Mass Spectroscopy

Mass spectrometry is the study of systems generatingthe formation of gaseous ions, with or withoutfragmentation; which are then characterized by theirmass to charge ratios (m/z) and relative abundances.

In MS compounds are ionized, the ionized moleculedecomposes into smaller ions/radicals/radical-ions/neutrals. One way to ionize molecules is to extractelectrons from a molecule by high energy collisons.

The positively charged species thus produced areseparated, based on their mass/charge (m/z) ratio.

Parent ion/ daughter ions, radicals, neutral, …Molecular ion

Most of the ions has z=+1 m/z = mass of the fragment.A plot of relative abundance of ions vs. m/z of thecharged particles is presented as the Mass Spectrum.

ionization fragmentationM M+. M+

1 + M+.2+..+N1+ N2

. + …..

MolecularIon peakM+.

Base peak

Spectral output is presented as a histogram.

Nominal mass

Fragmentation:

M1+ + N1

.

M2+. + N2

M+. M1+ M3

+ + N4

M+. Radical ion (odd e)N. Neutral radical (odd e)N Neutral (even e)

M+ (even e) would not break up into a radical ion….

Actual signalhas peaks witha line width.

Isotope peaks:In mass spectroscopy the actual mass of the fragmentsgenerated are determined. Therefore fragments withdifferent isotopes are distinguished, e.g. Lead metal.

For molecular fragments, the isotope peak abundanceis dependent on the molecular constitution and thenatural isotope abundance of the constituent elements.

In mass spectroscopy the masses of individual ‘ions’are measured.

Mass and ‘abundance’ of ions of each isotopiccomposition is measured!! – not the averagemolecular mass.

4

.

.

13 + 2 +4 3

2 13 + 2 +3 2 2

CH peak MCH , HCH isotope peak M+1

H CH , H CH isotope peak -negligible M+2

Cl-CH2-CH2-S-CH2-CH2-Cl = 159

Isotope peaks

Unit massspacing Cluster

Different peaks,because there aresome molecules with13C, 2H etc. Also M lossof or gain of H+ or H..Especially significant forCl, Br

Peaks are spaced by aunit mass

All peaks in a clustercan be of samemolecular formulam/z

The Nominal massis m/z of the lowestmass isotopomer,i.e. the member ofthe isotopes clusterthat has all the C’sas 12C, all protons as1H, all N’s as 14N, ….

The existence of isotopes generates a cluster of peaks(isotope peaks).

Isotopomers (isotopic isomers) are isomers having the samenumber of each isotopic atom but differing in their positions.

Mass spectrometry is highly sensitive as a detectiontechnique of analytes. The total ion current (TIC) orindividual ion current (SIM) generated by an analyteis proportional to the analyte amount in a sampleanalyzed (or concentration of the analyte in thesample). Thus MS makes an excellent detector forchromatography.

Further the spectra which are intensity vs. m/z valuesof ionized and fragmented analytes is unique to eachanalyte. Thus MS spectra are extremely helpful forqualitative analysis.

Mass Spectrometer:

Mass spectrometer has devices for each of the following;

Sample IntroductionCreate gas-phase ions of the sampleSeparate ions in space or time based on m/z ratio;

accomplished by mass analyzersDetect the of ion current from each m/z ratio ion

Ionization: If a quantity of energy is supplied to amolecule greater than the ionization energy of themolecule, a molecular ion is formed M+ •.

Electron Ionization (Electron Impact, EI)Chemical Ionization (CI)Electrospray Ionization (ESI)Matrix Assisted Laser Desorption Ionization (MALDI)Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Photo-ionization (APPI)Atmospheric Pressure Laser Ionization (APLI)Fast Atom Bombardment (FAB)Inductively Coupled Plasma (ICP)

Ion Detection:Faraday CupElectron MultiplierPhotomultiplier Conversion Dynode

Mass Analyzers:Magnetic Sector Mass Analyzer (Single/DoubleFocusing )Quadrupole Mass FiltersTime-of-flight (TOF) Mass AnalyzerIon Trap Mass AnalyzerFourier-Transform Mass Spectrometry (FTMS)

Electron impact ionizationMagnetic sector separationsingle focusing

Mass Spectrometer

Evacuatedsystem- 10-6 torr

B

-iF

The interior of the mass spectrometer must beevacuated. The ion source, mass filter/analyzer, anddetector are under vacuum so that the ions wouldmove from the ion source to the detector withoutcolliding with other ions and molecules. The mean-freepath of a charged particle should be greater than thedistance between ionization and detection regions.

A high vacuum is created with two pumps where alow-vacuum pump is connected to the output of ahigh-vacuum pump.

Diffusion pump(s) + rotary-vane rough pumpTurbo-molecular pump(s) + rotary-vane rough pump

Pressure (Torr) Mean Free Path (m)760 6.0x10-8

1 4.5x10-5

10-3 4.5x10-2

10-5 4.510-7 4.5x102

10-9 4.5x104

Electron impact ionization (EI)

- +

Ekin = zeV = mv2/2

VIon optics

70eV – high energy electrons,molecular ion - very energetic, low/no abundance.

70V

Volatilized compound is ionized by electron impact.An electron beam is generated by a accelerating theelectrons from a heated filament through an appliedvoltage.

The electron energy is defined by the potentialdifference between the filament and the sourcehousing and is usually set to 70 eV.

A field keeps the electron beam focused across theion source.

Upon impact with a 70 eV electron, the gaseousmolecule may lose one of its electrons to become apositively charged radical ion, daughter ions, etc.

All ions are subsequently accelerated out of the ionsource by an electric field produced by the potentialdifference applied to the ion source and a groundedElectrode, V.

A 'repeller' serves to define the field within the ionsource.

Depending on the lifetime of the excited state,fragmentation will either take place in the ion sourcegiving rise to stable fragment ions, or on the way tothe detector, producing metastable ions.

Ion source accelerates ions to a KE

KE = ½ mv2 = zeV

In the magnet F = mv2 /r = Bzev,

Upon rearrangement r = mv/zeB = (2Vm/ze)1/2/B

m/z = (eB2r2)/(2V)

Magnetic sector mass analyzer:

B

r

V

note: slits

Each m/z beam follows it’s own path (r) for agiven B and V in the magnetic sector (60o/900).

For specific V and B ions of unique m/z pass thro’the magnetic sector and reaches the stationarydetector. Variations of V and/or B causes fragmentsof different m/z value to reach the detector.

Usually B is scanned to allow different m/z’s toreach the detector sequentially generating thecomplete mass spectrum keeping V constant.

2 2

2

m r

z

e B

V

The m/z ratio of the ions that reach the detector canbe varied by scanning either the magnetic field (B)or the applied voltage of the ion optics (V).

i.e. by varying the voltage or magnetic fieldof the magnetic-sector analyzer, the individual ionbeams are separable spatially, radius of curvatureis held constant.

The distribution of a given mass by way of energydistribution of kinetic energy.

e(V-V)

e(V+V)

-

+E

B

2 2

22 m

r indepen

mv mvzeE and

dent of in E; focussing!z

zeV=r

Vr ;

E

Magnetic Sector Mass Analyzer: Double Focusing (EB)

V

Ionizationtechnique

TypicalAnalytes

SampleIntroduction

MassRange Comments

Electron Impact(EI)

Relativelysmallvolatile

GC orliquid/solid

probe

upto1,000Daltons

Hard methodversatileprovides

structure info

ChemicalIonization (CI)

Relativelysmallvolatile

GC orliquid/solid

probe

upto1,000Daltons

Soft methodmolecular ionpeak [M+H]+

Electrospray(ESI)

PeptidesProteinsnonvolatile

LiquidChromatography

or syringe

upto200,000Daltons

Soft methodions oftenmultiplycharged

Fast AtomBombardment(FAB)

CarbohydratesOrganometallics

Peptidesnonvolatile

Sample mixedin viscous

matrix

upto6,000Daltons

Soft methodbut harderthan ESI or

MALDIMatrix AssistedLaserDesorption(MALDI)

PeptidesProteins

Nucleotides

Sample mixedin solidmatrix

upto500,000Daltons

Soft methodvery high

mass

Sample introduction/ionization method: : Resolving Power1.

Defined in terms of the overlap (or ‘valley’)between two peaks. For two peaks of equal height, massesm1 and m2, when there is overlap between the two peaks toa stated percentage of either peak height (10% isrecommended), then the resolving power is defined as[m1/|m1 – m2|].

The percentage overlap (or ‘valley’) concernedmust always be stated.

Actual signalhas peaks with a linewidth.

Imposes a limitation onthe resolvability ofconsecutive peaks

10%

Example

MR

M

Minimum resolution necessaryto separate N2

+ and CO+

peaks?

Exact masses:N2

+ = 28.006158 amuCO+ = 27.994915 amu

282490

0.011241

MR

M

2.

State the method ofcalculation whenexpressing resolvingpower, and the positionof the lower peak.

Example

The molecular ion (dominant) is formed by theremoval of the least tightly bound electron.

Ionization EI: Chemical ionization (CI):

Interaction of the molecule M with a reactive ionizedreagent species (gaseous Bronsted acids). e.g..,EI of methane, generates CH4

+· which then reacts togive the Bronsted acid CH5

+;

CH4+· + CH4 CH5

+ + CH3·

If M in the source has a higher proton affinity than CH4,the protonated species MH+ will be formed by theexothermic reaction.

M + CH5+ MH+ + CH4

CI is a softer ionization process.

CI is a lower energy process than EI, results in lessfragmentation and therefore a simpler spectrum with theparent/molecular ion intact. M+. nearly nonexistent.

Abundant M+.

Fast Atom Bombardment Ionization:

The sample droplet is bombarded with energetic atoms (Ar, Xe) of 8-10keV kinetic energy. Ions (e.g., Cs+) can be used as the bombardingparticle in a similar technique termed liquid secondary ion massspectrometry (LSIMS)

Beam collides with the sample and matrix molecules, producing positiveand negative sample-related ions that can be accelerated into the massspectrometer.

Used for polar organic compounds, acidic and basicfunctional groups.

Basic groups run well in positive ionization mode andacidic groups run well in negative ionization mode.

FAB analytes: peptides, proteins, fatty acids,organometallics, surfactants, carbohydrates,antibiotics, and gangliosides.

Fast Atom BombardmentQuadrupole Mass Analyzer (spectrometer):

Diagonal electrodes have potentials of the same signU= DC voltage, V=AC voltage and ω= angular velocity of

alternating voltage in RF range.

4 parallel, polished metal rods

-U-Vcos(ωt)

+U+Vcosωtx

y

z

Behavior of electrical charges in electric fields.The path of charged particles depends on size/mass differences.

Larger masses have a higher inertia than a small mass.

xy

z

http://www.webapps.cee.vt.edu/ewr/environmental/teach/smprimer/icpms/icpms.htm

2r0

Parameters affecting motion: m/z, U, V, r0 and .

+ [U+Vcosωt]

-[U+Vcos(ωt)]

Positive plane

Negative plane

+ Apply a DC; make it + on twodiagonal rods.

+ ions

+

xy

z

Apply a DC; make it (+) on the twodiagonal rods.Superimpose an AC; V sin twith an amplitude V anda frequency .

+ ions

+ U + V sin t

+ U + V sin t

Lighter ions spirals out of the quadrupole (filters out).

+ ions

+ U + V sin t

+ U + V sin t

Apply a DC; make it (+) on the tworods.Superimpose an AC; V sin twith an amplitude V anda frequency .

Ions repelled from rods, AC component act on small m/z more effectively..Heavier ions travel straight to the detector because of their high inertia.

m/z

High Pass FilterLower m/z crashes

For a given U and V there will be m/z belowwhich ions would not pass through the rods.

+ ions

- U - V sin t

- U - V sin t

Ions attracted from rods, AC component act on large m/z more effectively.Heavier ion spirals out of the quadrupole (filters out).

+ ions

- U - V sin t

- U - V sin t

Lighter ions travel straight to the detector.

m/z

Low Pass Filterhigher m/z crashes

For a given U and V there will be m/z abovewhich ions would not pass through the rods.

Quadrupole Mass Analyzer (Spectrometer):

Ions oscillate under the influence of the variablefields.

Combined DC and RF potentials on the quadrupolerods create a stable ‘linear’ path and passes only aselected m/z ratio (resonant ion) at a time. All otherm/z ions acquire unstable paths and spirals out.

The mass spectrum is obtained by varying thevoltages on the rods and monitoring which ions passthrough the quadrupole rods.

Quadrupole mass (QM) analyzer is a "mass filter".

The solution of equations of motion of ions travelingthrough a QM analyzer shows that for an ion with aparticular m/z to pass through at certain combinationsof U and V must be obtained.

Varying rod voltages (scanning the spectrum):

a. scan ω while holding U and V constantb. scan U and V but keep the ratio U/V fixed

If U and V are scanned such that U/V = constant,with V>U then successive detection of ions ofdifferent m/z is achieved.

2 2 2 2 2 20 0

4 2ze ze =

m r

Vq

m r

Ua

Two functions a and q define a stable trajectory forwhich ions do not collide with the rods across arange of values of U and V.

In principle QM analyzer can be operated for a rangeof U and V values.

2a U

q V

(U)

(V)

A given m/z ion travels thro’ the quadrupole if the values of Uand V are in a segment of the operating line and bounded bythe stability curve .

Stability curve for an ion - m/z

0V

U

Of all possible U,V coordinates there exist combinationswhere the ion path through the quad is stable (ions reachesthe detector); other combinations are unstable.

Graphically, e.g. the three stability curves representvalues of U and V for which the masses m1, m2 andm3 have stable trajectories through the quadrupole.

Only those mass values on the operating linetransmits.

(U)

(V)

(U)

(V)

The resolution is determined by the magnitudeof U/V ratio, i.e the slope.

Resolution of the mass analyzer can be increasedby increasing the slope of the line, U/V (= held const.),and that if U = 0 then ions of all m/z are transmitted.

Because quadrupoles operate at lower voltages, theycan be scanned at faster rates (~1000 a.m.u./s) thanmagnet based spectrometers. QMs are betterdetectors for LC-MS and GC-MS implementation. m/z

High Pass Filter Low Pass Filter

Narrow window pass

OVERLAPPEDREGION

Use a high pass filter and a low pass filtersimultaneously and judiciously.

m/z

Holding U, V allows ions of only one m/z ion move down tothe detector. Simultaneous ramping of both U, V fields (withthe suitable ratio U/V held constant); ions of various m/z ionspasses through the mass filter to the detector sequentially.SCANNING!!

m/z

DC fields tend to focus positive ions in the positive plane anddefocus them in the negative plane. Ions oscillate withincreasing amplitudes until they collide with the electrodes.The lighter the ion in mass, the smaller the number of cyclesbefore it is annihilated.

z

y

x

y

+

Viewed down y

x

y

z

MS/MS

QqQ

MS1 MS2

Dissociation region

Scan MS1 (Q1 only) turned on – entire MS spectrum.

Set (e.g. proper U and V) MS1 to filter fragment ofinterest, dissociate further in q (Q2) the fragmentselected in MS1 by collision with Ar or N2. Mass analyzethe products from Q2 by scanning in MS2 (Q3).

Time-of-Flight Mass Analyzers (spectrometer):

TOF measures the mass-dependent time required forions of different masses to move from the ion sourceto the detector.

This requires that at the starting time t=0, (time ionsleaves the ion source) to be well-defined.

Ions are created by a pulsed method (MALDI), orby rapid electric field switching that serves as a 'gate'to release the ions from the ion source in a veryshort time.

Lv

t

2

2

2m Vt

ze L

1

2

mt L

eV z

2

2

mvKE zeV

L

Time-of-Flight Mass Spectrometry, Principle

dtof

m/z

dsource

Probe(Start)

+U

Field Free DriftTube

MALDIIon Source

Ion Detector

ttotal = tsource + ttof

Laser

+/- U

Ion Source - MALDI

Metal plate

ions

Acceleration region

Time-of-Flight Mass Spectrometers,

Prism

Pumps (Turbo)

Fore Pump

Valves

BA1

BA2

TC2

Inlet

TC1

Grids

SamplePlate

Camera

ReflectorIon Selector

Detectors

LaLaser

Flight Tube

Ion

Signals

M1M2

time

+/- U

Linear TOF:

Ionizing Probe (start)

M3 Ion detector(MCP)

output

ion mirror

Reflex MALDI TOF Mass Spectrometer

Laser

Reflection time-of-flight mass spectrometer

Parent/Molecular Peak M:

An molecular ion that has not lost/gained atoms.The nominal mass of which is calculated with themass numbers of the predominant isotopes of atoms.

Base peak:

Base peak is the peak from the most abundant ion,which is often the most stable ion.

High Resolution MS:

Using mass number for isotopes of atoms isapproximate. Actual mass of a given isotope deviatesFrom this integer by a small but unique amount(E = mc2). Relative to 12C at 12.0000000, the isotopicmass of 16O is 15.9949146 a.m.u., etc.

High resolution mass spectrometers that candetermine m/z values accurately to four/moredecimal places, making it possible to distinguishdifferent molecular formulas having the samenominal mass.

http://www.chem.queensu.ca/FACILITIES/NMR/nmr/mass-spec/mstable3.htm

IsotopeAccurateMass

1-H2-H

1.0078252.014102

12-C13-C

12.00000000013.0033548

14-N15-N

14.003074015.0001090

16-O17-O18-O

15.994914616.999130617.9991594

Very short list. MF Unsaturation

C2H2O3 2.0

CH2N2O2 2.0

C6H2 6.0

C3H3FO 2.0

C2H3FN2 2.0

C3H6O2 1.0

C2H6N2O 1.0

C4H7F 1.0

C4H10O 0.0

C3H10N2 0.0

m/z=74

MF Unsaturation Exact Mass

C2H2O3 2.0 74.00040

CH2N2O2 2.0 74.01163

C6H2 6.0 74.01565

C3H3FO 2.0 74.01679

C2H3FN2 2.0 74.02803

C3H6O2 1.0 74.03678

C2H6N2O 1.0 74.04801

C4H7F 1.0 74.05318

C4H10O 0.0 74.07316

C3H10N2 0.0 74.08440 MF finder

m/z=74

CHEMCALC

Isotope Peaks

The peaks from the isotopes.

The intensity ratios (relative intensities) in the isotopepatterns arises from the natural abundance of theisotopes, thus are valuable to ascertain the atomiccomposition of ions.

M+1 peaks are primarily due the presence of 13Cin the sample.M+2 peaks useful for N, O calculation (< seven C cpds)M+2, M+4, .. indicative of presence of Br, Cl, S;(79Br : 81Br 1:1, 35Cl : 37Cl 3:1)

1000.8014.52

Isotope peak abundance depends on the molecularconstitution. Example, halogens Cl, Br.

e.g. (Nominal mass, M =100%)

%M+1= 0.012nH+1.08nC+0.369nN+0.038nO+5.08nSi+0.801nS

%(M+2) = 0.005nC(nC-1)+0.205nO+3.35nSi+4.52nS+32.0nCl+97.3nBr)

X=M

Calculating isotope peak abundances (%) to confirmfragment and parent peaks.

Nominal mass, M =100%

%M+1= 0.012nH+1.08nC+0.369nN+0.038nO+5.08nSi+0.801nS

%(M+2) = 0.005nC(nC-1)+0.205nO+3.35nSi+4.52nS+32.0nCl+97.3nBr)

Chromatography – MassSpectroscopy

GC-MS, LC-MS are hyphenated analytical techniques.Two techniques are combined to form a single methodfor analyzing mixtures of chemicals. Chromatographyseparates the components and MS detects (the analytes inthe effluent) and characterizes each of the components.

Combination of the techniques allows both qualitative andquantitative evaluation of analytical samples.

The MS spectrometer is highly selective of analyte of interest.

http://www.shsu.edu/~chm_tgc/sounds/flashfiles/GC-MS.swf

http://www.shsu.edu/~chm_tgc/sounds/flashfiles/SIM.swf

IonSource

MassAnalyzer

IonDetector

Inlet DataSystem

VacuumPumps

ChromatographOutput

DataOutput

All fragment ion (total)current (TIC) /ora single ion detected.

LC-MS:Ionization viaElectrospray (API)APCI (API)

QM

GC-MS:Ionization viaEI or CI

Each analyte’s MS produced

Total (reconstructed) ion current (TIC) vs time; Chromatogram

http://www.gmu.edu/departments/SRIF/tutorial/gcd/gc-ms2.htm

Total current of all ions of all masses from a GC peak is detected abovea selected value of time (solvent peak) for a separated analyte.

Total ion current (TIC) ChromatogramGC-MS,LC-MS takes data in three dimensionssimultaneously, recording the number of ionscreated along with their masses over time.

This information is generally represented byexamining the total ion chromatogram (TIC)and ‘slicing’ along the third dimension (m/z) of agiven peak to look at the mass spectrum at achosen time.

3D data sett

t MS – analyte 1

MS – analyte 2

MS – analyte 3

m/z

3D data sett

t MS – analyte 1

MS – analyte 2

MS – analyte 3

TIC MS requires high vacuum ~10-3 – 10-6 torr. Theeluate from a chromatographic system has muchmore carrier (gas/eluent) than the analyte. If eluatefrom the chromatograph is allowed to enter directlyinto the MS it would overwhelm the vacuum system(especially in LC – liquid vaporizes into a largevolume).

To alleviate the strain on the system, differentstrategies are employed at the sampleintroduction/ionization stage.

GC-MS; EI LCMS - ESI; Electrospray interface

Coaxial flow

For positive ion MS

Strong electric field+ nebulization;ions vaporize

+

One of the softest ionization techniques.

Low collisional dissociation; minimal fragmentation.Positive ions accelerateand collide with N2 molecules;few fragments form. Changingskimmer voltage to larger –vewould produce more fragmentation.

Atmospheric Pressure Chemical Ionization APCI (API)

Coaxial flow

Electrons formed at corona, injects into aerosol.

This technique produces single charged ions. Littlefragmentation, therefore less structural information.

.2 2

. .2 2 4 2

. .4 2 2 2

. .2 2 3

3

3 2

2 3 2

2

2

2

2

( )

( 1)( )

n

n

N e N e

N N N N

N H O N H O

H O H O

MHH

OH H O

H O nH O H O H O

M nH O HO O

Selected ion monitoring possible with the quadrupole massanalyzer with appropriate tuning.

SIM leads to highly selective detection of analytes.

Selected Ion Monitoring

SIM describes the operation of the mass spectrometer wherethe intensities of one or several specific ion beams (Multipleion monitoring) are monitored rather than the entire massspectrum.

The SIM more sensitive than the full scan experimentbecause the mass spectrometer can dwell for a longer timeover a smaller mass range; reduces background.

3D data sett

t MS – analyte 1

MS – analyte 2

MS – analyte 3

Selected ion chromatogramQM filter tuned to select one ion

Sam Houston University Applet

Selectivity and S/N ratio increased markedly withQqQ triple quad arrangement. Selected Reaction Monitoring (SRM)

SRM delivers a unique fragment ion that can be monitoredand quantified in the midst of a very complicatedmatrix. Plots are simple, usually a single peak. Thereforethe SRM plot is ideal for sensitive and specific quantitation.

The SRM experiment is accomplished by specifying theparent mass of the compound for MS/MS fragmentation andthen specifically monitoring for a single fragment ion. Similarto the SIM of a fragment ion. The specific experiment inknown as a "transition" and can be written (parent massfragment mass) For example 534375.

Pick the mass of interest

Fragment via collisioncontrol collisionalenergy to optimizefragmentation

Choose a stable peak

Quantitative analysis with MS can be done with thecalibration curve method.

Internal standard method gives better results; a fixed amountof an IS is incorporated to both samples and calibrationstandards. The ratio of the peak intensity of the analytestandard to that of the IS, is plotted as a function of standardanalyte concentration. Main advantage of the use of IS, isthe minimization of uncertainties arising at the samplepreparation and introduction stage.

A suitable IS would be a stable, isotopically labeled analogof the analyte; where one or more atoms of 2-H, 13-C or15-N have been incorporated.

It is reasonable to assume that in the mass spectrometerthe labeled molecules would be subjected to same processas do the unlabeled analyte molecules.

Quadrupole Ion Trap Mass Analyzer.

Lv

t

2

2

2m Vt

ze L

1

2

mt L

eV z

2

2

mvKE zeV

L

MALDI MALDI

Lasers are used to deliver a focused high density ofmonochromatic radiation to a sample target, which isvaporized and ionized.

The yield of ions is often increased by using asecondary ion source or a matrix. Mass Spectroscopy

Bioanalytical Perspective

Reflex Time-of-Flight Mass Spectrometry

Not all ions gets desorbed at the same time and inexactly the same location, leads to slightly differentvelocities for identical ions. Degrades the resolutiondue to peak broadening (lower resolution).

Reflex can detect post source decay.

opposing EDE Linear Mode DE Reflex Mode

MALDI is incompatible with continuous operation.It is a pulsed technique. Not for tandem methods asGC-MS, LC-MS and CE-MS.

Peaks such as [M+2H]+2, [M+H]+, [M+Na]+ is typical.

Used for analysis of proteins, peptides, fragmentsof peptides and their mixtures (molecular weights withpractically no fragmentation).

Fast, mixtures handled, more powerful thanelectrophoresis, chromatography.

MALDI tolerates moderate concentrations of buffersand salts.

Identification of proteins via the “peptide fingerprint”.

Proteinenzyme

products from cleavage at specificplaces of protein.

Spectrum = “MS fingerprint”

MALDI-TOF/MS

ESI – MS

Electrospray Ionization

Electrospray Ionization is a versatile ionization method.capable of analyzing compounds directly from aqueousor aqueous/organic solutions, mol wt ~200,000Da;peptides, proteins, carbohydrates, DNA fragmentsand lipids.

It is compatible with continuous operation.

http://www.chemsoc.org/ExemplarChem/entries/2004/warwick_robinson/ESI.htm

Analyte molecules in solution reach the end of astainless steel needle which is held at a high voltage inrelation to the counter electrode.

A fine spray of micro droplets is formed when theanalyte leaves the needle. This spray is highlycharged due to the high potential of the needle.

On traveling to the counter electrode, the highlycharged droplets lose the solvent molecules, the microdroplets shrinks due to solvent evaporation.

The region between the needle and the counterelectrode is held at atmospheric pressure, whilst theother side of the counter electrode is in vacuum.

Evaporation of analyte from the micro droplet surfacetakes place continuously. The diameter of the dropletdecreases whilst the total amount of charge remainsthe same.

Coulombic forces are exerted and induce instability onthe droplet surface, and as a result the droplet breaksup into smaller droplets.

This process leads to very small droplets containingonly an analyte ion with a lot of solvent around it.

When the diameter becomes less than 10nm theremaining solvent leaves the analyte ion, and freeions is formed.

The resulting increase in charge density of the droplet,forces the charged analyte ion out of the solution,leaving highly charged analyte molecules.

The charge on the species due to the protons bonded.

[M+nH+] type ions produces for a given analyte; n~+50.

Electrospray ionization produces multiply charged ions,(up to +30 to +50 charges). Therefore high molecularweights can be measured using mass analyzers with alimited weight range.

The number of charges an ion can depends stronglyupon its 3D dimensional structure.

When a protein assumes a tight conformation, onlysurface sites are available for protonation making theobserved charge states are relatively low.

The electrospray process produces a distribution ofcharge states, used in the interpretation of the spectra.

http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html

z increases,m/z decreasesfor the samemolecule

ESI-MS Spectrum of a biomolecule; Monoisotopic Masspeaks considered here

Isotope peaks

The m/z values can be expressed as follows:

Because z number of H+ ions are added to the moleculem/z = (MW + zmp )/z

where m/z = the mass-to-charge ratio (from plot)MW = the molecular mass of the samplez = the integer number of charges on the ionscomes from the attached protonsmp = the mass of a proton = 1.008 Da.

But we do not know z!!

z

z+1

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For example, if the ions appearing at m/z 1431.6in the lysozyme spectrum have “z" charges,then the ions at m/z 1301.4 will have “z+1" charges.

For these two consecutive peaks using:

m/z = (MW + zmp )/z

(m/z)1 1431.6 = (MW + zmp)/z

(m/z)2 1301.4 = [MW + (z+1)mp] /(z+1)

Solve; MW = 14,305.9 Da and z = 10

Based on two consecutive peaks

Assume the m/z values for two peaks are m1 and m2and that z2 is j charges less than z1.

Example:z1

z2

m/z

In general the following relationships can be used:

11

1 21 2

1 2

1 1 1 2 2 2

2 1

21

2 1

( )

. .

( )

( )

p

p p

+p p p

1

p

MWMW can be calculated from m m

z

MW z m MW z mm values on x-axis; m and m

z z z

i e MW M

MW M m z z m m z z m where m mass of H

and z z j

j m mz

m

requires

m

z

m1 is the value for the peak on the ‘m/z-axis

21

2 1

1

1 1

( ) 6(1372.5 1.008)

( ) (1372.5 939.2)

19

( ) 19(939.2 1.008) 17825.5

p

p

j m mz

m m

yields z

MW z m m Da

m/z

z1

z2j=6

Based on two consecutive peaks

z our notation

The result corresponds to the average molecularweight of the protein.

Identifying one charge state effectively identifies themall, as they increase or decrease by one by going leftor right respectively.

Charges can only be integers.