Mass Spectrometry

PRINCIPLES OF PMASS

NCIPLES OF PRINSS SPECTROMETRY

HCMC 2013

Prepared by HUYNH KHANH DUY - HCMUT

(C) HKD 2013

BACKGROUND

� Mass spectrometry (Mass Spec or MS)uses high energy electrons to break amolecule into fragments.

� Separation and analysis of the fragments provides information about:� Molecular weight� Structure

(C) HKD 2013

BACKGROUND

� The impact of a stream of high energyelectrons causes the molecule to lose anelectron forming a radical cation.� A species with a positive charge and

one unpaired electron

+ e-C H

H

HH H

H

H

HC + 2 e-

Molecular ion (M+) m/z = 16

(C) HKD 2013

BACKGROUND

� The impact of the stream of high energyelectrons can also break the molecule orthe radical cation into fragments.

(not detected by MS)

m/z = 29

molecular ion (M+) m/z = 30

+ C

H

H

H

+ H

HH C

H

H

C

H

H

H C

H

H

C

H

H

H C

H

H

+ e-H C

H

H

C

H

H

H

(C) HKD 2013

BACKGROUND

� Molecular ion (parent ion):� The radical cation corresponding to the

mass of the original molecule

� The molecular ion is usually the highestmass in the spectrum� Some exceptions w/specific isotopes� Some molecular ion peaks are absent.

H

H

H

HC H C

H

H

C

H

H

H

(C) HKD 2013

BACKGROUND

� Mass spectrum of ethanol (MW = 46)

M+base peak

(C) HKD 2013

BACKGROUND

� C3H6O and C3H8O have nominal masses of 58 and 60, andcan be distinguished by low-resolution MS.

� C3H8O and C2H4O2 both have nominal masses of 60.

� Distinguish between them by high-resolution MS.

C2H4O2

C3H8O60.0211260.05754

60

60

MolecularFormula

Nominal Mass

PreciseMass

� High resolution MS can replace elemental analysis forchemical formula confirmation.

MASS RESOLUTION

(C) HKD 2013

BACKGROUND

Basic components of mass spectrometer

Ionization

Source

Mass

AnalzyerDetector

Inlet all ionsselected

ionsData

System

Vacuum pumps system(C) HKD 2013

BACKGROUND

� Electron impact (EI): vapor of sample is bombarded withelectrons: M + e → 2e + M.+

→ fragments

� Chemical ionization (CI): sample M collides with reagent ionspresent in excess e.g.

CH4 + e → CH4.+

→ CH5+

M + CH5+

→ CH4 + MH+

� Fast Atom/Ion Bombardment (FAB): Softer than EI. Ions areproduced by bombardment with heavy atoms. Gives (M+H)+ ionsand litle fragmentation. Good for more polar compounds.

� Laser Desorption & Matrix-Assisted Laser Desorption(MALDI): hit the sample with a laser beam.

� Electrospray Ionization (ESI): a stream of solution passesthrough a strong electric field (106 V/m).

WAYS TO PRODUCE IONS

(C) HKD 2013

BACKGROUND

EI ESI(C) HKD 2013

BACKGROUND

MALDI FAB(C) HKD 2013

BACKGROUND

ICP

(C) HKD 2013

BACKGROUND

IONIZATION METHODS

1. Electron Ionization (EI): most commonionization technique, limited to relatively lowMW compounds (<600 amu).

2. Chemical Ionization (CI): ionization with verylittle fragmentation, still for low MWcompounds (<800 amu).

3. Desorption Ionization (DI): for higher MW orvery labile compounds.

4. Spray ionization (SI)

for LC-MS, biomolecules, etc.(C) HKD 2013

BACKGROUND

Electron ionization

� vaporized sample is bombarded with highenergy electrons (typically 70 eV).

� “hard” ionization method leads to significantfragmentation.

� ionization is efficient but non-selective.

M

Neutral molecule

Molecular ion

M+

e-

E << 70 eV

++

+ ++

Fragment ions

fragmentationM + e- M+. + 2e-

M+. ABCD+ + N

ABCD+ ABC+ + C

ABC+ BC+ + A

e- E = 70 eV

(C) HKD 2013

BACKGROUND

Electron ionization

Advantages

• inexpensive, versatile and reproducible.

• fragmentation gives structural information.

• large databases if EI spectra exist and are searchable.

Disadvantages

• fragmentation at expense of molecular ion.

• sample must be relatively volatile.(C) HKD 2013

BACKGROUND

Chemical ionization (CI)

� Vaporized sample reacts with pre-ionizedreagent gas via proton transfer, chargeexchange, electron capture, adduct formation,etc.

� Common CI reagents: methane, ammonia,isobutane, hydrogen, methanol.

� “soft” ionization gives little fragmentation.

� Selective ionization-only exothermic orthermoneutral ion-molecule reactions will occur.

� choice of reagent allows tuning of ionization.(C) HKD 2013

BACKGROUND

Positive Chemical ionization (CI)

� Uses reagent gas (methane, isobutane or ammonia)

� Soft ionization method � only little fragmentations

� Produces positive ions

� Detects pseudo molecular ions � molecular weight information can be obtained

� Lower ion source temperature will produce more pseudo molecular ions. Becareful that lower ion source temperature can cause more contamination.

MMH+

C2H4

AnalytesProtonation Reaction

CH4

C2H5+

e-

High energy

electrons

Reagent gas

CH4

CH4+

CH5+CH3

+

CH2+

CH4

CH4

C2H3+

Primary

reagent ionsSecondary

reagent ions

C2H5+

CH4

+

(C) HKD 2013

BACKGROUND

Negative Chemical ionization (CI)

� Uses reagent gas (methane, isobutane or ammonia)

� Soft ionization method � only little fragmentations

� Produces negative ions

� Higher reagent gas pressure will produce more low energy electrons, and in turn,more molecular ions.

� Lowering the ion source temperature can produce more resonance capturereaction and increase molecular ions.

CH4

CH4+ CH3

+

H

CH4

Reagent gas Low energy electrons

CH4CH4

MX

M +

Associative resonancecapture

MX MXe-

e-High energy

electrons

(from the

filament)

X-

MX-

Dissociativeresonance capture

Analytes

(C) HKD 2013

BACKGROUND

MASS ANALYSER – MAGNETIC SECTOR

m/z = B2r2/2V(C) HKD 2013

BACKGROUND

� Classical mass spectra.

� Very high reproducibility.

� Best quantitative performance of all MS analyzers.

� High resolution.

� High sensitivity.

� 10,000 Mass Range.

� Requires Skilled Operator

� Usually larger and higher cost than other mass analyzers.

� Difficult to interface to ESI.

� Low resolution MS/MS without multiple analyzers.

MASS ANALYSER – MAGNETIC SECTOR

(C) HKD 2013

BACKGROUND

MASS ANALYSER – QUADRUPOLE

(C) HKD 2013

BACKGROUND


m1+

m2+

m3+

m1+

m1/z=kV1

m2/z=kV2

m3/z=KV3

m2+

m3+

1

1

m/z = k.V

m mass number

z charge

k constant

V :voltage applied to rods

We can select mass

+

-

+

-Two opposite rods will have a potential of

+(U+Vcos(ωt)) and the other two -(U+Vcos(ωt))

where U is DC voltage and Vcos(wt) represents a

radio frequency (RF) field of amplitude V and

frequency w. In general, for mass section U and V

are varied keeping the ratio U/V constant.

(C) HKD 2013

BACKGROUND


m/z = K.V/(r2ω2)(C) HKD 2013

BACKGROUND

� Easy to use ,simple construction, fast.

� Good reproducibility.

� Relatively small and low-cost systems.

� Able to separate ions at lower vacuum levels (10-2 to 10-3 Pa).

� Quadrupoles are now capable of routinely analyzing up to a m/q ratio

of 3000, which is useful in electrospary ionization of biomolecules.

� Low resolution(<4000).

� Slow scanning.

� Low accuracy (>100ppm).


(C) HKD 2013

BACKGROUND

MASS ANALYSER – ION TRAP

(C) HKD 2013

BACKGROUND

� Cannot perform SIM measurements for transmission type mass

spectrometry.

� Only a limited quantity of ions can be trapped, resulting in a narrower

dynamic range than quadrupole MS systems.

� All trapped ions are detected → higher sensitivity in scanning analysis

than quadrupole models.

� Enables trapping specific ions, then fragmenting them and detecting

the resulting fragment ions → a mass spectrometer specialized for

qualitative analysis.


(C) HKD 2013

BACKGROUND

� Poor quantitation.

� Less sensitive.

� Non-classical spectrum.

� Electrodes tend to be contaminated.

� Limited column flow rate.


(C) HKD 2013

BACKGROUND

MASS ANALYSER – Time-Of-Flight

m/z = 2eUt2/L2(C) HKD 2013

BACKGROUND


resolution = 2dt/t(C) HKD 2013

BACKGROUND

� Good for kinetic studies of fast reactions and for use with gas

chromatography to analyze peaks from chromatograph.

� High ion transmission.

� Can register molecular ions that decompose in the flight tube.

� Extremely high mass range (>1MDa).

� Fastest scanning.

� Requires pulsed ionization method or ion beam switching (duty cycle is

a factor).

� Low resolution (4000).

� Limited precursor-ion selectivity for most MS/MS experiments


(C) HKD 2013

BACKGROUND

Fourier Transform Ion Cyclotron Resonance (FT ICR) analyzers

(C) HKD 2013

BACKGROUND

Fourier Transform Ion Cyclotron Resonance (FT ICR) analyzersy

m/z (A) < m/z (B)

If the frequency of the applied field is the

same as the cyclotron frequency of the ions,

the ions absorb energy thus increasing their

velocity (and the orbital radius) but keeping a

constant cyclotron frequency. Ions having a

different cyclotron frequency are not

accelerated.

(C) HKD 2013

BACKGROUND


ω = qB/m = v/r

� The decay over time of the image current resulting after applying a

short radio-frequency sweep is transformed from the time domain into a

frequency domain signal by a Fourier transform(C) HKD 2013

BACKGROUND

� The highest recorded mass resolution of all mass .spectrometers

(>500,000).

� Very good accuracy (<1ppm).

� Well-suited for use with pulsed ionization methods such as MALDI.

� Non-destructive ion detection; ion remeasurement.

� Stable mass calibration in superconducting magnet FTICR systems.


(C) HKD 2013

BACKGROUND

� Expensive.

� Requires superconducting magnet.

� Subject to space charge effects and ion molecule reactions.

� Artifacts such as harmonics and sidebands are present in the mass

spectra.

� Many parameters (excitation, trapping, detection conditions) comprise

the experiment sequence that defines the quality of the mass

spectrum.

� Generally low-energy CID, spectrum depends on collision energy,

collision gas, and other parameters.


(C) HKD 2013

BACKGROUND

MASS ANALYSER COMPARISION

(C) HKD 2013

BACKGROUND

� Only cations are detected.� Radicals are “invisible” in MS.

� The amount of deflection observeddepends on the mass to charge ratio(m/z).� Most cations formed have a charge of

+1 so the amount of deflectionobserved is usually dependent on themass of the ion.

(C) HKD 2013

BACKGROUND

Mass, as m/z. Z is the charge, and for doubly charged ions (often seen in

macromolecules), masses show up at half their proper value

High

mass

[M+H]+(CI)

Or M•+ (EI)

“molecular ion”

Unit mass

spacing

Fragment IonsDerived from

molecular ion

or higher

weight

fragments

In CI, adduct ions,

[M+reagent gas]+

(C) HKD 2013

BACKGROUND

M + e- � M+ + 2e-Molecule High Energy

ElectronMolecular

Ion(Radical Cation)

1009080706050403020100In

tens

ity (%

of B

ase

Peak

)

20 30 40 50 60 70 80 90m / z

1-Pentanol - MW 88CH3(CH2)3 – CH2OH

CH2OH+M - (H2O and CH2=CH2)

M - (H2O and CH3)

M - H2O

M+ - 1Molecular Ion Peak

Base Peak


ElectronMolecular

Ion(Radical Cation)


ElectronMolecular

Ion(Radical Cation)

1009080706050403020100In

tens

ity (%

of B

ase

Peak

)

20 30 40 50 60 70 80 90m / z

1-Pentanol - MW 88CH3(CH2)3 – CH2OH

CH2OH+M - (H2O and CH2=CH2)

M - (H2O and CH3)

M - H2O

M+ - 1Molecular Ion Peak

Base Peak

(C) HKD 2013

BACKGROUND

� A partial MS of dopamine showing all peakswith intensity equal to or greater than0.5% of base peak.

(C) HKD 2013

BACKGROUND

� Most elements occur naturally as amixture of isotopes.� The presence of significant amounts of

heavier isotopes leads to small peaksthat have masses that are higher thanthe parent ion peak.

� M+1 = a peak that is one mass unithigher than M+

� M+2 = a peak that is two mass unitshigher than M+

(C) HKD 2013

BACKGROUND

� Bromine:� M+ ~ M+2 (50.5% 79Br/49.5% 81Br)

M+ ~ M+2

2-bromopropane

(C) HKD 2013

BACKGROUND

� Chlorine:� M+2 is ~ 1/3 as large as M+

M+2

M+Cl

(C) HKD 2013

BACKGROUND

� Sulfur:� M+2 larger than usual (4% of M+)

M+

Unusually large M+2

S

(C) HKD 2013

BACKGROUND

� Iodine� I+ at 127� Large gap Large gap M+

ICH2CN

(C) HKD 2013

FRAGMENTATION PATTERNS

1) Cleavage of f s bondThere are 3 type of fragmentations

---- C – C ---- ---- C + . C ----+

---- C – Z ---- ---- C + . Z ----+At heteroatom

+ .

+ .

� to heteroatom

---- C - C – Z ---- C=Z + ---- C .++ .

---- C - C – Z ---- Z + . ---- C = C ++ .

(C) HKD 2013


2) Cleavage of 2 s bond (rearrangements)There are 3 type of fragmentations

---- HC – C – Z ---- ---- C=C + HZ+

Retro Diels-alder

+ .

+ .

CH2

CH2

CH2

CH2+

+ . + .

McLafferty

ZH

Z R

CH2

CH2

ZH

Z R

+ .

3) Cleavage of Complex rearrangements(C) HKD 2013

1. Intensity of M.+ is Larger for linear chain than for branched compound

2. Intensity of M.+ decrease with Increasing M.W. (fatty acid is an exception)

3. Cleavage is favored at branching→ reflecting the Increased stability of the ion

Stability order: CH3+ < R-CH2

+ <


RR CH+ < C+

R

RR

RR”

CHR’

Loss of Largest Subst. Is most favored

RULES

(C) HKD 2013

(C) HKD 2013

CH3CH3

CH3CH3

CH3CH3

CH3

MW=170

M.+ is absent with heavy branchingFragmentation occur at branching: largest fragment loss

Branched alkanes

(C) HKD 2013

Illustration of first 3 rules(Linear alkane with Smaller MW)

Molecular ion is stronger than

in previous sample

(C) HKD 2013

Illustration of first 3 rules (Branched alkane with Smaller MW)

Molecular ion smaller than

linear alkane

Cleavage at branching is

favored

43

(C) HKD 2013

Rule 3 ALKANES

Cleavage Favored at branchingLoss of Largest substituentFavored

Rule1: intensity of M.+is smaller with branching

(C) HKD 2013


4. Aromatic Rings, Double bond, Cyclic structures stabilize M.+

5. Double bond favor Allylic Cleavage→ Resonance – Stabilized Cation

CH2+

CH CH2 R- R

.CH2

+CH CH2

CH2 CH CH2+

RULES

(C) HKD 2013

Aromatic ring has stable M.+

(C) HKD 2013


RULES

6. a) Saturated Rings lose a Alkyl Chain (case of branching)

b) Unsaturated Rings → Retro-Diels-Alder

CH2

CH2

CH2

CH2+

+ . + .

R+ . +

-R.

(C) HKD 2013

Retro Diels-alder+ . + .

(C) HKD 2013


RULES

7. Aromatic Compounds Cleave in β → Resonance Stabilized

Tropylium ion

C

CH+

R

-R.CH+

CH2 CH2+

+Tropylium ion

m/z 91(C) HKD 2013

+

+

(C) HKD 2013


RULES

8. C-C next to Heteroatom cleave leaving the charge on theHeteroatom

R CH2 CH2 Y Rx CH2 Y R

+

CH2+ Y R

x

R2

C

R1

O

C

R1

O+

C+

R1

O

- [RCH2]�

- [R2]�

larger

(C) HKD 2013


RULES

9. Cleavage of small neutral molecules (CO2, CO, olefins, H2O ….)results often from rearrangement.

x

CH2

CH2

H

CH2

O

C

Y

Y as H, R, OH, NR2

Ion Stabilized by resonance

x

CH2

CH2

H

CH2

O

C

Y

- CH2=CH2

x

CH2

O

C

Y

H

McLafferty

(C) HKD 2013


SUMMARY

� Alkanes� Fragmentation often splits off simple

alkyl groups:� Loss of methyl M+ - 15� Loss of ethyl M+ - 29� Loss of propyl M+ - 43� Loss of butyl M+ - 57

� Branched alkanes tend to fragmentforming the most stable carbocations.

(C) HKD 2013


SUMMARY

(C) HKD 2013


SUMMARY� Alkenes:

� Fragmentation typically forms resonance stabilized allylic carbocations

(C) HKD 2013

AlkenesMost intense peaks are often:

m/z 41, 55, 69

Rule 4: Double Bond Stabilize M�+

Rule 5: Double Bond favor : Doubl

Allylic

le Bond favole B

cc cleavage

CH2 CH CH�+

Et

EtMe

-Et�+CH2 CH CH

EtMe

CH2 CH CH +

EtMe

-29

M�+ = 112 m/z = 83

(C) HKD 2013


SUMMARY� Aromatics:

� Fragment at the benzylic carbon, forming a resonance stabilized benzylic carbocation (which rearranges to the tropylium ion)

M+

CH

H

CH Br

HC

H

H

or

(C) HKD 2013


SUMMARYAromatics may also have a peak at m/z = 77 for the benzene ring.

NO2

77M+ = 123

77

(C) HKD 2013


SUMMARY

� Alcohols� Fragment easily resulting in very small or

missing parent ion peak� May lose hydroxyl radical or water

M+ - 17 or M+ - 18� Commonly lose an alkyl group attached to

the carbinol carbon forming an oxoniumion.� 1o alcohol usually has prominent peak at

m/z = 31 corresponding to H2C=OH+

(C) HKD 2013


SUMMARY

M+M+-18

CH3CH2CH2OH

H2C OH

(C) HKD 2013


SUMMARY� Amines

� Odd M+ (assuming an odd number ofnitrogens are present).

� �-cleavage dominates forming animinium ion.

CH3CH2 CH2 N

H

CH2 CH2CH2CH3 CH3CH2CH2N CH2

H

m/z =72

iminium ion(C) HKD 2013

86

CH3CH2 CH2 N

H

CH2 CH2CH2CH3

72


SUMMARY

(C) HKD 2013


SUMMARY� Ethers

� �-cleavage forming oxonium ion

� Loss of alkyl group forming oxonium ion

� Loss of alkyl group forming acarbocation

(C) HKD 2013


SUMMARY

MS of diethylether (CH3CH2OCH2CH3)

H O CHCH3

CH3CH2O CH2H O CH2

(C) HKD 2013


SUMMARY

� Aldehydes (RCHO)� Fragmentation may form acylium ion

� Common fragments:

� M+ - 1 for

� M+ - 29 for

RC O

R (i.e. RCHO - CHO)

RC O

(C) HKD 2013


SUMMARY

M+ = 134C C C H

H

H

H

H

O

133

105

91

(C) HKD 2013


SUMMARY

� Ketones� Fragmentation leads to formation of

acylium ion:

� Loss of R forming

� Loss of R’ forming RC O

R'C O

(C) HKD 2013


SUMMARY

CH3CCH2CH2CH3

O

M+

CH3CH2CH2C O

CH3C O

(C) HKD 2013


SUMMARY

� Esters (RCO2R’)� Common fragmentation patterns include:

� Loss of OR’: peak at M+ - OR’

� Loss of R’: peak at M+ - R’

(C) HKD 2013


SUMMARY

M+ = 136

C

O

O CH3

105

77 105

77

(C) HKD 2013

RULES OF THIRTEEN

� The “Rule of Thirteen” can be used toidentify possible molecular formulas for anunknown hydrocarbon, CnHm.

� Step 1: n = M+/13 (integer only, useremainder in step 2)

� Step 2: m = n + remainder from step 1

(C) HKD 2013

RULES OF THIRTEEN

� Example: The formula for a hydrocarbonwith M+ =106 can be found:

� Step 1: n = 106/13 = 8 (R = 2)

� Step 2: m = 8 + 2 = 10

� Formula: C8H10

(C) HKD 2013

RULES OF THIRTEEN

� If a heteroatom is present,� Subtract the mass of each heteroatom

from the MW.� Calculate the formula for the

corresponding hydrocarbon.� Add the heteroatoms to the formula.

Example: A compound with a molecular ionpeak at m/z = 102 has a strong peak at1739 cm-1 in its IR spectrum. Determineits molecular formula.(C) HKD 2013

Mass Spectrometry

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