Mass Spectroscopy

Mass spectrometry is the study of systems causingthe 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, ionized moleculedecomposes into smaller ions/radicals/radical-ions/neutrals. One way to ionize molecules is to extractelectrons from a molecule.

The positively charged fragments 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 vs. m/z of the chargedparticles is presented as the Mass Spectrum.

ionization fragmentationM M+. M+

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

. + …..

MolecularIon peakM+.

Base peak

Spectrum 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 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

Isotope peaks

Unit massspacing Cluster

Different peaks,because there aresome molecules with13C, 2H etc. Especiallysignificant for Cl, Br

Peaks are spaced by aunit mass

All peaks (cluster) areof the same molecularformulam/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 Spectrometer:

Mass spectrometer has devices for each of the following;

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

accomplished by mass analyzers.Detect of the quantity of ions/ 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

2m er Bz 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 mr indepen

mv mvzeE and

dent of in E; focussing!z

zeV=r

Vr ;E

Magnetic Sector Mass Analyzer: Double Focusing (EB)

V

Resolving Power1.

Defined in terms of the overlap (or ‘valley’)between two peaks. For two peaks of equal height,masses m1 and m2, when there is overlapbetween the two peaks to a stated percentage ofeither peak height (10% is recommended), thenthe resolving power is defined as [m1/(m1 – m2)].

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

Actual signalhas peaks with aline width.

Imposes a limitationon the resolvabilityof consecutivepeaks

10%

Example

MRM

Minimum resolution necessaryto separate N2

+ and CO+

peaks?

Exact masses:N2

+ = 28.006158 amuCO+ = 27.994915 amu

28 24900.011241

MRM

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:Electrons in molecules occupy molecular orbitalsand hence acquire the energy associated withsuch orbitals. To remove electrons from suchorbitals and ionize the molecule energy is required.

The energy required depends on the orbital of electronoccupation namely the HOMO. Thus the ease ofionization will depend on the “types of electrons” inthe molecule.

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 inless fragmentation and therefore a simplerspectrum with the parent/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 andbasic functional groups.

Basic groups run well in positive ionizationmode and acidic groups run well in negativeionization mode.

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

Fast Atom Bombardment

Quadrupole Mass Analyzer (spectrometer):

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

alternating voltage

4 parallel, polished metal rods

-[U+Vcos(ωt)]

+ [U+Vcosωt]x

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

2r0

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

+ [U+Vcosωt]

-[U+Vcos(ωt)]

+

Apply a DC; make it (+) ontwo diagonal rods.

+ ions +x

y

z

Superimpose an AC; V sin twith an amplitude V anda frequency .

+ ions

+ U + Vsin 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 .

Heavier ions travel straight to the detector

m/z

High Pass FilterLower m/z crashes

High masspass through

+ ions

- U - V sin t

- U - V sin t

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

Low masspass through

m/z

High Pass Filter Low Pass Filter

Narrow window pass

m/z

High Pass Filter Low Pass Filter

Narrow window pass

m/z m/z

z

y

x

y

+Viewed down y

xy

z

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, 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, V>Uthen successive detection of ions of different m/z isachieved.

2 2 2 2 2 20 0

4 2ze ze =m r

Vqm 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 Uq V

(U)

(V)

A given m/z ion travels thro’ the quadrupole if thevalues of U and V are in a segment of the operating lineand bounded by the stability curve .

Stability curve for an ion - m/z 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.

MS/MS

QqQ

MS1 MS2

Dissociation region

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

Set (e.g. U and V) MS1 to filter fragment of interest,dissociate further in q (Q2) collision with Ar or N2 thefragment selected in MS1 and mass analyze byscanning with MS2.

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.

Lvt

2

2

2m Vtze L

12

mt LeV z

2

2mvKE 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, General Organization

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 MassSpectrometer

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

Isotope AccurateMass1-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