introduction to mass spectrometry introduction to mass spectrometry eddy esmans may 2004 3 th...

Introduction toMass Spectrometry

Introduction toMass Spectrometry

Eddy EsmansMay 2004

3th EU-Meeting on Cobalaminsand Mimics

Antwerp - Belgium

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NICI

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analyzers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

The Components of a Mass Spectrometer

Inlet system

Ion Source AnalyzerIon

Detector

ComputerMass Spectrum

m/z

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NIC

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analysers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

II. Ionization methods1. Electron impact

E

70 eV electron

M+. Fi+.

kr

Unimolecular type

kr = E – E0

E

N-1E = internal energy of e.g. M+.

E0 = activation energy of a particular fragmentation

N = degrees of freedom

= frequency factor

IONIZATION EFFICIENCY : ca. 1/1000

QET : Quasi Equilibrium Theory

a. A polyatomic molecule does not fragment immediately but during ionization period of 10-16 sec it undergoes a few vibrations.

fragmentation is a “relative slow” process.

b. The energy transferred to M is not localised but is statistically spread over the molecule.

c. If the event occurs than this energy is concentrated at one particular bond. This bond will break here.

d. The probability of breaking a particular bond in not a function of abundance.

e. Metastable ions are formed : ions with a life time of > 10-6 seconds.

AB0

1

2

3

3

3

AB+.

”0

”1

”2

”3

”4

”5Interconversion

’5

AB+.’0

’1

’2

’3

’4

-Ip (theoretical)

E-impact-ionisation occurs according to the Frank-Condon-principle(vibration is 100 times slower than ionisation)

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NICI

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analysers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

M(g) + reagent gas [MH]+

benefit: producing molecular mass information

proton affinity !!!

proton affinity PA of M > proton affinity PA of the reacting species

Classical reagent gasses:Methane: CH5

+

NH3: NH4+ (NH3

+. + NH3 NH4+ + NH2

.)Isobutane: C4H9

+

PS : if PA(M) PA(reagent gas) [MH]+ + ADDUCT FORMATION

[M + NH4]+

[M + C2H5]+

if PA(M) < PA(reagent gas) only adducts bad sensitivity

2. Chemical ionization (CI) and DCI, NICI

Desorption chemical ionization

Negative Ion Chemical Ionization

Principle : ion souce is filled with CH4 and 70 eV electrons are slowed down to thermal energy.

These electrons can be “captured” by molecules containing sulphur (cfr. Electron capture GC)

formation of M°--ions

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NICI

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analysers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

Fast atom bombardment (FAB) andSecundary Ion Mass Spectrometry (SIMS)

Principle: Ions (Cs+)Neutrals (Ar, Xe, …)

IONS analysedSample

1. FAB : Ar + e Ar+ acceleration (5-15 KeV)

Ar+ + Ar Ar + Ar+

fast slow slow+ 8 KeVfast

2. SIMS : Cs+ generated (35 KeV)

3. LSIMS : Sputtering yield (number of particles ejected/incident particle)

Dependent on mass and velocity of impinging particle

Matrix properties

1. Good solubility

2. Vapour pressure must be sufficiently low to maintain vacuum conditions

3. Viscosity must allow diffusion of the analyte from the bulk to the surface

4. Polar : to solvate and separate preformed ion

glycerol, 3-nitrobenzylalcohol, mixture of1,4-dithiothreitol/1,4-dithioerythitol 5:1 (magic bullet)

Laser Desorption & Matrix Assisted Laser Desorption

A few lasers:

N2 –laser : 337 nmNd-Yag laser : 354 & 266 nmE: 20mJ/cm2

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NICI

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analysers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

Field desorption

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NICI

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analysers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

++

+++

++

++

++

+++ + +

--

-

--

----

-

-

---

-

+

++

+

++

+

++

++

++++

++ ++

++++

++

+++

+

+++++

+

++++

+++

++

+++

++

++

+

Picofrit columns™Picofrit columns™

- injection: 1 l- flow-rate: 500 nl/min

- isocratic 20/80 NH4Ac (0.01 M) / MeOH- column: AQUASIL C18, 75 m x 4.9 cm (15cm 2cm), tip 5 m

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NICI

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analyzers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

I. Ion source : Ions get kinetic energy

V 8 KV

zVmv 2

2

1m

zVv

22

V = tensionm = massv = speedz = charge

II. Electrostatic sector :

E = electrostatic field =

a

V

zEr

mv

2

d

V

zE

mvr

2

2

2

12mvx

zEr

KinEzE

r .2

Ions with the same Ekin will travel with the same r and leave the electrostatic sector at the same point(This is independant of their mass !!!)

III. Magnetic sector :

Hzvr

mv

2

H

mvr

z

m

H

vr .

22

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NIC

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analyzers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

a. Quadrupole filter

Quadrupole field: E = E0 (x + y + z)Independent field in x,y,z-directions.

Ions entering this field will undergo a force

F = eE

Quadrupole field subjected to the restraites imposed by theLaplace-equations:

Physical meaning : the Laplacean is a measure for the distorsion of the E-field

0. E

0

Ezyx

or

zE

yE

xEbecause zyx

...xzyx

0...2

2

2

2

yx

0.2

zyx EEEEthen0E.if )zyx(EEand 0 0

)2or( 0

zE

yE

xEbecause zyx

02

z02

y02

x

0z0y0x

zzyyxx

Ez2

1Ey

2

1Ex

2

1zzEyyExxE

zEyExE

0if 20

20 yE

2

1xE

2

1

220 yxE

2

1

2

0

220

r2

yx

Hyperbolean !!!

Applied potential1. Equation of motion of the ions entering this field

mx = eEx¨

2

0

220

xr2

yx

xxE

20

0

r2

x2

20

0

r2

x2e

mx mx 0.

r

e02

0

x 0.mr

e02

0

y 0.mr

e02

0

xz and yz motionof ions in plane

mz = 0¨ ctet

z

Velocity in z-direction is cte but ions are accelerated in x and y-directions !

-

Stability diagram

0 = U + V.cost ( = 2f)

x

y

Matthieu-equations

U = 500-2000 VV = 0-3000 V

0)tcosVU.(mr

e2

0

0)tcosVU.(mr

e2

0

Stability diagram

20

2rm

eU4a

20

2rm

eV2q

!resolutionV

U2

q

a

scanning : changing U and V

cteV

Ubut

keeping

what if U = 0 resolution = 0

Rf-quadrupole only will be able to pass m/z-values > certain m/z-value as long as V is

in stability area.

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NIC

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analyzers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

Ion cyclotron resonance Fourier Transform MS

Ion can be trapped in a H-field

circular motion with frequency

Relation between and m/z-value

each m/z-value will move with its typical frequency/radius

zvHF:forcelcentripeta

r

mv'F:forcelcentrifuga

2

The ion will have a stable trajectory when :

r

mvzvH

2

r

mvzH

r2

v

H.m

z

r

v

r2

v22

Simultaneously excite all ions by electromagnetic pulse (1µs). Depending on their m/z-values ions will absorb energy at their frequency and subsequently get hifgher trajectories close to the receive plates. All the frequencies detected in this time ellaps by the receive plates at the same time.

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NIC

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analyzers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

Time of Flight

Source : ion

Resolution > 10.000Mass range 50.000

zVmv2

1E 2

kin flight tube : L

v

dt

V2

d.

z

mt

22

Time of Flight

Reflectron : corrects for energy dispension

Time an ion spends in reflectron

2 ions with mass M

correct energy E

energy E’

)definitionby(aE

'E 2

2/122

m

E2vmv

2

1E

2/122

m

'E2'v'mv

2

1'E

2/122

m

Ea2'v

av'v

av

d

'v

d't

v

dt

a

t't

t, t’ = flight time in the field free region of TOF

Ions come in the reflectron : penetrate a distance x or x’

a>1 E’kin>Ekin t’flight<tflight but x’ > x

x’ = a2x

Conclusion :

a<1 E’kin< Ekin t’flight>tflight but x’ < x

zE

Ea

zE

'E'x

zE

Ex kin

2kinkin

Tandem Mass Spectrometry

MP MF

2FMFkin

2PMPkin v.m

2

1Ev.m

2

1E

F

P

kinMF

kinMP

m

m

E

E

penetration depth:zE

Ex kin

zE

Ex

zE

Ex kinMF

FkinMP

P

P

FPF M

Mxx

Time in reflectron to penetrate a distance n

vi v0

x

2

vvv 0i

v

xtn

in v

x2t

Total time in reflectron to cover a distance of 2x

iv

x4t

i

FMFreflectron

i

PMPreflectron v

x4t

v

x4t

i

P

FP

MFreflectron v

mm

.x4t

?vv

xtn

I. Introduction

II. Ionization methods

1. Electron impact

2. Chemical ionization and DCI, NIC

3. FAB, SIMS, LD and MALDI

4. Field desorption

5. Electrospray ionization

III. Analyzers1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods