Introduction to Mass Spectrometry Introduction to Mass Spectrometry Eddy Esmans May 2004 3 th...
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Transcript of 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