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An Introduction to Mass SpectrometrybyScott E.Van BramerWidener UniversityDepartment of ChemistryOne University PlaceChester,PA 19013svanbramscience.widener.eduhttp:/science.widener.edu/svanbramrevised:September 2,1998 Copyright 1997TABLE OF CONTENTSINTRODUCTION.4SAMPLE INTRODUCTION.5Direct Vapor Inlet.5Gas Chromatography.5Liquid Chromatography.6Direct Insertion Probe.6Direct Ionization of Sample.6IONIZATION TECHNIQUES.6Electron Ionization.7Chemical Ionization.9Fast Atom Bombardment and Secondary Ion Mass Spectrometry.10Atmospheric Pressure Ionization and Electrospray Ionization.11Matrix Assisted Laser Desorption/Ionization.13Other Ionization Methods.13Self-Test#1.14MASS ANALYZERS.14Quadrupole.15Magnetic Sector.17Electric Sector/Double Focusing Mass Spectrometers.18Time-of-Flight.19Quadrupole Ion Trap.21Ion Cyclotron Resonance.22Self-Test#2.23DETECTORS.23VACUUM SYSTEM.24DATA SYSTEM.24INTERPRETATION.24Molecular Ion.25Fragmentation.26Isotope Abundance.31Exact Mass.33ACKNOWLEDGMENTS.34END OF PAPER QUESTIONS.35ANSWERS TO SELF-TEST QUESTIONS.36LITERATURE CITED.37Data SystemDetector MassAnalyzerVacuum SystemSourceRegionInletMore precisely mass spectrometry determines the mass of a molecule.*The mass to charge ratio(m/z)is used to describe ions observed in mass spectrometry.By convention,m*is the numerical value for the mass of the ion and z is the numerical value for the charge of the ion.The unifiedatomic mass(u)and the elementary charge units(e)are used for these values.The unified atomic mass is defined as1/12 the mass of an atom of C.Older terms still in use but not accepted as SI units include the atomic mass unit12(amu)and the dalton(Da).The amu is no longer acceptable because there are conflicting definitions.The dalton isfrequently used for polymers,peptides and other large molecules.The elementary charge unit is defined as z is aninteger equal to the number of electrons lost(or gained for negative ions).For most experiments one electron is lostduring ionization so z is+1 and the m/z value is equivalent to the relative molecular mass of the ion.Because theunified atomic mass and the charge number are pure numbers the mass-to-charge ratio is a number and does not haveany units.For calculations of the physical behavior of ions it is often necessary to use the actual mass(SI units ofkilogram)and charge(SI units of coulomb).Figure 1.Mass Spectrometer Block DiagramINTRODUCTION:Mass Spectrometry is a powerful technique for identifying unknowns,studying molecularstructure,and probing the fundamental principles of chemistry.Applications of massspectrometry include identifying and quantitating pesticides in water samples,it identifyingsteroids in athletes,determining metals at ppq(Parts Per Quadrillion)levels in water samples,carbon-14 dating the Shroud of Turin using only 40 mg of sample(1),looking for life on Mars,determining the mass of an Si atom with an accuracy of 70 ppt(2),and studying the effect of28molecular collision angle on reaction mechanisms.Mass spectrometry is essentially a technique for weighing molecules.Obviously,this*is not done with a conventional balance or scale.Instead,mass spectrometry is based upon themotion of a charged particle,called an ion,in an electric or magnetic field.The mass to chargeratio(m/z)of the ion effects this motion.Since the charge of an electron is known,the mass to*charge ratio a measurement of an ions mass.Typical mass spectrometry research focuses on theformation of gas phase ions,the chemistry of ions,and applications of mass spectrometry.This paper covers the basics of mass spectrometry instrumentation and introduces theinterpretation of mass spectra.It is only an introduction and interested readers are encouraged toconsult more specialized books and journal articles for additional details.The articles and booksreferenced in this paper should be available at most college and university libraries.Figure 1 is a block diagram that shows the basic parts of a mass spectrometer.The inlettransfers the sample into the vacuum of the mass spectrometer.In the source region,neutralsample molecules are ionized and then accelerated into the mass analyzer.The mass analyzer isthe heart of the mass spectrometer.This section separates ions,either in space or in time,according to their mass to charge ratio.After the ions are separated,they are detected and the5signal is transferred to a data system for analysis.All mass spectrometers also have a vacuumsystem to maintain the low pressure,which is also called high vacuum,required for operation.High vacuum minimizes ion-molecule reactions,scattering,and neutralization of the ions.Insome experiments,the pressure in the source region or a part of the mass spectrometer isintentionally increased to study these ion-molecule reactions.Under normal operation,however,any collisions will interfere with the analysis.SAMPLE INTRODUCTION:The selection of a sample inlet depends upon the sample and the sample matrix.Mostionization techniques are designed for gas phase molecules so the inlet must transfer the analyteinto the source as a gas phase molecule.If the analyte is sufficiently volatile and thermallystable,a variety of inlets are available.Gases and samples with high vapor pressure areintroduced directly into the source region.Liquids and solids are usually heated to increase thevapor pressure for analysis.If the analyte is thermally labile(it decomposes at hightemperatures)or if it does not have a sufficient vapor pressure,the sample must be directlyionized from the condensed phase.These direct ionization techniques require specialinstrumentation and are more difficult to use.However,they greatly extend the range ofcompounds that may be analyzed by mass spectrometry.Commercial instruments are availablethat use direct ionization techniques to routinely analyze proteins and polymers with molecularweights greater than 100,000 dalton.Direct Vapor Inlet.The simplest sample introduction method is a direct vapor inlet.Thegas phase analyte is introduced directly into the source region of the mass spectrometer through aneedle valve.Pump out lines are usually included to remove air from the sample.This inletworks well for gases,liquids,or solids with a high vapor pressure.Samples with low vaporpressure are heated to increase the vapor pressure.Since this inlet is limited to stable compoundsand modest temperatures,it only works for some samples.Gas Chromatography.Gas chromatography is probably the most common technique forintroducing samples into a mass spectrometer.Complex mixtures are routinely separated by gaschromatography and mass spectrometry is used to identify and quantitate the individualcomponents.Several different interface designs are used to connect these two instruments.Themost significant characteristics of the inlets are the amount of GC carrier gas that enters the massspectrometer and the amount of analyte that enters the mass spectrometer.If a large flow of GCcarrier gas enters the mass spectrometer it will increase the pressure in the source region.Maintaining the required source pressure will require larger and more expensive vacuum pumps.The amount of analyte that enters the mass spectrometer is important for improving the detectionlimits of the instrument.Ideally all the analyte and none of the GC carrier gas would enter thesource region.The most common GC/MS interface now uses a capillary GC column.Since the carriergas flow rate is very small for these columns,the end of the capillary is inserted directly into thesource region of the mass spectrometer.The entire flow from the GC enters the massspectrometer.Since capillary columns are now very common,this inlet is widely used.However,wide bore capillaries and packed GC columns have higher flow rates.This M is the molecular ion produced by removing a single electron to form a radical cation.M is the*+?molecule,is the charge of the cation,and is the remaining unpaired electron of the radical.+?Adduct ions are produced by a chemical reaction between an ion and a neutral molecule.Many of these*reactions cause the addition of a proton(H)to the molecule(M)and produce an adduct ion(MH).+6significantly increases the pressure in the mass spectrometer.Several inlet designs are availableto reduce the gas flow into the source.The simplest design splits the GC effluent so that only asmall portion of the total flow enters the mass spectrometer.Although this inlet reduces the gasload on the vacuum system,it also reduces the amount of analyte.Effusive separators andmembrane inlets are more selective and transport a higher fraction of the analyte into the sourceregion.Each of these methods has efficiency and resolution drawbacks but they are necessary forsome experiments.Liquid Chromatography.Liquid chromatography inlets are used to introduce thermallylabile compounds not easily separated by gas chromatography.These inlets have undergoneconsiderable development and are now fairly routine.Because these inlets are used fortemperature sensitive compounds,the sample is ionized directly from the condensed phase.These inlets are discussed in greater detail in the section on ionization techniques.Direct Insertion Probe.The Direct Insertion Probe(DIP)is widely used to introduce lowvapor pressure liquids and solids into the mass spectrometer.The sample is loaded into a shortcapillary tube at the end of a heated sleeve.This sleeve is then inserted through a vacuum lockso the sample is inside the source region.After the probe is positioned,the temperature of thecapillary tube is increased to vaporize the sample.This probe is used at higher temperatures thanare possible with a direct vapor inlet.In addition,the sample is under vacuum and located closeto the source so that lower temperatures are required for analysis.This is important for analyzingtemperature sensitive compounds.Although the direct insertion probe is more cumbersome thanthe direct vapor inlet,it is useful for a wider range of samples.Direct Ionization of Sample.Unfortunately,some compounds either decompose whenheated or have no significant vapor pressure.These samples may be introduced to the massspectrometer by direct ionization from the condensed phase.These direct ionization techniquesare used for liquid chromatography/mass spectrometry,glow discharge mass spectrometry,fastatom bombardment and laser ablation.The development of new ionization techniques is anactive research area and these techniques are rapidly evolving.Direct ionization is discussed ingreater detail in the next section.IONIZATION TECHNIQUES:A variety of ionization techniques are used for mass spectrometry.Most ionizationtechniques excite the neutral analyte molecule which then ejects an electron to form a radicalcation(M).Other ionization techniques involve ion molecule reactions that produce adduct+?*ions(MH).The most important considerations are the physical state of the analyte and the+*ionization energy.Electron ionization and chemical ionization are only suitable for gas phaseionization.Fast atom bombardment,secondary ion mass spectrometry,electrospray,and matrixassisted laser desorption are used to ionize condensed phase samples.The ionization energy isFilamentCollectorMe-e-e-e-M+e-e-Molecules(M)Injected Into SourceIons(M )Acceleratedto Mass Analyzer+MMolecular ions are the intact ionized analyte molecule.Fragment ions are formed by subsequent*fragmentation of molecular ions.Some older literature will refer to EI as electron impact,but this term is not considered accurate.Electron*Ionization is the currently accepted term.7Figure 2.Electron Ionization Source.significant because it controls the amount of fragmentation observed in the mass spectrum.Although this fragmentation complicates the mass spectrum,it provides structural informationfor the identification of unknown compounds.Some ionization techniques are very soft and onlyproduce molecular ions,other techniques are very energetic and cause ions to undergo extensive*fragmentation.Although this fragmentation complicates the mass spectrum,it providesstructural information for the identification of unknown compounds.Electron Ionization.Electron Ionization(EI)is the most common ionization techniqueused for mass spectrometry.EI works well for many gas phase molecules,but it does have*some limitations.Although the mass spectra are very reproducible and are widely used forspectral libraries,EI causes extensive fragmentation so that the molecular ion is not observed formany compounds.Fragmentation is useful because it provides structural information for interpreting unknown spectra.The electrons used for ionization are produced by passing a current through a wirefilament(Figure 2).The amount of current controls the number of electrons emitted by thefilament.An electric field accelerates these electrons across the source region to produce a beamof high energy electrons.When an analyte molecule passes through this electron beam,a valenceshell electron can be removed from the molecule to produce an ion.e-e-e-e-e-MMMMMM+e-A)B)C)D)E)F)e-The SI unit for energy is the Joule.The energetics of chemical reactions are typically expressed in*kJ/mole.In many gas phase experiments(like mass spectrometry),the mole is not a convenient unit.The electronvolt is frequently used as an energy unit for single molecules or atoms.1 eV=1.602 177 33(49)x 10 J.So that:-191 eV(per molecule or atom)=96.415 220 6 kJ/mole.8Figure 3 Electron Ionization Process.A)Ionizing electron approaches the electron cloud of a molecule;B)Electron cloud distorted by ionizing electron;C)Electron cloud further distorted by ionizing electron;D)Ionizingelectron passes by the molecule;E)Electron cloud of molecule ejecting an electron;F)Molecular ion and ejectedelectron.Ionization does not occur by electron capture,which is highly dependent upon molecularstructure.Instead,EI produces positive ions by knocking a valence electron off the analytemolecule(Figure 3).As the electron passes close to the molecule the negative charge of theelectron repels and distorts the electron cloud surrounding the molecule.This distortion transferskinetic energy from the fast-moving electron to the electron cloud of the molecule.If enoughenergy is transferred by the process,the molecule will eject a valence electron and form a radicalcation(M).+?Since the ionization is produced by a single electron that is accelerated to 70 V,this iscommonly referred to as 70 eV EI.This is enough energy to cause extensive fragmentation,andat this level small changes in the electron energy do not significantly effect the fragmentationpatterns.The amount of energy transferred during this process depends upon how fast theelectron is traveling and how close it passes to the molecule.In most 70 eV EI experiments,approximately 1400 kJ/mole(15 eV)of energy is transferred during the ionization process.*There is,however,a distribution of energy and as much as 2800 kJ/mole(30 eV)is transferred tosome molecules.Since approx