsow Arrapid USreferencestutorialsimstheocaistheohtml Static SIMS Low current 1010 A cm2 of keV primary ions Art Cs impact the solid analyte surface Low probability of area being struck by mulitiple ions less than 110 of atomic monolayer consumed Primary ion beam focused to less than 1 um enables high resolution mapping Often pulsed beam TOFMS Sensitive technique for the ID of organic molecules Spectra show high abundance of protonated or cationized molecular Ions Yields depend on substrate and primary beam as high as 01 ions per incident ion Elemental yields vary over many orders of magnitude FAB and liquidSIMS Sample is dissolved in nonvolatile liquid matrix is bombarded with beam of neutrals FAB or ions Shock wave ejects ions and molecules from solution Generally eject ions that already exist in solution Presence of charge has little effect on the desorption process Neutrals used out of convenience for coupling to some instruments Use of liquid allows high primary ion currents while maintaining molecular Ions Solution presents mobile constantly renewed surface to beam Disadvantage substantial background of matrix eg Glycerol ions and matrix adducts Flow FAB Continuous flow of liquid into mass spectrometer at rate of l 20 uLmin Allows more use of more volatile solvent eg water methanol or acetonitrile Can be coupled to separation Matrix Assisted Laser Desorption Ionization MALDI Analyte molecules are embedded in a crystalline matrix composed of a low molecular weight organic species Dried mixture is struck with a short intense laser pulse having a wavelength that is strongly absorbed by the matrix often UV Rapid heating of matrix causes sublimation and expansion into gas phase Intact analyte molecules carried with little internal energy Most widely accepted ionization mechanism is gas phase proton transfer Efficient soft and relatively universal wavelength independent of analyte Matrix isolates analyte molecules preventing clusters Allows analysis of large 100s of kDa intact biopolymers 2002 Nobel Prize IEEDDD laser analytelmatmt spot near matrix inns l l l l 39 To mass 1 spectrometer l l l l l cation extraction fGCussing gnd lens sample piste Image from Univ of Bristol http wwwchmbrisacukmstheorymaldiionisationhtml Example of MALHDlI Data 10039 Spectra contain mostly singlecharged ions n C Relative intensity IEEUUU MH 149190 3M2H22MH 50000100000 200000 300000 O Fragmentation due to m excess energy imparted on Ami00 analyte during DI process is gm possible Prompt Fast or 75 Post Source f I I 50 will 5 I I l Optimized conditions 2539 39 39 determined empirically I I I I I I I 4000 5000 6000 7000 8000 10000 Figure115 The MALDI spectra of a monoclonal antibody top and polymethy1 methoacrylate of average mass 7100 Da bottom Reproduced modi ed from Ref 24 and from From De HOffmann Finnigan MAT documentation with permission MALDITOF Mass Spectrometer DUB Analyser From Barker MicroChannel Focus Source plate detector Ion beam l I 1 I n Rotable source E I I J U stage l l igt Start detector Window ll N2Laser e Mirror I Nd Focusing Beam filter lens splitter Figure 263 Schematic representation of a MALDI TOF mass spectrometer MALDI conveniently coupled to ToFMS 1 Pulsed source pulsed analyzer 2 Large mz large mass range See lab 1 HW 3 and next lecture Atmospheric Pressure Desorption Ionization IDDD 2000 AP MALDI is first atmospheric pressure ionization technique for condensedphase analyte Burlingame et al A Chem 652 2000 2004 Desorption Electrospray Ionization DESI Cooks et al Science 2004 471473 2005 Direct Analysis in Real Time DART Cody et al A Chem 2005 2297 2005 Present Rapid development application and evolution of these methods Desorption Electrospray Ionization Image from Cooksela Soleme 2004 4717473 l HV 339 WW Atmospheric lnlel of Solvonl 39 mass spectromelev N 7 Inn translar llne Nebulizer capillary 13 Gas jet Desorbed 1 ions A Spray 7 7 7 Surface L g g 4 Freely moving s sample stage in air Photos from mm MNva prosolla cornindex hlml Advance DESI and DART Analysis in free ambient environment sample potentially subject to arbitrarily chosen processes during MS analysis Applicable to small molecule organics and large biomolecules in solids liquids and adsorbed gases Natural products in plant material High throughput of pharmaceuticals oBiological tissue and fluids Forensics Public safety Typically no pretreatment Sample is sprayed with an electrically charged aqueous mist Released ions are transported through air to MS interface which may be long transfer line Desorption Electrospray Ionization DDI Solution can be tuned to make processselective Relative movement of sample and spray enables spatial resolution down to 50 um for imaging of surfaces I I I w power supply Afmosphevlc mm ol The momentumtransfer collisions of DI In vacuum smwm WV mquot 39 m 39 5 Ion Irnnsfor line are not applicable as prolectlles have low kinetic mummnm Two mechanisms ESIlike DESI produces multiply charged ions from biological macromolecules Ionization believed to occur through formation of charged droplets after liquid spreads across surface APCIIike For molecules that cannot be analyzed by ESI eg nonpolar compounds see singly charged species indicative of charge transfer between liquid and surface or ionmolecule reactions Requires potential 2kV between sprayer and surface 53 9 Desurbed I inns 4 Freely moving E sample stage In air Image from CookSeIa Soleme 20044717473 Summary of DI Techniques IEDDD TABLE 1314 Summary of Characteristics of DI methods Sample type Nonvolatile thermally labile Basic technique Prompt delivery of energy to sample by energetic beam Matrix Organic liquid or solid enhances ionization minimizes fragmentation Ionization mechanisms Direct ion emission Cationization and ionmolecule reactions Energy deposition Broad both a soft and a hard method Ionization efficiency 104 in FAB to 10 4 in MALDI sample can be recovered Mass range 104 Da FAB liquid SIMS to 106 Da MALDI From Lambert Elemental Analysis Notes IDDB Need to decompose analyte into vapor phase ATOMS Eg to determine isotope ratios Souces are also used by optical spectroscopy MS tends to produce less complex signals and can be more sensitive Thermal Ionization One of the earliest ionization techniques for MS Remains the most precise and accurate method in MS to determine isotope ratios of solid samples Sample may be gas phase or deposited on filament surface One or more filaments are heated to high T by passage of a current Eectron transfer between atom and filament produces intense stable beams of positive andor negative atomic ions Multiply charged ions are not observed and clusters are rare Multipe filaments allows separate control of vaporization and ionization parameters IEEDDD JENIZM39EON FL LEHEHT FFtL MEHT SHiEiLD 7 SAMPLE FiLAMENTE 7 w DEFUEUESING SLIT W was BHlM lHlATING BUT w l WFDEUBSME SLLT mimq g LLlM TtMG BUT w 39 BJEAM CENTEHIMB H I ll l2 amp II IL 1 r jij ESWLLIHATHNG SLIT 294 r a H I I m 139 IGN BEAM EEGTIDH THRGU 6H h A Figure from lnghram and Chupka Rev Sci Inst 24 518 1953 Thermal Ionization cont d IDDD filaments lon ield range from 1 to 10 anddepend on electron affinity of the analyte the work unction of the filament material and the temperature of the filament Work function may depend on treatment of filament surface See Heumannetal Analyst 120 1291 1995 Positive Ions compounds with low first ionization potentials high work function Negative ions High electron affinities low work function filaments Thermal ionization cavity source offers order of magnitudes improvements in efficiency Metal tube is heated by high energy electron bombardment As the sample evaporates inside the crucible gaseous analyte atoms are produced which interact with the inner surface of the crucible walls to produce positive ions through surface ionization Ion beam l gt HH Metal tube till V Electron bombardmek Figure 131 Schematic representation of a thermal ionization cavity source From de Ho mann Spark Ionization Source Most transition metals difficult to ionize thermally Spark developed by Dem pster to extend MS analysis to metals Pulsed 1MHz rfvoltage of several kilovolts produces discharges between two rod electrodes Sampe may serve as one electrode or be mixed with carbon and placed in cup electrode onization occurs within plasma Sineg and multiply charged atomic ions polymer ions and heterogeneous compounds ntensity of major constituent peaks decreases with charge number Approximately equal sensitivity for all elements Useful for analysis of trace impurities DLs in ppb range Ions have very wide energy distribution and the spark is subject to random fluctuations in intensity Insulator EIGCTFOde UL liii Vaccum pump From de Hoffmann Figure 132 Typical spark ion source From Dempster RevhSci Inst 7 46 1936 1 by electrostatic and magnetic de ections 317 Fm 4 Ions will high frequency spark analyzer a I right angles a Platinumplatinum Ib tungstensteel Glow Discharge Source PIN CATHCJDE R INSULATDR AND LIIHINGE SEAL GAS GAS PIN SAMPLE EDUFICE From King Teng and Steiner J Mass Spec 30 1061 1995 J PLANAH SAMPLE SOURCE F39FID EII TIP IDDD 39 mamquot mow 1 v I JF39 39i V Produces singly charged atomiccations Used primarily for bulk metal analysis Discharge between cathode sample and anode in Ar at low pressure ca 0110 Torr 5 mm gap 1 kV 12 mA Sputtering Ions and electrons from plasma accelerate toward electodes Ar attacks cathode surface releasing M neutral and M 80 of potential drop occurs in nonluminous dark space cathode fall extending 1 mean free path from cathode M produced by sputtering cannot escape this region Effectively decoupling atomization and ionization steps and reducing matrix effects on ionization Colisions and ionization abound in Negative Glow Neutral M ionized by multiple pathways GD Collisional Ion Formation Processes D I Electron Ionization o I Penning Ionization Within Negative Glow electron ionization and Penning Ionization account for 90 of the observed M Penning Ionization U ABC gtABCe O O J ABC ABCe BC A Fig It The cleclronytrunsfer proccss in Penning ionization From Fallbert et a1 IJMS 1993 6977 Electron transfer reaction Occurs if ionization potential of BC is less than excited state energy of A Inductively Coupled Plasma IDDU ICP source was originally developed for Atomic Emission Spectroscopy of solutions CP MS is capable of trace multielement analysis of solids or liquids often at the part per trillion level Can detect all elements except F Ne and He Ionization efficiency gt 90 for 54 elements Plasma Load Coil Ground FIGURE 31 Plasma With torch assembly and load coil Hardware 3 concentric quartz tubes Argon flowing through each at atmospheric pressure Cooled induction coil surrounds the top of largest tube powered by Rf Generator An Initial Tesla sparks ionizes flowing argon Rf field accelerates these ionsL leading to collisions and more Ions Equulibrium between Ionization and recombination Plasma reaches 10 000 K Photo from httpewrceevteduenvironmentalteachsmprimericpmsi cpmshtm ICP Source IDDDD High LOW From de Hoffmann vacuum vacuum pump pump rf Generator Ar 39 l L l min 1 OOi lg g Ion beam R if 5 39 quot r 355 9 000 I Peristaltic um Coil p p Plasma ml min1 Sample solution Figure 134 Schematic diagram of an inductively coupled plasma source Sample vapor or droplet carried into plasma through central tu be High T desolvates vaporizes atomize and ionize sample Close to 100 efficiency As for other atmospheric pressure ionization sources transfer to MS requires differentially pumped interface ICPMS VS ICPOES ill 08 Pl 35 ppm Mass Spectrum of sludge sample by ICPMS 4s Ti 1m 3 quot1quot 5 111m 115 ppm as Sr In 15 ISIS ppm 13 PP 159 Th 18 ppm 11 115 12 l 4 l I I I I so 12 l l 2m 41 Ar Ar NH H H 33 Ar Spectrum of 100 ppm CE quot C Ce by ICPDES I q Courtesy of Selby 1 Hireftje Amer Lab Aug 1987 E C N Ar 3 i2 E a a E 0H 39 I quot i 39 l continuum l I I I 2m 3m 4m 5m ism Wmleng l 11m IDDE MS much simpler than the optical emission spectra Most heavy elements exhibit hundreds of emission lines but they have only 110 natural isotopes in the mass spectrum lCPOES suffers from many overlapping spectral interferences from other elements and a very high background emission from the plasma itself limiting detection limits Detection limits of lCPMS are three orders of magnitude better than lCPOES Comparison of Molecular Ionization Techniques Table 23 Comparison of various ionization methods IDDU Ionization Separation Method Ion Type Sample Type Technique El M39 fragments Nonpolar and some polar organic GC compounds Cl M H M H Mquot Nonpolar and some polar organic GC compounds Thermospray M H M H Polar compounds LC M NH4 FAB M l H M H Peptides proteins lipids LC CE carbohydrates oligosaccharides nucleotides oligonucleotides APCI M H M H Polar compounds drugs LC ESI M nH M nH Peptides proteins lipids LC CE carbohydrates oligosaccharides oligonucleotides MALDI M H M Hr Peptides proteins lipids LC CE carbohydrates oligosaccharides oligonucleotides Desorption Ionization Summa ry IDUD TABLE 1311 Procedures Used in Desorption Ionization Ionization Energy Mass Method Source Flux Matrix Analyzer Comments Static secondary keV ions 10 10 A cm 2 None solid Any Surface sensitive ion MS SIMS Art Cst etc nondestructive low signal Liquid SIMS keV ions 10 6 A cm 2 Liquid Any Higher longer 1013 ions cm 2 s lasting signal Cst etc Fast atom bom keV atoms 1013 atoms cm 2 3 1 Liquid Any High longlasting bardment Xe atoms signal high FAB background Plasma desorp MeV ions 103 particles cm 2 s 1 Nitrocellulose Timeof flight High ionization tion PD efficiency low signals Laser desorption Photons 2106 Solid matrix Usually time CW lasers can LD and ma watt cm 2 absorbs offlight cause thermal triXassisted radiation in degradation laser desorp MALDl tion MALDI From Lambert Igor Intro Fall 2007 Chem 5161 and 5181 Donna Sueper Igor IS Made by Wavemetrics wwwwavemetricscom Used for data analysis generating publicationquality graphs data acquisition less common Much like Mathematica Origin Sigma Plot C programming language Key Igor Concepts Wave 1 23 or 4 dimension ordered set of values text or numeric individual values within a wave are referred to as points naming best to use only the characters aZ and 09 Command window place to enter commands for execution also serves as a history of previously executed commands Experiment contains all parts in one file data code graphs tables etc pxp extension Procedure filewindow p ace where user created code lives XOP external operation file code modules that adds functionality Not case sensitive Starts counting from 0 Igor Tasks for Today A Demonstrate basic tasks Create modify simple data Create modify simple graphs B Begin to step through aWavemetrics tutorial Open up a demo experiment to viewmanipulate Gauss Lorentzian and Voigt lineshapes Task A Demonstrate Basic Tasks Make a wave called xwave of101 points Make a table ofthis wave Change the values in xwave to be 50 49 4950 xwave 50 Duplicate this wave name it ywave Change the values in ywave to be ywave 1 3exp xwave20quot2 Plot ywave vs xwave Create another wave named ywave2 and add to plot ywave2 1 3exp xwave30quot2 Modify the graph by adding legends axis labels and displaying points and markers Add cursors to graph to view individual values 10 Save graph macro 11 Recreate graph via graph macro 12 Save the experiment 0 Task B Step Through a Wavemetrics Tutorial 1 Open up le Programs Wavemetricslgor Pro FolderExamples Curve FittingMultipeak tpxp 2 Follow Tutorial 3 Play For More Igor Help Go through the HelpGetting Started tutorial Don t be afraid to explore Contact me Office Eckley 141 next to Maggie Tolbert s of ce Of ce hours Tues 1 2 Email donnasuegercoloradoedu Or Ingrid Ulbrich Email ingrid ulbrichcoloradoedu Mass Analyzers lll Quadrupoles Chem 5181 Fall 2007 Joel Kimmel Announcements Please reenter your iClicker is spreadsheet Forgot to save last time Journal Skim 1 is due today Handin printed copy in class or email to Joel by tonight Lab Times Group 1 Wednesday 1 4P Group 2 Friday 9A Noon Mass Spec Facility Tour Postponed Until Next Tuesday 911 Lab Group 1 930 to 1005A Lab Group 2 1010 to 1045A Science Journal Presentations Date Presenter Oct 4 Coburn Oct 16 Herzenach Oct 23 Thalman Oct 30 Axson amp Craven Nov 27 Robinson Dec 4 Tienes A B H I O quot 1 m H mlz 3441433 mlz 3441069 Suppose you are attempting to separate these two compounds by LOMS The first compound to appear in your chromatogram has an intense peak at mlz 3441421 You know that your mass spectrometer has mass accuracy greater than 7 ppm What conclusion can you make Compound A is the first to come off of the column Compound B is the first to come off of the column You are measuring an average of the two compounds that contains mostIyA Nothing You do not have sufficient mass accuracy to determine which compounds you are measuring Q How many of the following statements are TRUE 1 To increase them2 range of a TOFMS one must lengthen the drift axis 2 The magnitude of an ion s velocity is equal when entering and exiting a reflectron 3 A TOFMS can simultaneously measure positive and negative ions but it cannot distinguish between ions having mZ ofequal magnitude and opposite sign eg mz 100 and mz 100 4 Peak shapes in an averaged TOF mass spectrum are affected by differences in the initial positions of ions within the region where the accelerating potential is appHed A fact not discussed in class is that the resolution ofa TOFMS can often be increased by lengthening the distance that ions drift D Suppose D is increased from 1 meter to 10 meters in a TOFMS having a chamber pressure of 9e6 Torr Should any change be made to the vacuum system to maintain TOFMS sensitivity ie ion transmission A Yes The pressure should be increased by a factor of 10 collisions help keep ions focused B No Less than or equal to 1e 5 Torr is ideal for TOFMS C Yes The pressure should be decreased by a factor of 10 so that ions can drift the additional distance with low probability of collision Yes The pressure should be decreased by a factor of 100 so that ions can drift the additional distance with low probability of collision E No 9e 6 Torr is the lowest pressure that can be achieved in a Measured mz 3441421 mass accuracy greater than 7 ppm mz 3441433 mz 3441069 7ppm iX166 gt Ami 24mu 3441433u WMw 40219191quot 3441069u lee6349ppm 3441433u I A Compound A is the first to come off of the column I Q How many of the following statements are TRUE 1 To increase the mz range of a TOFMS one must lengthen the drift axis 2 The magnitude of an ion s velocity is equal when entering and exiting a reflectron 3 A TOFMS can simultaneously measure positive and negative ions but cannot distin uish between ions having mz of equal magnitude and OppOSI e sign eg mz 100 and mz 00 4 Peak shapes in an averaged TOF mass spectrum are affected by differences in the initial positions of ions within the region where the accelerating potential is applied A fact not discussed in class is that the resolution of a TOFMS can often be increased by lengthening the distance that ions drift D Suppose D is increased from 1 meter to 10 meters in a TOFMS having a chamber pressure of 9e6 Torr Should any change be made to the vacuum system to maintain TOFMS sensitivity ie ion transmission L 495 9 Mean free path is inversely proportional to pressure Want trajectory to be controlled by voltages The pressure should be decreased by a factor of 10 so that ionscan dn39ft the additional distance with equal probability of collision TOFMS A mass spectrometer determines the mass tocharge ratio mz of gasphase ions by subjecting them to known electric or magnetic fields and analyzing their resultant motion How does TOFMS fit this definition What MUST we know in orderto calibrate a TOFMS For purposes of calibration instrument parameters Voltage and Distance can be bundled into a constant 2 Fortunate Exact measurement ofdistance and voltage would be tedious if not impossible Recall that these equations are an ideal system Groups modify calibration t in order to accommodate nonidealities TOFMS Pulse packet ofions introduced into analyzer All mz in packet reach detector simultaneous detection mz determination based on dispersion Based on static DC fields Quadrupole MS Continuous introduction of ions into analyzer Transmit only specific mz value to detector mz determination based on bandpass ltering Based on timevary RF elds Quadrupole Geometry Quadrupole consists of four parallel rods Typical length might be 10 s of cm Precise dimensions and spacing Rods connected diagonally in pairs From Watson 4 RF OSCILLATOR i l gt 4 D FIG 46 Schematic diagram showing arrangement of quadrupole rods and electrical connec tion to RF generator a DC potential not shown is also impressed on the rods The inset illus trates the ideal quadrupole electriofield dispersion lon Motion inside a Quad Animation Voltages applied to rods define timevarying fields between rods and determine the mz that is transmitted Quadrupole Voltages Voltage of all rods have a DC component U All rods have RF component of voltage with MHz frequency l2p and amplitude V0 Potentials on the two sets are out of phase Quadrupole fields cause no acceleration along 2 axis V1V339Fo V2V4Fo UVocost U Vocos t 1 v e 3 ZX h hi IOl s 4 2 I an i Q i 15 39 LE unstable 03 U V m rods 3 V0 U I or E a o l t e U v gt i rods I U V 39 u D 05 it Number of Cycles From Steel and Henchman J Chem Ed 758 1049 19980 Note that U is now an applied voltage unlike in discussion of TOFMS An Stable lon in a Quadrupole Mass Spectrometer U VD m rods 3 V0 U l on E d O i E i e u v 3339 i rods I U V 39 quot D 05 to Number of Cycles OUT OF PHASE Symmetric voltages create clearly defined wells ridges At any given moment one axis focuses ion to center while the other pulls off center Rapid alternation between polarities From De Hoffmann Figure 28 A positive ion represented within a dotted circle is at the center of quadrupole rods the potential signs of which are indicated It goes down the potential valley with respect to the negative rods and acquires some kinetic energy in that direction However the potentials quickly change so that the kinetic energy is converted into potential energy and the ion goes back to the center of the rods as would happen for a ball on a horse saddle that is turned quickly The name saddle eld is an allusion to this phenomenon Stability apex of stability region Stability diagram for fixed Rf frequency g 8 3 fixed mlz 3 23 D 50 xStabIe q 2 Xunstable An ion will have stabe trajectory through 4o quotS ab39e gvstab39e quadrupole if X and y are always less than 3 2 C D 39E radius of quadrupole D to XandYS ab39e o 160 360 I 560 I 760 RF amplitude VO volts Sim A With no RF and positive U positive ion XAxis Y39Af is stable along X repelled to center A 399 F attracted to negative Y rod causes instability B 39 aortas 7 Sim C RF field has stabilized Y trajectory C Note that with increased U need greater VO BMW Waste I to achieve this stability Y sA Ellill Sim E Instable along xaXIs er Note that as U increases lower VO will Key U induce this instability From Steel and HenchmanJ Chem Ed 758 1049 1998 Increasing V Forces Generalizing for all mz The Laplace Equationleads to me x2 y2U V0 commr02 Acceleration in X and y directions IS described based ApplyingNewton39s Second Law on the terms a or a and q F ma qE Note that dzx Fx m dig eZ 26UV0commr02 a Is proportional to Um While Substituthg P cot2 q Is proportional to Volm 8eZU 2 2 r0 ma 4eZV0 For given quadrupole ro is r02me constant is held constant and V and U will be variables Yields dzx 2 12 0032 x dq q 1 2 21 a2qcosZ y Stability Diagram for a Quadrupole au au a 8 zeU qu qu u m a 2 r0 2 Stable along X 4 ZeV au Stable along y q u m a 2 r0 2 a and q are used to define generalized stability plots Intersections of x and y stability in auqu space define stability in quad Region A used for most quadrupole mass spectrometers Note that earlier slide was Region A for fixed mass From De Hoffmann 1O Mass Filter Many conditions U V m fall within stability region there is more than one way for ion to pass through For selectivity must also consider stability of other mass values Apex of generalized stability diagram is at a 0237 q 0706 To select transmit narrow mass window adjust U and Vosuch that 0237 q 0706 eg lon B For any value m aq 2UNo To scan values of m through narrow transmission window hold other parameters constant and scan U and Vowith constant ratio UNo 120233 0706 A B he C X Axis 1 le WA vlvAv m 1 Av WW NW WW quotW 7 WM lm71 M W m202 m199 m197 0 25 I 3 020 A C 3 B E 3 015 8 x 39 n 010 gee U 105 I 0 l I I l 02 04 08 10 0 6 q 4eV0mr02m2 From Steel and Henchman J Chem Ed 758 1049 1998 Figure limited to singly charged ions hence lack ofz in expression Mass Filter For ANY value m aq 2UVo For example Reduce U Hold V Still stable slope of scan line is reduced What effect does this have on resolution A J3 C X Axis V Vivi 39AV wily ile m m llv quotmi W ml W a 3 W f Y aXIs 7 7 KW m202 m199 m197 025 3 A quotC N o 020 x L 0 B E v quot 3 015 D a 00 we 039 I 010 7ng x 005 I o 1 l l I l 02 04 08 10 0 6 q 4eVGmr02m2 From Steel and Henchman J Chem Ed 758 1049 1998 Figure limited to singly charged ions hence lack ofz in expression 11 Mass Filter A B C qunnwlrwmmnunnm Yaxis Scan line shows UNo 120233 0706 0425 Increase in mass requires 01 proportional increases in U Ni 020 and V0 to maintain this ratio E and these a and q values 5 015 3 6 II 010 Example bringing 202 G into stability apex 0135 requires increases in U and V a W mMWmmm m20i2 m109 m197 i I l I I I 02 04 00 03 10 q 4eVOmr02m2 From Steel and HenchmanJ Chem Ed 758 1049 1998 Figure limited to singly charged ions hence lack ofz in expression mz Scanning in a Quadrupole MS an 0233 mwro qu 220706 Scan line shows UVo 120233 0706 Increase in mass requires proportional increases in U and V0 to maintain this ratio and these a and q values From De Hoffmann Figure 27 Stability areas as a function of U and V for ions with different masses m1 lt 1442 lt m3 Changing U linearly as a function of V we obtain a straight operating line that allows us to observe those ions successively A line with a higher slope would give us a higher resolution as long as it goes through the stability areas Keeping U 0 no direct potential we obtain zero resolution All of the ions have a stable trajectory as long as V is within the limits of their stability area Reproduced modi ed from Ref 6 with permission 12 Animation httpwwwchemagilentcomScriptsPDS asplPage41723 Start at 1 min Quad Source ESI APCI APPI Quadrupole Notes Maximum mz 4000 Resolution 3000 Quadrupoles are low resolution instruments Usually operated at Unit Mass Resolution Small lightweight Easy to couple with chromatography RfOnly Quadrupoles 02 04 06 q 4eV0mr02m2 pass filter Often denoted with small q Operated with U O quadrupole becomes a broad band Such rfonly quads are an important tool for transferring ions between regions of mass spectrometers Collisional Cooling A common application of rfonly multipoles involves collisional cooHng In an ESI source the expansion into vacuum produces a ion beam with broad energy distribution lon optics and TOFMS experiments rely on precise control of ion energies Desire strategies to dampen energy from external processes Rfinduced trajectory in high pressure region yield collisions and reduction in energy i 2 l Mam Ctiiinllxn D TOF lli L h Pu39rp AF H w quot39 I IIMI39I39I39I H Fm rum in N7 Figure 1 Rhemat ic diagram of the timeofFlight instrument TClF III 1 1131 ion 50mm 3 lteatecl capillary 3 fncu5ing electrode 4 1st aperture p e S rt39 quadrupole second aperture plate T grids S Q tlte storage region ll extraction electmdes ll acceleraljon column l2 electmstatic mirror 13 de ection plates 14 detector 3 Ken Standing et al JASMS 1998 9 569579 Collisional cooling mu m m m aw muwam 1 mm igum A H stngnuns at Llquot 1m mix n mmpnnunu m Anal a live Inpul nl tho qmdm mle Inn gm in Am cm m u Almdlsmnwvil me m 1 admpnlexmtgnide n n 5 a n5 5 n1mmminmmmimnpumzhe qludmpulcmnguidc nDl 3W 1 M Radialveinmamhsumor l5m imidelhe quadrupnlulon smutV ism Comma mum mums mthequmtmpole a 5mm girlhmimulAFmpxtmnnlim lmiixluneson w rim lmmngzommm um m qua ale ux15wlleie a my um 359 5 2 2 00 0 Q1 q2 Q3 Q1 selects parent q2 CID fragmentation inside RF only quad Q3 fragment analysis Fragment Ion Scan Park Q1 on speci c parent mz scan Q3 through all fragment mzto determine makeup on1 Parent Ion Scan Park Q3 on speci c fragment mz scan Q1 through all parent mz to determine source offragmen Neutral Loss Scan Scan Q1 and Q3 simultaneously with constant difference a between transmittedmz values a MQ7 Signal recorded if ion ofmz MQ7 has undergone fragmentation producing a neutral m Clicker Q Q 1 2 3 Which of the followmg are true A quadrupole MS can only transmit ions of onemz value at a time A quadrupole MS has a larger mass range than a TOFMS In determining whether a quadrupole will transmit an ion of given mz value one must knowthe applied voltages the frequency of the RF voltage and the dimensions of the quadrupole The timevarying fields of a quadrupole accelerate an ion in the x y and z axes A triple quadrupole is a powerful tool for MSMS analysis A 12345 B 134 C l345 D l45 E 35 Clicker Q Q l 2 3 Which of the following are true A quadrupole MS can only transmit ions of onemz value at a time A quadrupole MS has a larger mass range than a TOFMS In determining whether a quadrupole will transit an ion of given mz value one must know the applied voltages the frequency of the RF voltage and the dimensions of the quadrupole The timevarying fields of a quadrupole accelerate an ion in the x y and z axes A triple quadrupole is a powerful tool for MSMS analysis A 12345 B 134 C l345 D l45 E 35 Mass Analyzers Timeof flight Mass Spectrometry CUBouwer CHEM 5181 Mass Spectrometry amp Chromatography Joel Kimmel Fall 2007 An ion with mz m has charge equal a 1 Coulombs b m Coulombs 0 16022 x 103919 Coulombs d e Coulombs A mass spectrometer with a resolution of 5000 should be capable of resolving isotopic peaks for singly charged species with m a Of any value b Less than 5000 u 0 Greater than 5000 u d It depends on the type of mass spectrometer Arbitrary Units Calculate resolution and accuracy 0 4 Argon Atomic Weight Da 39948 399600 Atomic mass mau Abundance 03 3596754552 033 379627325 006 39 9623837 996 0 2 e 70 0 l i l l l l l l l l l l 39 88 39 90 39 92 39 94 39 96 39 98 40 00 40 02 40 04 40 06 mlz Which ofthe following pairs requires the greatest mass resolving power to distinguish A Art from Ar2 B CO from N2 C CH from CDH2 Ar 39 9623837 u C 12 u 0 159949 u N 14003 u H 1007 u and D 20141 u Ion Motion in Electrostatic Fields Electrical force on an ion Electric Field Lines Electric field is the force on a unit positive charge Q What effect does this field have on a positive ion Q How does the voltage vary across the space between the and electrodes Q Would ions originating at A and B be affected equally Same E Same V TimeofFlight Mass Spectrometry To determine mz values A packet of ions is accelerated by a 4 known potential and the flight times lt of the ions are measured over a h F m known distance 5quotquot mil R455quot Newquot Q What are V e and Z 1 CC Key Performance Notes Based on dispersion in time IMeasures all mz simultaneously implying potentially high duty cycle 3 Unlimitedquot mass range IDC electric fields In 2V t 2 Small footprint e f Relatively inexpensive T39me Force F qE Electric Field Lines Effective Voltage V Es Potential U Vq Potential U Esq TOFMS Source 8 Drift Region D La m Detector v J J E wequot E 0 39 U an qESa lons accelerated by strong field E within short 1 source region S 2 qESa EmVD Drift times recorded across long fieldfree drift region D Zqua VD T VD depends on starting position of ion ideally all ions start from same plane t 7 D 7 D 12 VD quSam Q What else is ideally assumed U0 E D Q What figures of merit will nonideality affect Drawing adapted from p20 ofCotter reference on next Slide Actual Picture More Complex TOFt0tatDtd TOF total recorded flight time of an ion to lon formation time after T0 of TOF measurement t3 Time in acceleration region Which depends on initial position and initial energy tD Time in drift region which depends on initial position and initial energy td Response time of detector Furdetalled dlscusslun see Gullhaus Mass Speq lElB lBBE Cutter TlrnerufrflghtMa Spe mmaery lnsimmematiun and Applications in Biological Research ACS lBB7 Resolution For any mz in a timeof flight mass spectrum the recorded peak will be the sum of signals corresponding to multiple independent ion arrival events Each ion arrival will be recorded at a unique TOF as determined by expression on previous slide TOF39 which is the center of the peak in the mass spectrum will be an average of all individual ion arrival TOFs The width of TOF 7t will depend on the distribution ofthe individual ion arrival TOFs and other factors R TOF ZAZ Improving Resolution TOFMS was first commercialized in 19505 Early instruments had limited resolution Speed ofelectronics Energy distribution Recent Renaissance Delayed lon Extraction TimeLag Focusing Continuous extraction hv Source Flight tube CD 2 E E y 39 J ft 3 7 quot IL 2 U tilti39iiiiii l lift 0 O I g g a i ll 9 g I I i l g Q mz 20 W 0 W O kV 20 W D Desorption Extraction 3 Flight Delayed pulsed extraction hv Z O M 0 5 5 a o g I I I I Modified from Cotter E E E Z g m httpwwwhopkinsmedicineorgmamsMAMSmiddleframe lesteachin 20W 20W 0W OKVZOKV gfilesME3308842005MSZOO5Lecture 5lnstrumentationpdf i i iiAmplitude of the pulse From De HO mann 39l39im e39delay i i V 7 g detector plane if Delay between ionization and extraction mien events At ionization U UO Initial Energy of Ion a v a m 39 grid a 7 quot r r 39 A mg I quot r i At exxit of extraction U UO Eextxq Fi nu e 1 e 235quot E beginning of drift U UO Eextxq VI V2q adeiayw 5 i i I acceleratian Tune surce voltages andor dely to We compnsate for U0 and create space focus at detector Mass dependent From Guilhaus J Mass Spec 1519 1995 Reflectron Detector Ion Mirror I I I I I I I I I I I I i 4 I 1 l g ll III l h quot 2Lquot i r 39 quot Innenurce Reflectron consists of a series of electrodes forming a linear fiel in irection opposite of initial acceleration Potential En ergy Ions are slowed by this field eventually turning around and accelerating back in direction of Li etector Penetration depth depens on USI which is functin of U0 and acceleration field E Distance From httpwwwchemistrywustledumsfdamonre ectronshtml Vltages are tu spce focus t the plane of the detector


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