mass-spec [MASS SPECTROMETRY] BY P.RAVISANKAR,VIGNAN PHARMACY COLLEGE, VADLAMUDI,GUNTUR,A.P,INDIA.
Mass spectrometry(Mass spec)Mass spectrometry is super important technique.Mass spectrometry was previously known as mass spectroscopy.But use of the term mass spectroscopy is now discouraged because of probableConfusion with the light spectroscopy.Unlike IR,NMR mass spec.does not use absorption of and interaction of light.There fore, it is not appropriate to call it” mass spectroscopy” rather, it should beCalled mass spectrometry or must mass spec.Mass spectrometry is usually represented as mass-spec or simply MS.Prof. RavisankarVignan Pharmacy collegeValdlamudiGuntur Dist.Andhra PradeshIndia.firstname.lastname@example.org
Still spectroscopy?• the interaction of electric and/or magnetic fields (i.e.eletromagnetic radiation) with matter to determine weightor mass.• Measures mass, not absorption or emission ofelectromagnetic radiation
The concept of mass spectrometry was first put forth bySir J.J Thomson, English Physicist Who discovered the electron in 1887.He got 1906 Nobel Laureate in Physics.We have to first ionize and impart the charged on those particles ..In order to respond electric field and magnetic field..Quickly remind you Thomson-II experiment a beam of electronsJust use magnetic field close to the beambecause the beam is charged .. Its just gets deflected ..How much the beam(that Particles are) is deflected depends on how heavy they are andTheir charge is..We use the exact principle,(same basic technique )in Mass-spec.
Cathode rays are stream of –ve chargesCathode rays bend towords +ve charged plateCathode rays bend near magnets.
Mass spectrometry is essentially a technique for "weighing" molecules.* Obviously, thisis not done with a conventional balance or scale. Instead, mass spectrometry is based uponthe motion of a charged particle, called an ion, in an electric or magnetic field. The massto charge ratio (m/z)** of the ion effects this motion. Since the charge of an electron isknown, the mass to charge ratio a measurement of an ions mass.The concept of MS is to form ions from a sample,separate the ions based on their mass-to-charge ratio (this can beconsidered to be the same as the mass because the ion has onlya single charge in most cases), and measure the abundance ofthe ions.
History:In the early 1900’s while working on electromagnetic radiations, the ions generatedBy the gases in the cathode ray tube led to the discovery of Mass spectrometer.Francis William Aston a physicist working in Cambridge England 1919 worked onMass spectrometry and established technique for the measurement of atomic mass.Francis William Aston, English physicist,student of Thomson, and He was awarded1922 Nobel prize in Chemistry.1948-52 - Time of Flight (TOF) mass analyzers introduced1955 - Quadruple ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins1989 Nobel Prize)1968 - Tandem mass spectrometer appearsMass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY.
What does a mass spectrometer do?Mass –spec or simply MS is a super important techniqueMass spec is easy technique to give you Molecular weight(from molecular ion (M+)You can get Molecular formula (we talk elements about by HRMS)and some of the fundamental things.One thing that Mass spec can easily,easily,easily talk to youabout elements presenNearly in the periodic table can be determined by mass spectrometry.MS is really important technique because that You can easily see Br,Cl and youCan see S and Si you are looking for..NMR is not going to be a technique that talks to you about elementslike that and IR is not going to be a technique that talks to you …This is why we should study about Mass spec..MS is incredible valuable in getting structure (from fragments) .Infact Bio molecular MS to sequencing peptides and proteins and alsonatural products and also organic structures.Structures (from fragmentation proces)Hard ionization(slash techniques) MSMS( taking ions anddeliberately dashing in to them smashing them and see how they look like.It measures mass better than any other technique.It can give information about chemical structures.
MS was originally used to determine the existence of the stable isotopes of theelements in the periodic table..1/ [The word isotope was suggested by Frederick Soddy(1877–1956) as a student and collaborator of Ernest Rutherford at McGillUniversity in Montreal, Canada, in 1913. Isotopes are different forms of the sameelement that have the same atomic number, but differ in their relative atomicmass due to a difference in the number of neutrons present in thenucleus of the atom. The word is derived from the Greek words isos (equal) andtopos (places), ‗having the same place‘ in the periodic table.MS plays an important role not only in organic and biochemistry but also in inorganicchemistry such as the determination of metal contaminants in siliconewafers(silicone crystal,in electronics), drinking water, soils, industrialwaste, etc. To quantitate unambiguously identified sub picogram amounts ofthe pesticide Malathion on orange peel, age of artefacts [isotope-ratiomass spectrometry (IR MS)], contamination of the surface of metal and compositeIt is used to determine the airplane wings [secondaryion mass spectrometry (SIMS)], and the components that give fresh-baked bread itsdelightful aroma.One of most powerful analytical tools MS is sensitive (10-6 to <10-13 g)But Complex instrumentation, expensive,structure obtained indirectly• complex spectra/fragmentation for hard ionization sources• simple spectra for soft ionization sourcesThe list of applications are endless.
It is also used for the identification and quantitation of various organic substances from thesimplest gases such as methane and halomethanes to complex biomoleculessuch as proteins, oligonucleotides, and noncovalent complexes.Mass spectrometers are not only found in analyticallaboratories but also inside the helmets of space suits (todetermine the level of gases that may pose a hazard),in tanks and other battlefield vehicles and on ships andaircraft (to detect the presence of chemical and biologicalwarfare agents), and as more conventional field-portableinstrumentation for use at crime scenes, hazardous.SIMS technique was used to study the impurities in materialsuch as the aluminium used for airplane wings and germaniumwafers used in earlysolid-state electrical devices.SIMS and its variants are widely used in the analysis and study ofsurfaces of alltypes of material – from papers used in laser printers, tomicroprocessor devices, to the wings of aircraft manufactured frommany new polymeric composites.
What are mass measurements good for?To identify, verify, and quantitate: metabolites, recombinantproteins, proteins isolated from natural sources, oligonucleotides,drug candidates, peptides, synthetic organic chemicals, polymers
Assigning numerical value to the intrinsic propertyof ―mass‖ is based on using carbon-12, 12C, as areference point.One unit of mass is defined as a Dalton (Da).One Dalton is defined as 1/12 the mass of a singlecarbon-12 atom.Thus, one 12C atom has a mass of 12.0000 Da.How is mass defined?
Exact Masses of Some Common Elements and Their Isotopes:Element Symbol Exact Mass (u) Rel. Abundance %Hydrogen 1H 1.007825037 100.0Deuterium 2H or D 2.014101787 0.015Carbon 12 12C 12.00000 100.0Carbon 13 13C 13.003354 1.11223Nitrogen 14 14N 14.003074 100.0Nitrogen 15 15N 15.00011 0.36734Oxygen 16 16O 15.99491464 100.0Oxygen 17 17O 16.9991306 0.03809Oxygen 18 18O 17.99915939 0.20048Fluorine 19F 18.998405 100.0Sodium 23Na 22.9897697 100.0Silicon 28 28Si 27.9769284 92.23Silicon 29 29Si 28.9764964 5.0634Silicon 30 30Si 29.9737717 3.3612Phosphorus 31P 30.9737634 100.0Sulfur 32 32S 31.972074 100.0Sulfur 33 33S 32.9707 0.78931Sulfur 34 34S 33.96938 4.43065Sulfur 36 36S 35.96676 0.02105Chlorine 35 35Cl 34.968854 100.0Chlorine 37 37Cl 36.965896 31.97836
14.Mass DefinitionsMolecular masses are measured in Daltons (Da) or mass units (u).One Dalton = 1/12 of the mass of a 12C atom. TheInternational Union of Pure and Applied Chemistry (IUPAC) suggests theunified atomic mass unit (abbreviated u), which is based on 12C . Thedalton (abbreviated Da) is identical in size to uMonoisotopic mass = sum of the exact masses of the most abundantisotope of each element present, i.e., 1H=1.007825, 12C=12.000000,16O=15.994915, etc.This is the most accurately defined molecular mass and is preferred if ameasurement of it can be determined.Average mass = sum of the abundant averaged masses (―atomic weights‖)of the constituent atoms of a given molecule.The result is a weighted average over all of the naturally occurring isotopespresent in the compound. This is the common chemical molecular weightthat is used for stoichiometric calculations (H=1.0080, C=12.011, O=15.994,etc.). The average mass cannot be determined as accurately as themonoisotopic mass because of variations in natural isotopic abundances.
Vaccum system reduces collisions b/nIons and gas molecules.
Sample introduction-Vaporize sample)Ion source- ionizeAnalyte gasmoleculesMass analyzer:Separates ionsAccording to M/ZCount ions(DetectIons) A detector countsthe number of ions atdifferent deflections andthe data can be plotted asa ‘spectrum’ of differentmasses.
Mass SorIllustration of the basic components of a mass spectrometry system.Solid• Liquid• VaporIonizationSourceMassAnalzyerDetectorInletForm ionscharged moleculesselectedionsDataSystemSort or separatesions by M/ZWhen ions strikeDetector it Detect ionsBlock 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,neutral sample molecules are ionized and then accelerated into the mass analyzer. Themass analyzer is the 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 areseparated, they are detected and the signal is transferred to a data system for analysis.High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the ions.Neutral samplemolecules
Different elementscan be uniquelyidentified by their massMS principles
Basic principle:MS basic principle is super, super, super simple ….A charged particle moving in a magnetic field is deflected.The ionized molecule is moving along some sort of magnetic field…As the particle moves in to the magnetic field its path gets bent..The degree of deflection depends up on the mass /charge(M/Z) ormass to charge ratio.In other words any given particle whether it has 20AMU or 1 charge or40 AMU and 2 charges is going toget deflect in to the same amount.When a compound is exposed to a high voltage electric current it loses electrons andgets converted to positively charged ions.The basic magnetic or electric field acting on these moving charged ions (+ve ions)Deflects them along circular path on a radius that is a function of their mass to chargeRatio i.e. (m/z or m/e ratio)M+Ionized molecule(Charged particle)As the particle movesIn to the magneticField its path gets bentThe degree of deflection dependsup on the M/Z ratio and getInformation about M/Z. UltimatelyWe can figure out the mass.Lighter components or components with more ionic charge will deflect in the field more than heavier orless charged components
How basic technique works?MIonized molecule is moveAlong some sort of magnetic fieldAs the particles moves in the magnetic field its path gets bent.The degree of deflection depends on mass to charge ratio( M/Z) .Heavier particles deflects less (bend less)more charge -----deflected moreLighter particles deflects more.The mass spectrum is a plot of relative intensities (which represent the ion abundances)onthe ordinate versus the m/z values of the ions on the abscissa.
The purpose of MS or spectroscopy is to provide clues about the structure ofMolecules.In the chemistry test books you can see the picture of the molecules but youCan’t see the picture in the test tube but you can get some cluesYou have to see what is the types of clues we can get from mass spectrometry.MS stats with the original molecule.The original molecule is designaged as M (Capital M)Capital M stands for original molecule.In organic chemistry, a radical ion is typically indicated by a superscriptdot followed by the sign of the charge: and . In mass spectrometry,the signis written first, followed by the superscripted dot.
M + 1e M.+ 2e+MS starts with the original molecule (M) we call the original molecule capital M.When we hit(whack) with high energy electron .The high energy electron (does roughly speaking is it fetishize (with highintensity) collides with a neutral analyte molecule can knocks off anotherelectron. (2e) resulting in a positively charged ion.This high energy electron is going to knock another electron loose from amolecule, so what should be the correct symbol of the molecule nownow that the molecule has electron not loose, is a radical and radicals withunpaired electron and since it lost an electron it must have a +ve charge.The symbol we would use is radical cation. A radical cation.Original moleculeRadical cation or Molecular ion(M+)or parent ion.M is the molecular ion produced byremoving a single electron to form aradical cation. M is the molecule, + isthe charge of the cation, and (.) is theremaining unpaired electron of theradical.High energy electrons smashing into sample molecules andknocking electrons out of orbitals.
When the energy supplied is more than the ionized energy of the molecule, theMolecular ion (M+) undergoes further fragmentation to give smaller ions andFree radicals.M+ A1+ + A2 ( or ) A1 + A2+
Ions are formed continuously in the ion source of mass spectrometer andaccelerated toward the detector by an electrical potentialOr the accelerating voltage V.Once the accelerated ions then move in to the magnetic field B or H , whosedirection is perpendicular to their path.The magnetic field is constrained to force the ions to follow a circular path (arc)whose radius is r.In a magnetic field, an ion with mass m will experience a centripetalforce is given by mv2/r(one pulling the ion toward the centre of thecircle) equal to Bzvwhere B strength of the magnetic field, BZv = mv2/rz is the charge on the ion,Theory or concept of mass sectrometry (fragmentation)mass spec equation for magnetic sector or basis forSeparation of particle (relation b/n m,r,V,and H equation)
From the equation it is clear that the radius of the circular path of a particle isDepends on the magnetic field(H),accelerating voltage V and the m/z ratio.The radius of the ionized molecule is dependent on mass (m) as the charge(e)Voltage(V)and magnetic field (H) are constant.The relation between mass(m),radius of the circular path(r) accelerating voltage (V)And magnetic field(H) is shown in the above equation and is considered to beThe basis for separation of the particles with respect to their masses.
Atoms can be deflected by magnetic fields - provided the atom is first turned into anion. Electrically charged particles are affected by a magnetic field althoughelectrically neutral ones arent.The sequence is :Stage 1: IonisationThe atom is ionised by knocking one or more electrons off to give a positive ion. Thisis true even for things which you would normally expect to form negative ions(chlorine, for example) or never form ions at all (argon, for example). Massspectrometers always work with positive ions.Stage 2: AccelerationThe ions are accelerated so that they all have the same kinetic energy.
Accelerating platesLets start out by looking atThis pair of electrodes.Apply high voltage(potential)Across them.its going to generateHigh voltage electron beam.(HighEnergy source of particles.You can collide some sample that youAre interested in.Produce ions. Ions that we generateHere will be accelerated towards either-ve or +ve plates.Ions starts moving in the different directions+ve ions will be attrected towords -ve plates-ve ions will be attracted towords + ve plates.We are going to interested in the +ve ions only+-++-
Stage 3: DeflectionThe ions are then deflected by a magnetic field according to theirmasses. The lighter they are, the more they are deflected.The amount of deflection also depends on the number of positivecharges on the ion - in other words, on how many electronswere knocked off in the first stage. The more theion is charged, the more it gets deflected.Stage 4: DetectionThe beam of ions passing through the machine is detectedelectrically.
Understanding whats going onThe need for a vacuumIts important that the ions produced in the ionisation chamber have a free runthrough the machine without hitting air molecules
The vaporised sample passes into the ionisation chamber. The electricallyheated metal coil gives off electrons which are attracted to the electron trapwhich is a positively charged plate.The particles in the sample (atoms or molecules) are therefore bombardedwith a stream of electrons, and some of the collisions are energetic enough toknock one or more electrons out of the sample particles to make positiveions.Most of the positive ions formed will carry a charge of +1 because it is muchmore difficult to remove further electrons from an already positive ion.These positive ions are persuaded out into the rest of the machine by the ionrepeller which is another metal plate carrying a slight positive charge
The components of the mass spectrometer that cause ion formation,separation,and detection are contained in an ultraclean housing usually keptat moderately high vacuum (10-3–10-6 torrHigh vacuum ensures that, once the ions formed in the ion source begin tomove toward the detector, they will not collide with other moleculesbecause this could result in further fragmentation or deflect themfrom their desired path. Nearly all fragmentation reactions occurringunder these conditions are intramolecular (involving only the decompositionof individual ions) rather than intermolecular (involving the reaction of ionswith other species that may be present).High vacuum also protects the metal and oxide surfaces of the ionsource, analyzer, and detector from corrosion by air and watervapor, which could compromise the spectrometer’s ability to form, separate,and detect ions.1 torr = 1 mm Hg, which is equivalent to 133 pascal(Pa).
The positive ions are repelled away fromthe very positive ionisation chamber andpass through three slits, the final one ofwhich is at 0 volts. The middle slit carriessome intermediate voltage. All the ions areaccelerated into a finely focused beamWhen an ion hits the metal box, its charge is neutralised by an electronjumping from the metal on to the ion (right hand diagram). That leaves a spaceamongst the electrons in the metal, and the electrons in the wire shuffle along to fill it.A flow of electrons in the wire is detected as an electric current which can beamplified and recorded. The more ions arriving, the greater the current
Different ions are deflected by the magnetic field by different amounts. The amountof deflection depends on:the mass of the ion. Lighter ions are deflected more than heavier ones.the charge on the ion. Ions with 2 (or more) positive charges are deflectedmore than ones with only 1 positive charge.These two factors are combined into the mass/charge ratio.Mass/charge ratio isgiven the symbol m/z (or sometimes m/e).For example, if an ion had a mass of 28 and a charge of 1+, its mass/charge ratiowould be 28. An ion with a mass of 56 and a charge of 2+ would also have amass/charge ratio of 28.Assuming 1+ ions, stream Ahas the lightest ions, stream Bthe next lightest and stream Cthe heaviest. Lighter ions aregoing to be more deflectedthan heavy ones.
In the last diagram, ion stream A is most deflected - it will contain ions with thesmallest mass/charge ratio. Ion stream C is the least deflected - it contains ionswith the greatest mass/charge ratio.It makes it simpler to talk about this if we assume that the charge on all the ionsis 1+. Most of the ions passing through the mass spectrometer will have a chargeof 1+, so that the mass/charge ratio will be the same as the mass of the ion.Note: Youmustbeawareofthepossibilityof2+(etc)ions,butthevastmajorityofAlevel questionswillgive youmassspectrawhichonlyinvolve1+ions.Unlessthereissomehintinthequestion, youcanreasonablyassumethattheionsyouaretalkingaboutwillhaveachargeof1+.Assuming 1+ ions, stream A has the lightestions, stream B the next lightest and stream C theheaviest. Lighter ions are going to be moredeflected than heavy ones.
DETECTIONOnly ion stream B makes it right through the machine to theion detector. The other ions collide with the walls wherethey will pick up electrons and be neutralised.Eventually, they get removed from the mass spectrometerby the vacuum pump.
Detecting the other ionsHow might the other ions be detected - those in streams A and C which have been lostin the machine?Remember that stream A was most deflected - it has the smallest value of m/z (thelightest ions if the charge is 1+). To bring them on to the detector, you would need todeflect them less - by using a smaller magnetic field (a smaller sideways force).To bring those with a larger m/z value (the heavier ions if the charge is +1) on to thedetector you would have to deflect them more by using a larger magnetic field.If you vary the magnetic field, you can bring each ion stream in turn on to the detector toproduce a current which is proportional to the number of ions arriving. The mass of eachion being detected is related to the size of the magnetic field used to bring it on to thedetector. The machine can be calibrated to record current (which is a measure of thenumber of ions) against m/z directly. The mass is measured on the 12C scale.Note: The 12C scale is a scale on whichthe 12C isotope weighs exactly 12 units
What the mass spectrometer output looks likeThe output from the chart recorder is usually simplified into a "stick diagram". Thisshows the relative current produced by ions of varying mass/charge ratio.The stick diagram for molybdenum looks lilke this:You may find diagrams in which the vertical axis is labelled as either "relativeabundance" or "relative intensity". Whichever is used, it means the same thing. Thevertical scale is related to the current received by the chart recorder - and so to thenumber of ions arriving at the detector: the greater the current, the more abundantthe ion.As you will see from the diagram, the commonest ion has a mass/charge ratio of 98.Other ions have mass/charge ratios of 92, 94, 95, 96, 97 and 100.That means that molybdenum consists of 7 different isotopes. Assuming that theions all have a charge of 1+, that means that the masses of the 7 isotopes on thecarbon-12 scale are 92, 94, 95, 96, 97, 98 and 100The largest peak in the massspectrum (100% relativeintensity) is called the base peakThis molecular ion (M+) is very importantbecause it has virtually the same mass as that ofthe analyte molecule (the small mass of the lostelectron can be ignored).
The physics behind mass spectrometry is that a charged particle passing through a magneticfield is deflected along a circular path on a radius that is proportional to the mass to chargeratio, m/e.In an electron impact mass spectrometer, a high energy beam of electrons is used to displacean electron from the organic molecule to form aradical cation known as the molecular ion. Ifthe molecular ion is too unstable then it can fragment to give other smaller ions.The collection of ions is then focused into a beam and accelerated into the magnetic field anddeflected along circular paths according to the masses of the ions. By adjusting the magneticfield, the ions can be focused on the detector and recorded.
It is important to distinguish between the terms ions and peaks inmass spectrometry. Ions are particles that have both massand charge, and they can fragment to form other ions. There can belarge or small numbers of ions, so that it is appropriate to speak oftheir relative abundance.On the other hand, peaks in a mass spectrum correspond to localizedmaximum signalsproduced by the detector and have only m/z values associated withthem. These signals are either weak or strong (depending on thenumbers of ions produced) and therefore are best described as havingintensity. The abundance of peaks implies that there are manypeaks, not that a given peak is big or little.
Hard ion sources leave excess energy in molecule - extensivefragmentationSoft ion sources little excess energy in molecule - reducedfragmentation
A) External (Batch) Inlet Systems:Sample heated (<400 °C) in small external ovenVapour admitted to ionizer through valveGas stream added to entrain analyte(B) Direct Probe:Sample vial inserted through air-lock into ionizer chamberVial heated to vaporize sampleVial can be reduced to capillary or surface plate for smallquantities(C) Chromatography/Electrophoresis/Injection AnalysisCan be modified to directly flow into ionizer regionSample inlet system:
World War II. During thisperiod, Dempster developed EI.EI was originally called electronimpact.This is a process by which gas-phasemolecules ata pressure of >10-3 Torr are ionized bya beam of electrons, produced by ahot wire (filament), that havebeen accelerated by 70V (i.e. 70 eV).EI is used in modern massspectrometers where analytes are inthe gas phase.Electron Ionization (EI)
Also referred to as electron impact ionization, this is the oldest and best-characterized ofall the ionization methods. A beam of electrons passes through the gas-phase sample.An electron that collides with a neutral analyte molecule can knock off another electron,resulting in a positively charged ion.The EI source is most commonly a small chamber about 1 cc in volume, inwhich analyte molecules interact with a beam of highly energetic electrons thathave typically been accelerated through a potential difference of 50–70 volts (V)across the volume of the ion source [50–70 electron volts (eV); 1 eV = 23 kcal].This electron beam is produced by boiling electrons off a narrow strip or coil ofwire made of a tungsten-rhenium alloy. Between the filament and the center ofthe ion source is a metal plate with a slit called the electron aperture. This slit limitsthe size of the electron beam and confines ionization to a small volume within thecenter of the ion source. Opposite the filament is the collector, a metal plate held ata positive electrical potential (+V) that attracts and intercepts the electronbeam after it has passed through the source. a collimating magnet, which causesthe electrons in the beam to travel in a helical path. Although this helical trajectoryimproves the probability that the electrons and molecules will interact, sampleionizationis still very inefficient—less than one molecule in a thousand undergoesionization.What happens during ionization is complex. electronssmashing into sample molecules and knocking electrons out of orbitals.
one electron is ejected from one of the bonding or nonbonding orbitals of the moleculeIonization energies (IE) for most organic compounds range from about 5–15 eV.Bond dissociation energies are even smaller, so this method of ionization not onlycauses molecules to expel one or more electrons, it also provides enough energy forsubstantial fragmentation of the first-formed ion (the molecular ion, M+).Because of the excess energy present in 50–70 eV electrons, enough additionalenergy may be transferred to overcome the second, or even third, ionizationpotential of the molecule, leading to ions having +2 or +3 charges.Many different products form during ionization.Excited molecules can return to their neutral ground statethrough thermal vibrations or the emission of light, and becauseno ions are formed in the process, they are simply pumped awayfrom the ion source by the vacuum System.
Sometimes the analyte molecule absorbs an electron and a negative ion isformed (Table 1.2, product b). In order to be absorbed by the molecule, the electronmust be of very low energy (0.1 eV), and there are few electrons of this energy ina standard EI source. By reversing the polarity of the repeller, ion focusing plate,and extractor plate in the ion source, and by altering the detector so that it willdetect negative ions, a negative ion mass spectrum can be recorded. For mostcompoundsnegative ion MS offers few advantages over positive ion MS, and overall ittends to be less sensitive. There are some specific applications, however, mostnotably with halogenated compounds. In this book only positive ion products andtheir fragmentations will be covered.The remaining products listed in Table 1.2 are positive ions. The ion that isformed first results directly from ejection of a single electron from the neutral molecule(product c). This molecular ion (M+) is very important because it has virtuallythe same mass as that of the analyte molecule (the small mass of the lostelectron can be ignored). Indeed, mass spectrometry is one of the few analyticaltools available for determining the molecular mass of a compound.
Ion products d and e in Table 1.2 are formed by unimolecular dissociation ofM+. In the first case a single bond is broken and a neutral group of atomshaving an odd number of electrons (called a radical; is lost.The second process (dissociation with rearrangement) involves breakingsome bonds while attach same time forming new ones. This results inexpulsion of a fragment containing an even number of electrons, usually as aneutral molecule.Table 1.2 imply that such ions are formed in a concerted process in whichionization, bond making, and bond breaking all occur at about the same time.However, fragmentations that involve rearrangement of atoms usually occur ina stepwise fashion through one or more intermediates.If more than one electron is ejected from the analyte molecule, ions havingcharges of +2, +3, or even +4 may be formed (Table 1.2, products f ).Biopolymers such as peptides may have charge states of +10 or more fromprotonation of basic sites on the molecule. Since mass spectrometry actuallymeasures the mass-to charge ratio (m/z) of an ion, not its mass, an ion havinga charge greater than +1 is found not at the m/z value corresponding to itsmass (m), but rather at m/2, m/3, or m/4, depending on the number of chargestates. Further, if m is not evenly divisible by the number of charges z, m/z willhave a non integral value. For example, the double charged molecular ion(M2+) of a compound having a molecular mass of 179 is found at m/z 179/2 =89.5.
Neutral products are removed by the vacuum system, because the electric andmagnetic fields present in the ion source have no effect on their motion.Positive and negative ions, on the other hand, can be separated by appropriatelyplaced charged surfacesin the ion source (Figure 1.2). To accomplish this, the repeller is kept at a positivepotential (+V) both to attract and neutralize negative ion products and to repel positiveions. Conversely, the extractor plate and ion focusing plate (the ion optics) areboth kept at a negative electrical potential (V) to attract and accelerate the positiveions toward the m/z analyzer. Slits in the extractor and ion focusing plates allowpassage of the positive ions and help focus the ion beam as it approaches the analyzer
In the first case a single bond isbroken and a neutral group ofatoms having an odd number ofelectrons (called a radical; is lostbreaking some bonds whileattach same time forming newones. This results in expulsionof a fragment containing aneven number ofelectrons, usually as a neutralmoleculeIf more than one electron is ejected fromthe analyte molecule, ions having chargesof +2, +3, or even +4 may be formed
Chemical IonizationUnlike EIMS, in which molecules are ionized through interaction with high-energyelectrons, ionization in chemical ionization mass spectrometry (CIMS) depends oncollisions of ions and molecules. In positive ion CIMS the sample is ionized byreaction with ions generated within a large excess of a relatively low molecularmass reagent gas such as methane (as CH+5 ), isobutane [as (CH3)3C+], or ammonia(as NH+4), at a pressure of about 1 torr. Although some reagent gas ions arethemselves formed by ion/molecule reactions
This type of ion formation (often called soft ionization) imparts significantly lessenergy to analyte molecules than do interactions with high-energy electrons, so thatthe resulting ions have little excess internal energy. These ions therefore fragmentless than those formed by EIMS. As a result, although CIMS is useful for determiningthe molecular mass of compounds that do not produce a detectable M+. byEIMS
Fast Atom Bombardment and Secondary Ion Mass Spectrometry. (5) Fast AtomBombardment (FAB) and Secondary Ion Mass Spectrometry (SIMS) both use highenergy atomsto sputter and ionize the sample in a single step. In these techniques, a beam of raregas neutrals(FAB) or ions (SIMS) is focused on the liquid or solid sample. The impact of this highenergybeam causes the analyte molecules to sputter into the gas phase and ionize in a singlestep(Figure 6). The exact mechanism of this process is not well understood, but thesetechniqueswork well for compounds with molecular weights up to a few thousand dalton. Since noheatingis required, sputtering techniques (especially FAB) are useful for studying thermallylabilecompounds that decompose in conventional inlets (6, 7).
Fast atom bombardment:The most significant difference between FAB and SIMS is the sample preparation. InFAB the analyte is dissolved in a liquid matrix. A drop of the sample/matrix mixture isplaced at the end of an insertion probe and introduced to the source region. The fastatom beam is focused on this droplet to produce analyte ions. Glycerol or similar lowvapor pressure liquids are typically used for the matrix. Ideally, the analyte is solublein the liquid matrix and a monolayer of analyte forms on the surface of the droplet
SIMS experiments(8) are used to study surface species and solid samples.* Nomatrix isused and the ionizing beam is focused directly on the sample. Although this makessamplingmore difficult, it is useful for studying surface chemistry. High resolution chemicalmaps areproduced by scanning a tightly focused ionizing beam across the surface and depthprofiles areproduced by probing a single location(9, 10). Although SIMS is a very sensitive andpowerfultechnique for surface chemistry and materials analysis, the results are often difficultto quantitate
Sample introduction. heated batch inlet. heated direct insertion probe. gas chromatograph. liquid chromatograph (particle-beam interface)Benefits. often gives molecular weight information through molecular-like ions such as[M+H]+,even when EI would not produce a molecular ion.. simple mass spectra, fragmentation reduced compared to EILimitationssample must be thermally volatile and stable. less fragmentation than EI, fragment pattern not informative or reproducible enoughfor library search. results depend on reagent gas type, reagent gas pressure or reaction time, andnature of sample.Mass range. Low Typically less than 1,000 Da.
Desorption Chemical Ionization (DCI)SummaryThis is a variation on chemical ionization in which the analyte is placed on a filamentthat is rapidly heated in the CI plasma. The direct exposure to the CI reagentions, combined with the rapid heating acts to reduce fragmentation. Some samplesthat cannot be thermally desorbed without decomposition can be characterized by thefragments produced by pyrolysis DCI.Sample introduction. sample deposited onto a filament wire. filament rapidly heated inside the CI source.Benefits. reduced thermal decomposition. rapid analysis. relatively simple equipmentLimitations. not particularly reproducible. rapid heating requires fast scan speedsfails for large or labile compoundsMass rangeLow Typically less than 1,500 Da.
Negative-ion chemical ionization (NCI)SummaryNot all compounds will produce negative ions. However, many important compounds ofenvironmental or biological interest can produce negative ions under the rightconditions. For suchcompounds, negative ion mass spectrometryis more efficient, sensitive and selectivethan positive-ion mass spectrometry.Negative ions can be produced by a number of processes. Resonance electroncapture refers to the capture of an electron by a neutral molecule to produce amolecular anion. The electron energyis very low, and the specific energy required for electron capture depends on themolecular structure of the analyte.Electron attachment is an endothermic process, so the resulting molecular anion willhave excess energy. Some molecular anions can accommodate the excess energy.Others may lose the electron or fall apart to produce fragment anions. Electronattachment is an endothermic process, so the resulting molecular anion will haveexcessenergy. Some molecular anions can accommodate the excess energy. Others maylose the electronor fall apart to produce fragment anions.In negative-ion chemical ionization, a buffer gas (usually a common CI gas such asmethane) isused to slow down the electrons in the electron beam until some of the electrons havejust the
BENEFITSefficient ionization, high sensitivity. less fragmentation than positive-ion EI or CI. greater selectivity for certain environmentally or biologically important compoundsLimitations. not all volatile compounds produce negative ions. poor reproducibilityMass range. Low Typically less than 1,000 Da.Field
Field Desorption and IonizationThese methods are based on electron tunneling from an emitter that is biased at a highelectricalpotential. The emitter is a filament on which fine crystalline whiskers are grown. When ahighpotential is applied to the emitter, a very high electric field exists near the tips of thewhiskers.There are two kinds of emitters used on JEOL mass spectrometers: carbon emitters andsiliconemitters. Silicon emitters are robust, relatively inexpensive, and they can handle a highercurrentfor field desorption. Carbon emitters are more expensive, but they can provide about anorder ofmagnitude better sensitivity than silicon emitters.Field desorption and ionization are soft ionization methods that tend to produce massspectrawith little or no fragment-ion content.
Field Desorption (FD)SummaryThe sample is deposited onto the emitter and the emitter is biased to a high potential(severalkilovolts) and a current is passed through the emitter to heat up the filament. Massspectra areacquired as the emitter current is gradually increased and the sample is evaporatedfrom the emitterinto the gas phase. The analyte molecules are ionized by electron tunneling at the tipof theemitter whiskers. Characteristic positive ions produced are radical molecular ionsand cationattachedspecies such as [M+Na]+ and [M-Na]+. The latter are probably produced duringdesorptionby the attachment of trace alkali metal ions present in the analyte
Sample introductionDirect insertion probe.The sample is deposited onto the tip of the emitter by. dipping the emitter into an analyte solution. depositing the dissolved or suspended sample onto the emitter with a microsyringeBenefits. simple mass spectra, typically one molecular or molecular-like ionic species percompound.. little or no chemical background. works well for small organic molecules, many organometallics, low molecular weightpolymers and some petrochemical fractionsLimitations. sensitive to alkali metal contamination and sample overloading. emitter is relatively fragile. relatively slow analysis as the emitter current is increased. the sample must be thermally volatile to some extent to be desorbedMass range. Low-moderate, depends on the sample. Typically less than about 2,000 to 3,000 Da.. some examples have been recorded from ions with masses beyond 10,000 Da.
Field Ionization (FI)SummaryThe sample is evaporated from a direct insertion probe, gas chromatograph, or gasinlet. As thegas molecules pass near the emitter, they are ionized by electron tunneling.Sample introduction. heated direct insertion probe. gas inlet. gas chromatographBenefits. simple mass spectra, typically one molecular or molecular-like ionic species percompound.. little or no chemical background. works well for small organic molecules and some petrochemical fractionsLimitationsThe sample must be thermally volatile. Samples are introduced in the same way as forelectron ionization (EI).Mass range. Low Typically less than 1000 Da.
Different Ionization Methods• Electron Impact (EI - Hard method)– small molecules, 1-1000 Daltons, structure• Fast Atom Bombardment (FAB – Semi-hard)– peptides, sugars, up to 6000 Daltons• Electrospray Ionization (ESI - Soft)– peptides, proteins, up to 200,000 Daltons• Matrix Assisted Laser Desorption (MALDI-Soft)– peptides, proteins, DNA, up to 500 kD
86+++++++++ ++++++ ++++++++++++++++++++++++++++++MH+[M+2H]2+[M+3H]3+Electrospray Ion FormationDroplets formed in electric field have excess positive ions.Evaporation of neutrals concentrates charge.Droplets break into smaller droplets.Eventually one molecule + n protons is left.+++++++++++++Needle at High Voltage++++++++ ++++ ++++
High voltage appliedto metal sheath (~4 kV)Sample Inlet Nozzle(Lower Voltage)Charged droplets++++++++++++++++++ ++++++++++++++++++++++++++++MH+MH3+MH2+Pressure = 1 atmInner tube diam. = 100 umSample in solutionN2N2 gasPartialvacuumElectrospray ionization:Ion Sources make ions from sample molecules(Ions are easier to detect than neutral molecules.)
Most of the molecules don’t have a charge on them.Generally magnetic force effects the charge particles. A mass spectrometer worksby using magnetic and electric fields to exert forces on charged particles(ions) in a vacuum. Therefore, a compound must be charged or ionizedto be analyzed by a mass spectrometer.The first question isHow do you get it charged on the moleculesHistorically the first technique developed was called EI(Electron impact ionization)EIMSThe basic idea isThe electron gun is used to ionized the moleculeYou give it a good whack, you knock an electron you get a Molecular cation.For exCH4 + e- CH4+. +2e-Methane and you hit with an electron. You just take an electron out of itYou get a radical cation .What mass spectrometry called a molecular ion.
just prior toWorld War II. During this period, Dempsterdeveloped EI. [EI was originally called electron impact.This is a process by which gas-phase molecules ata pressure of >10-3Torr are ionized by a beam ofelectrons, produced by a hot wire (filament), that havebeen accelerated by 70V (i.e. 70 eV).] EI is used inmodern mass spectrometers where analytes are in thegas phase.
Several common modes differing by method of ion formation:Electrospray (ESI)Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Photo-Ionization (APPI)New dual sources (ESI/APCI) or (APCI/APPI)Which is best?It depends on the exact application.Increasing polarity and molecular weight andthermal instability favors electrospray.Most drugs of abuse are highly polarand are easily analyzed usingelectrospray.High molecular weight proteins alsorequire electrosprayLower polarity and molecular weight favorsAPCI or APPI.Lower background, but compoundsmust be more thermally stable.
Electrospray is a method of getting the solution phase ions into the gas phase so that theycan be sampled by the mass spectrometeThree Fundamental Processes:1. Production of charged droplets.2. Droplet size reduction, and fission.3. Gas phase ion formation
Orifice1. A large voltage ( up to 6kV) is applied between the end of a capillary carrying theLC mobile phase and the mass spectrometer entrance.2. Ions (of the same polarity) are drawn out toward the counter electrode (curtainplate) pulling the mobile phase along.3. When the excess charge at the tip of the capillary overcomes surface tension, adroplet is formed.
Electrospray ionizationEluent is sprayed (nebulized) into achamber at atmospheric pressure in thepresence of a strong electrostatic fieldand heated drying gas.The electrostatic field causes furtherdissociation of the analyte molecules.The heated drying gas causes the solventin the droplets to evaporate. As the dropletsshrink, the charge concentration in thedroplets increases.Eventually, the repulsiveforce between ions with like charges exceedsthe cohesive forces and ions are ejected(desorbed) into the gas phase. These ionsare attracted to and pass through a capillarysampling orifice into the mass analyzer.
Electrospray is especially useful for analyzinglarge biomolecules such as proteins, peptides,and oligonucleotides,but can also analyzesmaller moleculeslike benzodiazepinesand sulfatedconjugates.electrospray canbe used to analyzemolecules as largeas 150,000 ua typical LC/MSinstruments is around 3000 m/z. For example:100,000 u / 10 z = 1,000 m/zWhen a large molecule acquires many charges,a mathematical process called deconvolutionis often used to determine the actual molecular weight of an analyte.
In atmospheric pressure ionization, the analyte molecules are ionized first, at atmosphericpressure. The analyte ions are then mechanically and electrostatically separated from neutralmolecules. Common atmospheric pressure ionization techniques are• Atmospheric pressure chemical ionization (APCI)• Atmospheric pressure photoionization (APPI)Atmospheric pressure chemical ionizationIn APCI, the LC eluent is sprayed through a heated (typically 250°C – 400°C) vaporizerat atmospheric pressure. The heat vaporizes the liquid. The resulting gas-phase solventmolecules are ionized by electrons discharged from a corona needle. The solvent ionsthen transfer charge to the analyte molecules through chemical reactions (chemicalionization).The analyte ions pass through a capillary sampling orifice into the mass analyzer.APCI is applicable to a wide range of polar and nonpolar molecules. It rarely results inmultiple charging so it is typically used for molecules less than 2000 u. Due to this,and because it involves high temperatures, APCI is less well-suited than electrosprayfor analysis of large biomolecules that may be thermally unstableMass range. Low-moderate Typically less than 2000 Da.Benefits. good for less-polar compounds. excellent LC/MS interface. compatible with MS/MS methods
Atmospheric pressure photoionizationAtmospheric pressure photoionization (APPI)for LC/MS is a relatively new technique. Asin APCI, a vaporizer converts the LC eluent tothe gas phase. A discharge lamp generatesphotons in a narrow range of ionizationenergies. The range of energies is carefullychosen to ionize as many analyte moleculesas possible while minimizing the ionizationof solvent molecules. The resulting ions passthrough a capillary sampling orifice intothe mass analyzer.APPI is applicable to many of the samecompounds that are typically analyzed byAPCI. It shows particular promise in twoapplications, highly nonpolar compoundsand low flow rates (<100 ìl/min), where APCIsensitivity is sometimes reduced.In all cases, the nature of the analyte(s)and the separation conditions have a stronginfluence on which ionization technique:electrospray, APCI, or APPI, will generatethe best results. The most effective techniqueis not always easy to predict.
TerminologyMolecular ion The ion obtained by the loss of an electron from the moleculeBase peak The most intense peak in the MS, assigned 100% intensityM+Symbol often given to the molecular ionRadical cation +ve charged species with an odd number of electronsFragment ionsLighter cations formed by the decomposition of the molecular ion.These often correspond to stable carbcations.SpectraThe MS of a typical hydrocarbon, n-decane is shown below. The molecular ion isseen as a small peak at m/z = 142. Notice the series ions detected that correspondto fragments that differ by 14 mass units, formed by the cleaving of bonds atsuccessive -CH2- units
Matrix-Assisted Laser Desorption Ionization (MALDI)SummaryThe analyte is dissolved in a solution containing an excess of a matrix such as sinapinic acidordihydroxybenzoic acid that has a chromophore that absorbs at the laser wavelength. Asmall amount of this solution is placed on the laser target. The matrix absorbs the energyfrom the laser pulse and produces a plasma that results in vaporization and ionization ofthe analyte.Sample introduction. direct insertion probe. continuous-flow introductionBenefits. rapid and convenient molecular weight determinationLimitations. MS/MS difficult. requires a mass analyzer that is compatible with pulsed ionization techniques. not easily compatible with LC/MSMass range. Very high Typically less than 500,000 Da.
Direct laser desorption relies on the very rapid heating of the sample or samplesubstrate to vaporize molecules so quickly that they do not have time todecompose. This is good for low to medium-molecular weight compounds andsurface analysis. The more recent development of matrix-assisted laserdesorption ionization (MALDI) relies on the absorption of laser energy by amatrix compound. MALDI has become extremely popular as a method for therapid determination of high-molecular-weight compounds.The analyte is dissolved in a solution containing an excess of a matrix such assinapinic acid or dihydroxybenzoic acid that has a chromophore that absorbs atthe laser wavelength. A smallamount of this solution is placed on the laser target. Thematrix absorbs the energy from the laserpulse and produces a plasma that results in vaporization and ionization of the analyte.Sample introduction. direct insertion probe. continuous-flow introductionBenefits. rapid and convenient molecular weight determinationLimitations. MS/MS difficult. requires a mass analyzer that is compatible with pulsed ionization techniques. not easily compatible with LC/MSMass range. Very high Typically less than 500,000 Da.
Sinapinic acid -cyano-4-hydroxycinnamic acid (CHCA) 2,5-dihydroxybenzoic acid (DHB)HOCOOHOHCH3OCH3OHO CH=CH-COOH HO CH=C-COOHCNAnalyte is dissolved in solution with excessmatrix (>104).Sample/matrix mixture is dried on a target andplaced in the MS vacuum.Requirements for a satisfactory matrix:It must co-crystallize with typical analytemoleculesIt must absorb radiation at the wavelength ofthe laser (usually 337 nm)To transfer protons to the analyte it should beacidicTypical successful matrices for UV MALDI arearomatic carboxylic acids.
MALDI: Matrix Assisted Laser Desorption/Ionization.The sample is prepared by mixing the analyteand a matrix(Sinapinicacid,Dihydroxybenzoicacid that has a chromophore that absorbs at thelaser wave length) compound chosen to absorbthe laser wavelength. This is placed on a probetip and dried.A laser beam is then focused on this driedmixture and the energy from a laser pulse isabsorbed by the matrixThe matrix absorbs the energy from the laser pulseand produces a plasma that results in vaporizationand ionization of the analyte..(MALDI) is used to analyze extremely large molecules .MALDI is often used for the analysis of synthetic and natural polymers, proteins, andpeptides. Analysis of compounds with molecular weights up to 500,000 dalton is possible.Desorbed sampleions and neutralsPulsed laser (337 nm)
105±20 kVSample and matrix,crystallized on stageDesorbed sampleions and neutralsPulsed laser(337 nm)3.5 nsSample stageMass analyzerMatrix-assisted laser desorption ionization (MALDI)
106-+-++++--------------+++++++++++1. Laser pulse produces matrix neutrals, + and - ions, andsample neutrals: M --> M*, MH+, (M-H)- (M= Matrix)2. Sample molecules are ionized by gas-phase proton transfer:MH+ + A --> AH+ + M (A=Analyte)(M-H)- + A --> (A-H)- + MMALDI Ionization Mechanism
107Mass (m/z) analyzers can be divided into two broadcategories: (1) those that in some way isolate ionsof individual m/z values from a beam – beam-typeinstruments; and (2) those that store ions of all m/zvalues and detect ions through some process of single m/zisolation – traps. Magnetic-sector, double-focusing, TQ,and TOF mass spectrometers are beam-type instruments.QIT (both external and internal ionization variations)and ICR mass spectrometers are traps.the mass spectrometer used to separategas-phase ions according to their m/zvalues is the mass analyzer. Massanalyzer is the traditional terminology.
108(A) Magnetic Sector Analyzers:Magnetic-sector mass spectrometers useonly a magneticfield to separate ions according to their m/zvalues. These instruments are referred to assingle-focusing mass spectrometers. Theyare capable ofseparating ions that differ in one m/z unitover a rangefrom 1 to 700m/z.
Quadrupole Mass AnalyzerUses a combination of RFand DC voltages to operateas a mass filter.• Has four parallel metalrods.• Lets one mass passthrough at a time.• Can scan through allmasses or sit at onefixed mass.
110A quadrupole mass analyzer consists of four parallel rods arranged in a square.The analyte ions are directed down the center of the square.Voltages applied to the rods generate electromagnetic fields.These fields determine which mass-to-charge ratio of ions can passthrough the filter at a given time.Quadrupoles tend to be the simplest and least expensive mass analyzers.Quadrupole mass analyzers can operate intwo modes:• Scanning (scan) mode• Selected ion monitoring (SIM) modeThe analyzer consists of four rods or electrodes arranged acrossfrom each other . As the ions travel through the quadrupole they are filteredaccording to their m/z value so that only a single m/z value ion can strike thedetector. The m/z value transmitted by the quadrupole is determined by theRadio Frequency (RF) and Direct Current (DC) voltages applied to the electrodes.These voltages produce an oscillating electric field that functions as a bandpassfilter to transmit the selected m/z value.Quadrupole mass analyzer
111In scan mode, the mass analyzer monitors a range of mass-to-charge ratios.In SIM mode, the mass analyzer monitors only a few massto-charge ratios.SIM mode is significantly more sensitive than scan modebut provides information about fewer ions. Scan mode istypically used for qualitative analyses or for quantitationWhen all analyte masses are not known in advance.SIM mode is used for quantitation and monitoring of targetcompounds.
mass scanning modem1m3m4 m2m3m1m4m2single mass transmission modem2 m2 m2 m2m3m1m4m2Quadrupoles have variable ion transmission modes
CID and multiple-stage MSMultiple-stage MS (also called tandem MS or MS/MS or MSn) is a powerfulway to obtain structural information. In triple-quadrupole orquadrupole/quadrupole/time-of-flight instrumentsthe first quadrupole is used to select the precursor ion.CID(Collision-Induced Dissociation) takes place in the second stage (quadrupole oroctopole), which is called the collision cell.The third stage (quadrupole or TOF) then generates a spectrum of the resultingproduct ions.It can also perform selected ion monitoring of only a few product ionswhen quantitating target compoundsTo obtain structural information, analyte ions are fragmented by colliding themwith neutral molecules in a process known as collisioninduceddissociation (CID) or collisionally activated dissociation (CAD)
114The mass analyzer is the heart of the mass spectrometer. This sectionseparates ions, either in space or in time, according to their mass tocharge ratio.
116Double-focusing Mass SpectrometerDouble-focusing mass spectrometers use a magnetic fieldto select ions based on theirm/z values and an electric fieldto select ions based on their energy. These instrumentsbecame the workhorse of MS from the 1930s throughthe end of the 1970s. These instruments are capable ofseparating ions with very small differences in m/z valuesallowing for the determination of the elemental compositionof the ion based on these millimass measurements.CEC was the first commercial manufacturer of doublefocusingmass spectrometers beginning before World war II
118Quadrupole mass analyzers are often called mass filters because of the similaritybetween m/z selection by a quadrupole and wavelength selection by an optical filter orfrequency selection by an electronic filter.MASS ANALYZERS:After ions are formed in the source region they are accelerated into the mass analyzerby an electric field. The mass analyzer separates these ions according to their m/zvalue. The selection of a mass analyzer depends upon the resolution,** (26) massrange,*** scan rate**** and detection limitsAnalyzers are typically described as either continuous or pulsed. Continuous analyzersinclude quadrupole filters and magnetic sectors.Pulsed analyzers include time-of-flight, ion cyclotron resonance, and quadrupole iontrap mass spectrometers.They transmit a single selected m/z to the detectorand the mass spectrum is obtained by scanning the analyzer so that different mass tocharge ratio ions are detected.These (pulsed)instruments collect an entire mass spectrum from a single pulse of ions.This results in a signal to noise advantage similar to Fourier transform or multichannelspectroscopic techniques
119Time-of-flight (TOF)In a time-of-flight (TOF) mass analyzer,a uniform electromagnetic forceis applied to all ions at the sametime, causing them to acceleratedown a flight tube.Lighter ions travel faster andarrive at the detector first,so the mass-to-charge ratiosof the ions are determined bytheir arrival times.Time-of flight mass analyzers havea wide mass range and can be veryaccurate in their mass measurements.
122time-of-flight massspectrometer (TOF-MS). Ions of different m/z valuesaccelerated from a region such as an ion source intoan evacuated tube will have different velocities, andtherefore these ions will reach the end of this evacuatedregion at different times. By separating the times at whichion current is observed at a detector placed at the endof this evacuated region, it is possible to obtain a massspectrum. Ions of the lowest m/z will reach the detectorFirst.
124Applications of massspectrometry include identifying and quantitating pesticides in water samples, itidentifyingsteroids in athletes, determining metals at ppq (Parts Per Quadrillion) levels in watersamples,carbon-14 dating the Shroud of Turin using only 40 mg of sample (1), looking for life onMars,determining the mass of an 28Si atom with an accuracy of 70 ppt(2), and studying theeffect ofmolecular collision angle on reaction mechanisms.Mass spectrometry is essentially a technique for "weighing" molecules.* Obviously, thisis not done with a conventional balance or scale. Instead, mass spectrometry is basedupon themotion of a charged particle, called an ion, in an electric or magnetic field. The mass tochargeratio (m/z)** of the ion effects this motion. Since the charge of an electron is known, themass tocharge ratio a measurement of an ions mass.
Time-of-flight (TOF) Mass Analyzer++++Source Drift region (flight tube)detectorV• Ions are formed in pulses.• The drift region is field free.• Measures the time for ions to reach the detector.• Small ions reach the detector before large ones.
126++++Source Drift region (flight tube)detectorV•Ions formed in pulses.•Measures time for ions to reach the detector.Time-of-Flight (TOF) Mass Analyzer222LVtzm or zmt
127Quadrupole Ion Trap•Uses a combination of DCand RF fields to trap ions•Ions are sequentiallyejected by scanning theRF voltageLinear Trap•Essentially a quadrupole with end-caps•Advantage: Larger ion storage capacity, leading to better dynamic rangeIons in(from ESI)3D TrapEnd capsIons outto detectorRing electrode(~V)InsulatedspacerHe gas1x10-3 TorrRaymond E. March, JOURNAL OF MASS SPECTROMETRY, VOL. 32, 351È369 (1997)
128Ion trapAn ion trap mass analyzer consists ofa circular ring electrode plus two endcaps that together form a chamber. Ionsentering the chamber are ―trapped‖ thereby electromagnetic fields. Another fieldcan be applied to selectively eject ionsfrom the trap.Ion traps have the advantage of being ableto perform multiple stages of massspectrometrywithout additional mass analyzers
129Electron MultiplierFrom Detector Technolgy: http://www.detechinc.com/ B. Brehm et al., Meas. Sci. Technol. 6 (1995) 953-958.Multi-Channel Plate (MCP)
130Fourier transform-ioncyclotron resonance (FT-ICR)An FT-ICR mass analyzer (also called FT-MS)is another type of trapping analyzer.Ions entering a chamber are trapped in circularorbits by powerful electrical and magneticfields.When excited by a radio-frequency(RF) electrical field, the ions generate a timedependent current.This current is converted by Fourier transforminto orbital frequencies of the ions whichcorrespond to their mass-to charge ratios.Like ion traps, FT-ICR mass analyzers canperform multiple stages of mass spectrometrywithout additional mass analyzers.They also have a wide mass range and excellentmass resolution.They are, however, the most expensive ofthe mass analyzers.
131B0Detect+++++++++R CExcite+++++++++Fourier Transform Ion Cyclotron Resonance (FT-ICR)•Ions trapped andmeasured in ultrahigh vacuuminside a superconducting magnet.A.G. Marshallzm1
134Comparison of Analyzer TypesIon Trap/QuadrupoleTOF OrbiTrap FT-ICRSensitivity +++ ++* to +++ ++* +*MassAccuracy+** ++ +++ +++**ResolvingPower+** ++ +++ ++++**DynamicRange+ to +++** ++ +++ ++**Upper m/z + ++++ +++ ++*Sensitivity lowered due to losing ions on way to analyzer, rather than inherentsensitivity.**Can be improved by scanning narrower mass range or slower.
135Alexander Makarov, Anal. Chem. 2000, 72, 1156-1162OrbitrapTOF•Simultaneous excitationFTICR•Confined ion trajectory•Image current detection•Fourier transform data conversionUnique to Orbitrap•3D electric field trapping•No need for magnet•Easy access•Final detection device
136Image Current Detection in OrbitrapFrom Alexander Makarov‘s 2008 ASMS Award Address
137Three Important Properties to Assess Performance ofa Mass Spectrometer1. Sensitivity•Minimum quantity of sample needed (always estimate how muchsample you have, in femtomoles!)2. Mass Accuracy•Needed for identifying samplesby database searching or to determine elemental composition3. Resolving Power•Determine charge state. Resolve mixtures. High resolving canalso improve mass accuracy.
138Mass (Measurement) AccuracyMass Accuracy or Mass Measurement Error is the difference between theexperimental mass (Mexp) and the theoretical value (Mtheo), calculated fromelemental composition.In absolute term, , in Da or milli-DaIn relative term, , unit-less (ppm for high resolution MS)Example:Mexp = 1569.684Mtheo= 1569.66956Mass Measurement Error = 0.014Da or 9.2ppmtheotheoMMMMA exptheoMMMA exphttp://physics.nist.gov/PhysRefData/Elements/per_noframes.html
15.01500 15.01820 15.02140 15.02460 15.02780 15.03100Mass (m/z)1000102030405060708090100%IntensityISO:CH315.0229MFWHM = MR = M/ MHow is mass resolution calculated?
140Resolving Power•Measure of the ability to differentiate between components of similarmass.•Two definitions:•Valley Definition: Neighboring peaks overlap at 10% peak apex height.•Full Width Half Maximum (FWHM): Width of a single peak measured at50% peak apex. This is the most commonly used definition nowadays(because it is simpler).MMRPMM5%10%50%M10% Valley Definition FWHM Definition
141dynode electron multiplier, in which the entire surface of themultiplier is physically and electrically continuous. The interior surface of the electronmultiplier that is located near the entrance isheld at a highly negative potential (usually 1.2 to 3 kV); the exit end is referencedto ground (0 V).
142As each incoming ion collides with the multiplier surface,approximately two electronsare ejected from the surface.To the ejected electrons the remaining interior of the multiplier appears more positivethan the entrance does, so that they are attracted further into the multiplier where theycollide with the interior surface.Each electron ejected by the second collision also results in theejection of two electrons, and this process continues down to the exit or last dynodeof the multiplier.The total number of electrons ejected depends on the gain of the multiplier,which is roughly a function of the total potential difference between the entranceand exit to the multiplier surface. The gain can be adjusted daily during instrumenttune-up so that a standard quantity of a reference sample such as PFTBA will produceapproximately the same signal intensity. The total signalamplification is approximately 2n, where n is the total number of collisions withthe multiplier surface. Most multipliers provide about a 105- to 106-fold increasein signal—about 18–20 collisions. Electrons generated in the last collision withthe multiplier surface constitute the signal current output of the multiplier. Thiscurrent is sent to an external electronic signal amplification circuit and finally tothe data system.1.4.2.
143The most commonly used calibrationstandard for routine GC/MS work is perfluorotri-n-butylamine [(CF3CF2CF2-CF2)3N; PFTBA], which gives fragment ions over the range from m/z 30 – 600. Prominent peaks at m/z 69, 219, and 502 in the spectrum ofthis compound can be used to adjust settings for instrument variablesThis compound exhibits peaks of at least moderate intensity over the entiremass range normally used in GC/MS work
144The largestpeak in the mass spectrum (100% relative intensity) is called the base peakIt is important to distinguish between theterms ions and peaks in mass spectrometry. Ions are particles that have both massand charge, and they can fragment to form other ions. There can be large or smallnumbers of ions, so that it is appropriate to speak of their relative abundance. Onthe other hand, peaks in a mass spectrum correspond to localized maximum signalsproduced by the detector and have only m/z values associated with them. Thesesignalsare either weak or strong (depending on the numbers of ions produced) andtherefore are best described as having intensity. The abundance of peaks impliesthat there are many peaks, not that a given peak is big or little.
145EI mass spectrum of methane is shownEarth would contain 98.9% C atoms that were 12C and 1.1% that were 13C.It may seem surprising that 14C is missing from this list, because it is undoubtedlyfamiliar to many readers as the basis for radioactive C dating in archaeology.Although 14C is indeed a naturally occurring isotope of C, it undergoes continualradioactive decay, which makes it unsuitable for determining elemental compositions
147If this information is applied to methane, the MM of 12CH4 is calculated to be16 u (12 u for the C and 1 u for each H), whereas that of 13CH4 is 17 u (13 u for theC and 1 u for each H). Because ions are separated in mass spectrometry accordingto their m/z values, the mass spectrum exhibits a peak for each of these ions. Indeed,mass spectrometry offers one of the best ways to identify and quantify the presenceof different isotopes in a sample. The ratio of the intensities of the peaks at m/z 17and 16 are directly related to the natural abundances of the two C isotopes (1.1% for13C / 98.9% for 12C ¼ 1.1%).The difference between the actual atomic mass of anisotope (relative to 12C) and the nearest integral mass is called the mass defect,which is denoted by the capital Greek letter DELTA.
148Three of the elements (F, P, and I) occur without natural stableisotopes. This means that these elements will contribute only one peak at a singlem/z value for each ion in which they occur.The small amount of deuterium (2H) that occurs naturally (0.015%) is usually ignoredin the MS analysis of compoundshaving M < 500 u because its contribution falls at or below the normal limits ofdetection, which are often 0.1–0.5% of the base peak. This is not true for very largemolecules, however, because the 2H contribution for an ion containing even 100 Hatoms is 100 x0.015% = 1:5%.For compounds containing only H, F, P, and I, or only one atom of an elementthat has a naturally occurring isotope, isotopic abundance considerations are fairlytrivial.