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3 th EU-Meeting on Cobalamins and Mimics Antwerp - Belgium. Introduction to Mass Spectrometry. Eddy Esmans May 2004. I. Introduction. II. Ionization methods. 1. Electron impact. 2. Chemical ionization and DCI, NICI. 3. FAB, SIMS, LD and MALDI. 4. Field desorption.

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slide1

3th EU-Meeting on Cobalamins

and Mimics

Antwerp - Belgium

Introduction to

Mass Spectrometry

Eddy Esmans

May 2004

slide2

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. Analyzers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide3

Ion

Detector

Ion Source

Analyzer

Inlet system

The Components of a Mass Spectrometer

m/z

Mass Spectrum

Computer

slide4

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. Analysers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide5

E – E0

E

II. Ionization methods

1. Electron impact

kr

E

M+.

Fi+.

70 eV electron

Unimolecular type

E = internal energy of e.g. M+.

N-1

E0 = activation energy of a particular fragmentation

kr = n

N = degrees of freedom

n = frequency factor

IONIZATION EFFICIENCY : ca. 1/1000

slide6

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.

QET : Quasi Equilibrium Theory

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.

slide7

Interconversion

n”5

n”4

n”3

n”2

n’5

n”1

n’4

n”0

n’3

n’2

AB+.

n’1

n’0

AB+.

-Ip (theoretical)

n3

n3

n3

n2

n1

n0

AB

E-impact-ionisation occurs according to the Frank-Condon-principle

(vibration is 100 times slower than ionisation)

slide8

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. Analysers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide9

2. Chemical ionization (CI) and DCI, NICI

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

slide11

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

slide12

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. Analysers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide13

Fast atom bombardment (FAB) and

Secundary Ion Mass Spectrometry (SIMS)

Ions (Cs+)

Neutrals (Ar, Xe, …)

Principle:

IONS analysed

Sample

1. FAB :

Ar + e Ar+ acceleration (5-15 KeV)

Ar+ + Ar Ar + Ar+

fast

slow

+ 8 KeV

slow

fast

2. SIMS :

Cs+ generated (35 KeV)

3. LSIMS :

Sputtering yield (number of particles ejected/incident particle)

Dependent on mass and velocity of impinging particle

slide14

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 of

1,4-dithiothreitol/1,4-dithioerythitol 5:1 (magic bullet)

slide16

Laser Desorption & Matrix Assisted Laser Desorption

A few lasers:

N2 –laser : 337 nm

Nd-Yag laser : 354 & 266 nm

E: 20mJ/cm2

slide19

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. Analysers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide21

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. Analysers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide25

Picofrit columns™

  • injection: 1 ml
  • flow-rate: 500 nl/min
  • isocratic 20/80 NH4Ac (0.01 M) / MeOH
  • column: AQUASIL C18, 75 mm x 4.9 cm (15cm 2cm), tip 5 mm
slide26

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. Analyzers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide29

I. Ion source :

Ions get kinetic energy

V  8 KV

V = tension

m = mass

v = speed

z = charge

slide30

II. Electrostatic sector :

E = electrostatic field =

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 !!!)

slide32

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. Analyzers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide33

a. Quadrupole filter

E = E0 (x + y + z)

Quadrupole field:

Independent field in x,y,z-directions.

Ionsentering this field will undergo a force

F = eE

slide34

Quadrupole field subjected to the restraites imposed by the

Laplace-equations:

Physical meaning : the Laplacean is a measure for the distorsion

of the E-field

slide36

Applied potential

1. Equation of motion of the ions entering this field

mx = eEx

¨

-

mx

¨

mx

¨

¨

x

xz and yz motion

of ions in plane

¨

y

mz = 0

¨

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

slide37

Stability diagram

0 = U + V.cost ( = 2f)

¨

x

¨

y

Matthieu-equations

U = 500-2000 V

V = 0-3000 V

slide38

Stability diagram

scanning : changing U and V

but 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.

slide41

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. Analyzers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide42

Ion cyclotron resonance Fourier Transform MS

Ion can be trapped in a H-field

The ion will have a stable trajectory when :

circular motion with frequency

Relation between  and m/z-value

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

slide45

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.

slide46

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. Analyzers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods

slide47

Resolution > 10.000

Mass range 50.000

Time of Flight

Source : ion

flight tube : L

slide48

Time of Flight

Reflectron : corrects for energy dispension

slide49

Time an ion spends in reflectron

correct energy E

2 ions with mass M

energy E’

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

slide50

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

x’ = a2x

Conclusion :

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

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

slide53

Tandem Mass Spectrometry

MP MF

penetration depth:

slide54

x

vi

v0

Time in reflectron to penetrate a distance n

Total time in reflectron to cover a distance of 2x

slide60

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. Analyzers

1. Magnetic sector

2. Quadrupole – ion trap

3. Fourier transform

4. Time of flight

IV. MS/MS-methods