What is Mass Spectrometry

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hat is Mass Spectrometry?

Mass spectrometry is an analytical technique that involves the studyin the gas phase of ionized molecules with the aim of one or more of

the following:

• Molecular weight determination

• Structural characterization

• Gas phase reactivity study

• Qualitative and quantitative analysis of components in a

mixture

Mass spectrometry consists !asically of weighing ions in the gasphase "he instrument used could !e considered as a sophisticated!alance which determines with high precision the masses of individualatoms and molecules #epending on the samples chemical and physicalproperties$ different ionization techniques can !e used %ne of themain factor in choosing which ionization technique to !e used isthermola!ility &or samples that are not themola!ile and relativelyvolatile$ ionization such as 'lectron (mpact and)or *hemical(onization can !e effectively used &or samples that are thermola!ilesuch as peptides$ proteins and other samples of !iological interest$soft ionization techniques are to !e considered +mong the most usedsoft ionization techniques are 'lectrospray ,'S(- and Matrix +ssisted

.aser #esorption ,M+.#(- "he name given to a particular mass spectechnique is usually pointing to the ionization method !eing used

+tomic and molecular masses are assigned relative to the mass of thecar!on isotope$ /0*$ whose atomic weight is defined as exactly /0 "heactual mass of /0* is /0 daltons$ with one dalton is equal to /11//2304 g "he mass of a molecule or an ion can !e presented in daltons,#a- or 5ilodaltons ,5#a-

"he Mass Spectrometer

Mass spectrometry uses an instrument called a mass spectrometer"he main components of a mass spectrometer are:

• (nlet system ,.*$ G*$ #irect pro!e etc-

• (on source ,'($ *($ 'S($ +6*($ M+.#($ etc-

• Mass analyzer ,Quadrupole$ "%&$ (on "rap$ Magnetic Sector-

•

#etector ,'lectron Multiplier$ Micro *hannel 6lates M*6s-


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Samples can !e introduced to the mass spectrometer directly viasolids pro!e$ or in the case of mixtures$ !y the intermediary of

chromatography device ,eg Gas chromatography$ .iquidchromatography$ *apillary electrophoresis$ etc- %nce in thesource$ sample molecules are su!7ected to ionization (ons formed inthe source ,molecular and fragment ions- acquire some 5inetic energyand leave the source + cali!rated analyzer then analyzes the passingions as a function of their mass to charge ratios #ifferent 5ind ofanalyzer,s- can !e used$ Magnetic$ Quadrulpole$ (on trap$ &ourier"ransform$ "ime of &light$ etc"he ion !eam exiting the analyzerassem!ly is then detected and the signal is registered *ommonionization method acronyms include:

• '(8'lectron (mpact9

• *(8*hemical (onization9

• S(MS8Secondary (ons Mass Spec9

• &+8&ast +tom om!ardment9

• .#MS8.aser #esorption Mass Spec9

• 6#MS86lasma #esorption Mass Spec9

• "S8"hermospray9

• +S8+erospray9

• 'SMS8'lectrospray Mass Spec

*ommon mass analyzer acronyms include:

• '8'lectrostatic3Magnetic9

• ("8ion trap9


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• Q8Quadrupole9

• "%&8"ime of &light

(onization Methods

Selection of the proper ionization method for the analysis of yoursample is extremely important +lthough we can offer suggestions$ itis your responsi!ility to understand and select the method,s-appropriate for your research compounds

• 'lectron (mpact '( (onization

•

*hemical (onization *(

• ;egative (on *hemical (onization

• 'lectrospray (onization "echniques

• Matrix +ssisted .azer #esorption ,not offered in our facility$

!ut availa!le elswhere on campus-

• +tmospheric 6ressure *hemical (onization +6*(

'lectron (mpact (onization

M < e3,=2e>- 33333? M


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in order to get molecular ion information %ne helpful rule fordetermining whether an ion is a molecular ion is the ;itrogen @ule

;itrogen rule: +s indicated a!ove$ molecular ions formed in '(ionization are odd electron ions (f their o!served mass tocharge ratio is odd$ the molecule under investigation containsan odd num!er of nitrogen atoms (f that mass to charge ratiois an even num!er$ that molecule contains no or even ;itrogenatoms

*hemical (onization

&or organic chemists$ *hemical (onization ,*(- is especially usefultechnique when no molecular ion is o!served in '( mass spectrum$ andalso in the case of confirming the mass to charge ratio of themolecular ion *hemical ionization technique uses virtually the sameion source device as in electron impact$ except$ *( uses tight ion

source$ and reagent gas @eagent gas ,eg ammonia- is firstsu!7ected to electron impact Sample ions are formed !y theinteraction of reagent gas ions and sample molecules "his phenomenonis called ion3molecule reactions @eagent gas molecules are presentin the ratio of a!out /22:/ with respect to sample molecules6ositive ions and negative ions are formed in the *( process#epending on the setup of the instrument ,source voltages$ detector$etc- only positive ions or only negative ions are recorded

(n *($ ion molecule reactions occur !etween ionized reagent gasmolecules ,G- and volatile analyte neutral molecules ,M- to produceanalyte ions 6seudo3molecular ion MA< ,positive ion mode- or BM3AC3

,negative ion mode- are often o!served Dnli5e molecular ionso!tained in '( method$ MA< and BM3AC3 detection occurs in high yieldand less fragment ions are o!served

6ositive ion mode:

GA< < M 333333? MA< < G

;egative ion mode:

BG3AC3 < M 333333? BM3AC3 < G

"hese simple proton transfer reactions are true gas3phase +cid3aseprocesses in the ronsted3.owrey sense +EtightE ion source,pressure82/30 torr- is used to maximize collisions which results inincreasing sensitivity "o ta5e place these ion molecule reactionsmust !e exothermic 6roton transfer is one of the simple processeso!served in positive *(:

@A< < M 33333? MA< < @

%ne of the decisive parameter in this reaction is the protonaffinity &or the reaction to occur$ the proton affinity of themolecule M must !e higher that the one of the gas molecule "he mainreagent gases used in *( are: +mmonia$ Methane$ and (so!utane "he


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predominant reactant ions formed are given in the mechanisms shown!elow *hoice of reagent gas affects the extent of fragmentation ofthe quasi3molecular ion

Methane ,positive ion chemical ionization-:

• *A4 < e 33333? *A4


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;AF ,ammonia- is the most used reagent gas in *( !ecause of the lowenergy transfer of ;A4< compare to *A< for example With ;AF asreagent gas$ usually MA< and M;A4< ,/= mass units difference- areo!served

;egative (on *hemical (onization

"hree mechanisms can !e underlined:

/ 'lectron capture reaction due to attainment of slow moving$ lowenergy EthermalizedE electrons which may !e transfered moreefficiently to sample molecules

0 'lectron transfer from ionized reagent gas ,eg ;A03 maytransfer an electron to a molecule having a greater electronaffinity than ;A0-

F @eagent gas ions participate in true *( reactions ,eg protona!straction$ according to relative acidities-

Molecular ions o!served in negative ion chemical ionization massspectra are usually M3 or BM3AC3

'lectrospray (onization Method

+mong the most used spray ionization techniques is 'lectrospray(onization ,'S(- "his technique continues to !e the method of choicefor analyzing thermola!ile chemicals (ts capa!ilities are welldocumented (t uses an electrical stress !etween the 'S( pro!e exit

,eg capillary- and the counter electrode$ which is located fewmillimeters from the pro!e "he process results in the generation of


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highly charged droplets directly from the infused solution Multiplyand)or singly charged analyte molecules desor!e from the sprayeddroplets and sampled through the rest of the mass spectrometer 'S(has !een distinguished for its a!ility to produce multiply chargedmolecular ions from a large variety of polymers such as protein and#;+ fragments9 it allows also sensitive detection of singly chargedlow molecular weight polar species such as drugs and drugmeta!olites "he formation of positive or negative ions ,depending onthe sign of the applied electrical field- occurs in high yield (nthe positive ion mode protonated and)or al5ali adduct analytemolecules generally o!served in the mass spectra (n the negative ionmode operation pea5s corresponding to deprotonated analyte moleculesare o!served 'S( is descri!ed as a very EsoftE ionization techniquewhere the surrounding !ath gas has a moderating effect on theinternal and translational energies of desor!ed ions

+dvantages of 'S(:

• Soft ionization process so intact molecular ions are

o!served

• 'S( allows production of multiply charged ions "his

results in the a!ility of analyzing very high molecularweight species using the most availa!le mass analyzers,eg quadrupoles-

• 'S( is an atmospheric pressure process "his ma5es it

easy to use and easy to interface with A6.* and *'separation techniques

Matrix +ssisted .aser #esorption ,M+.#(-


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Matrix +ssisted .aser #esorption ,M+.#(- mass spectrometry techniquewas introduced !y Jaras and Aill5amp in /HII for the ionization ofpeptides and proteins Soon there after this technique was a!le toanalyze other type of !iomolecules$ such as oligosaccharides$glycolipids$ nucleotides$ and synthetic polymers (n this technique$samples are cocrystallized with a D>3a!sor!ing su!stance calledmatrix &or example for proteins$ the matrix of choice is oftensinapinic acid + FF= nm radiation from nitrogen laser is mostcommonly used "he laser helps introducing energy into the molecularsystem in such a way preventing thermal decomposition

M+.#( is often used with time3of3flights mass spectrometers , "%& -due to the pulsing nature of the technique$ and the mass rangecapa!ility Molecular weights up to few hundreds of daltons could !emeasured *omparison of M+.#( and 'S( ionization techniques wasattempted in the last few years (n my opinion these two techniquesare not competitive !ut complementary Kust to name a few$ for high

molecular weight species$ M+.#( leads to the formation of singlycharged molecular ions while 'S( allows the formation of multiplycharged molecular ions

6ractical considerations:

• "he final molar ratio sample)matrix is a!out or around

/)222

• &inal concentration of the sample is from / to /2

pmol)ul

• %ur experience with M+.#( point to a dynamic range of

/22 fmol)ul to few hundreds pmol)ul

• M+.#( is relatively ro!ust ionization technique that

tolerates the use of salts and surfactants and !uffers+lthough it is !est to remove them for !etterperformance

6eptide and 6rotein Standards for M+.#(:

• +ngiotensin (( ,human- MW: /2410

• Su!stance 6 ,human- MW: /F4==

• (nsulin ,!ovine- MW: =FF1

• *ytochrom c ,equine- MW: /0$F12/

• @;ase + ,!ovine- MW: /F$1I00

• +po3Myoglo!in ,equine- MW: /1$H/

• "rypsinogen ,!ovine- MW: 0F$HI2H


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+tmospheric 6ressure *hemical (onization

+6*( is a technique which creates ions at atmospheric pressure +sample solution flows through a heated tu!e where it is volatilizedin a mist and sprayed into a corona discharge with the aid ofnitrogen ne!ulization Sample molecular are ionized !y ion moleculereactions from the am!iant corona discharge ions (ons are producedin the discharge and extracted into the mass spectrometer +6*( is!est suited to relatively polar$ semi3volatile samples +n +6*( massspectrum usually contained the quasi3molecular ion$ BM3AC3 or BM < AC


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"he sector mass spectrometer was one of the most common types of massanalyser and pro!a!ly the most familiar to the everyday scientist (nthe /H2Ls$ the first commercial mass spectrometers were sectorinstruments "hey consist of some com!ination of a largeelectromagnetic$ and some 5ind of electrostatic focussing device#ifferent manufactures use differing geometries &igure / shows aschematic of a standard L'L geometry dou!le focussing instrument"he ' configuration is also called reverse geometry sector massspectrometer 3 that is$ a dual sector instrument consisting of amagnetic sector followed !y an electrostatic sector

(ons enter the instrument from the source ,!ottom left- where theyare initially focussed "hey enter the magnetic sector through thesource slit where they are deflected according to the left3hand rule

Aigher3mass ions are deflected less than lower3mass ions Scanningthe magnet ena!les ions of different masses to !e focussed on themonitor slit +t this stage$ the ions have !een separated only !ytheir masses "o o!tain a spectrum of good resolution where all ionswith the same m)z appear coincident as one pea5 in the spectrum$ ionshave to !e filtered !y their 5inetic energies +fter another stage offocussing the ions enter the electrostatic sector where ions of thesame m)z have their energy distri!utions corrected for and arefocussed at the dou!le focussing point on the detector slit

Sector instruments had huge commercial successes in the /H2Ls and/H12Ls as they were the only practical way of o!taining high3

resolution data (n the last 02 years or so$ with the decreasingprices of &"MS and the development of high3resolution alternatives,for example Q3"of- sector instruments are in decline

"ime3of3&light Mass Spectrometry ,"%&3MS-

+ time3of3flight mass spectrometer uses the differences in transittime through a drift region to separate ions of different masses (toperates in a pulsed mode so ions must !e produced or extracted inpulses +n electric field accelerates all ions into a field3freedrift region with a 5inetic energy of q>$ where q is the ion chargeand > is the applied voltage Since the ion 5inetic energy is 2mv0$lighter ions have a higher velocity than heavier ions and reach thedetector at the end of the drift region sooner


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"heory:

• J' 8 q>

• /)0 mv0 8 q>

• v 8 ,0q>)m-/)0

"he transit time ,t- through the drift tu!e is .)v where . isthe length of the drift tu!e

• t8. ) ,0>)m)q-/)0

Schematic of a linear "%&3MS

"his schematic shows a!lation of ions from a solid sample with apulsed laser "he reflectron is a series of rings or grids that actas an ion mirror "his mirror compensates for the spread in 5ineticenergies of the ions as they enter the drift region and improves theresolution of the instrument "he output of an ion detector isdisplayed on an oscilloscope as a function of time to produce themass spectrum

(on "rap


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(ons created !y electron impact ,'(-$ electrospray ,'S(-$ or matrix3assisted laser desorption ,M+.#(- ionization are focused using an

electrostatic lensing system into the ion trap +n electrostatic iongate pulses open ,3>- and closed ,- to in7ect ions into the iontrap "he pulsing of the ion gate differentiates ion traps fromE!eamE instruments such as quadrupoles where ions continually enterthe mass analyzer "he time during which ions are allowed into thetrap$ termed the Eionization durationE$ is set to maximize signalwhile minimizing space3charge effects Space3charge results from toomany ions in the trap that cause a distortion of the electricalfields leading to an overall reduction in performance "he ion trapis typically filled with helium to a pressure of a!out / mtorr*ollisions with helium dampens the 5inetic energy of the ions andserve to quic5ly contract tra7ectories toward the center of the ion

trap$ ena!ling trapping of in7ected ions "rapped ions are furtherfocused toward the center of the trap through the use of anoscillating potential$ called the fundamental rf $ applied to thering electrode +n ion will !e sta!ly trapped depending upon thevalues for the mass and charge of the ion$ the size of the ion trap,r-$ the oscillating frequency of the fundamental rf , w-$ and theamplitude of the voltage on the ring electrode , > - "he dependenceof ion motion on these parameters is descri!ed !y the dimensionlessparameter qz $ qz 8 4e>)mr0w0

Quadrupole


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+ quadrupole mass filter consists of four parallel metal rodsarranged as in the figure !elow "wo opposite rods have an appliedpotential of ,Dcos,wt-- and the other two rods have a potential of

3,Dcos,wt--$ where D is a dc voltage and >cos ,wt- is an acvoltage "he applied voltages affect the tra7ectory of ions travelingdown the flight path centered !etween the four rods &or given dc andac voltages$ only ions of a certain mass3to3charge ratio pass throughthe quadrupole filter and all other ions are thrown out of theiroriginal path + mass spectrum is o!tained !y monitoring the ionspassing through the quadrupole filter as the voltages on the rods arevaried "here are two methods: varying w and holding D and >constant$ or varying D and > ,D)>- fixed for a constant w

"andem Mass Spectrometry:

"andem Mass Spectrometry$ usually referred to as MS)MS$ involves theuse of 0 or more mass analyzers (t is often used to analyzeindividual components in a mixture "his technique adds specificityto a given analysis +lthough tandem mass spectrometry can !ereferred to MS)MS$ MS)MS)MS$ etc$ in this presentation ( am goingto descri!e only MS)MS

"he !asic idea of MS)MS is a selection of a m)z of a given ion formedin the ion source$ and su!7ect this ion to fragmentation$ usually !y

collision with inert gas ,eg +rgon- "he product ions are thendetected "his is a powerful way of confirming the identity of


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certain compounds and determining the structure of un5nown speciesSo MS)MS is a process that involves F steps: ionization$massselection$mass analysis

MS)MS could !e performed on instruments such as triple quadrupole,QQQ-$ ion trap$ time of flight$ fourier transform$ etc "he triplequadrupole is the most frequently used mass spectrometer for MS)MS$perhaps !ecause of the cost and ease of use among other factors

Separation Methods for *oupling with Mass Spec

• G*3MS: Sample mixtures are directly vaporized and enter !onded

fused silica columns *omponents of the mixture are separated!ased on their affinity difference with the !onded phaseSeparated compounds exit the column and enter the vacuum system

of the mass spectrometer Sample molecules are ionized ,'($ or*(-$ and accelerated into a precali!rated mass analyzer ,egQ$ (on "rap$ "%&$ &"MS etc- @etention times$ molecularmasses$ and fragmentation patterns are recorded %ne of themost important considirations of G*)MS is that the sample,s-must !e non thermola!le meaning thermally sta!le

• .*3MS: &or compounds that are thermally unsta!le$ .*)MS method

is considered "he separation is !ased on the diffrence inaffinity of samples with stationary phase and the mo!ile phaseeg hydropho!icity in case of @6 chromatography

• *'3MS: "his method is !ased on diffrences of electrophoretic

mo!ility of samples when the fused silica column is under apotential difference !etween in7ection side and detector side

•*('&3MS:

"his is a variant of *' (t is !ased on diffrerences

in isoelectric points of analytes

What is Mass Spectrometry

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