The way to ionize a
compound
and
MS Configurations
The way to ionize a
compound
and
MS Configurations
Mass Spectrometer
Schematic
Mass Spectrometer Schematic
Atmospheric Pressure
Ionization
•
ESI (Electrospray ionization)
•
APCI (Chemical ionization)
•
APPI (Photo ionization)
Positive / Negative Modes
2.5
5.0
min
0.0e6
5.0e6
10.0e6
Int.
2:TIC(1.00)
1:TIC(1.00)
m/z280~600, +/-; Scan speed: 1000
amu/s
S/N = 124
2.5
5.0
min
0.0e6
5.0e6
10.0e6
Int.
TIC(1.00)
m/z280~600, + only/ Scan speed: 500
amu/s
S/N = 136
22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5
min
500e3
1000e3
1500e3
2000e3
2500e3
3000e3
3500e3
4000e3
4500e3
5000e3
5500e3
6000e3
6500e3
Int.
2:TIC(1.00)
1:TIC(1.00)
-
negative scan
-
positive scan
P1
Peak
P1
could not be detected in positive ion mode
MS
analyze
r
vacuum
HPLC
N
2
gas
N
2
nebulizer
3-5 kV
Electrospray Ionization
(ESI)
Electrospray (ESI)
•
The mechanism for ion generation in electrospray mass
spectrometry was first announced by the work of Fenn and
Dole, although the original publication on electrospray
dates back to 1917 from the work by Zeleny.
•
The principal outcome of the electrospray process is the
transfer of analyte species, generally ionized in the
condensed phase, into the gas phase as isolated entities.
Nanospray
Online analysis
~ 20 µm tip ID
Interface with nanoLC
Flow rate: ~300nL/min
Offline analysis (static infusion)
~ 2 µm tip ID
Flow rate: ~40nL/min
Requires pure sample free from salt
New Objective, Inc.
⊿
5
⊿
16
⊿
17
⊿
18
⊿
14
⊿
９
(M+H)
+
MH
+0
(M+NH
4
)
+
MH
+17
(M+Na)
+
MH
+22
(M+K)
+
MH
+38
(M+H+H
2
O
)
+
MH
+18
(M+H+MeOH
)
+
MH
+32
(M+H+CH
3
CN
)
+
MH
+ ９９
Molecular Ions Often Observed
in ESI-MS
NH
O
HN
R
2
H
N
R
1
H
3
C
O
N
NH
OCH
3
CH
3
CH
3
O
CH
3
CH
2
COOH
CH
3
COOH
H
3
C
O
O
Protonated molecule
(M+H)
+
Molecule with added sodium ion (M+Na)
+
COOH → COONa
1x COONa (M-H+Na+H)
+
= (M+Na)
+
2x COONa (M-H+Na-H+Na+H)
+
= (M-H+2Na)
+
•
multiply charged ions can be generated
with
ESI interface if the chemistry fits
•
hardware mass range (- 2000 m/z) is
mathematically extended up to 100.000
amu
•
acquisition has to be in profile mode
•
following deconvolution
Multiply charged ions
Electrospray Mass Spectrum
of Bovine Ubiquitin
700
800
900
1000
1100
m/z
779.44
+11
856.9681
857.47
+10
952.63
+9
714.72
+12
659.75
+13
857
858
+0.1
+0.2
+0.3
+0.4
+0.5
+0.6
+0.7
+0.9
+0.8
+1.0
M
theo
=8559.611
2
M
exp
=8559.603
Z=+10
858.5
857.5
Charge State Determination
High Resolution
– isotope peaks resolved
(1) counting isotope peaks in ONE m/z unit
(2) if the measured spacing of neighboring isotopes is (m/z),
z=1/ (m/z) or more accurately z=1.00235/(m/z)
1.00235 is the average isotope spacing
Low Resolution
- isotope peaks are not resolved
Use neighboring charge states (m/z)
1
[higher charge] and (m/z)
2
[lower charge, higher m/z]
Solve the following linear equations
for z (for (m/z)
1
) and M
(neutral mass)
(m/z)
1
Xz – z =M
(m/z)
2
X(z-1) – (z-1) =M
Deconvolution
750
1000
1250
1500
1750
m/z
0e3
100e3
200e3
300e3
400e3
500e3
600e3
Int.
1131
1060
1212
998
1305
943
1414
893
1542
849
1696
1136
1418
1064
ESI mass spectrum of myoglobin
16800
16900
17000
Mas s
0.0e6
1.0e6
2.0e6
3.0e6
4.0e6
Int.
16953.2
+10
+12
+14
+20
Multiply charged ion
deconvolution
Advantages of ESI
•
High sensitivity to polar compounds
•
Produces multiply charged ions
•
Concentration dependent technique
•
Well suited to reverse phase solvents
Critical factors in ESI
•
Solvation plays an important role on ionization
efficiency. Surface tension effects will affect
sensitivity [phosphates].
•
Ion suppression must be carefully considered;
levels of trifluroacetic acid, sample matrix
•
Adduct ion generation must also be accounted
for in interpretation and quantitation.
MS
analyze
r
vacuum
HPLC
N
2
gas
N
2
nebulizer
Corona discharge needle 3-5 kV
200-500°C
Atmospheric pressure
chemical ionisation (APCI)
Analyte containing
aerosol
Charged
reagent gas
formed
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
+
+
+
+
+
Vapor
Heat
+
Analyte ions
Charge
transfer to
analyte
+ + +
+
APCI Process
APCI Ionisation
•
The sample needs to be thermally stable to
a certain extent
•
A protonic solvent is necessary to generate
reactant ions
•
Under normal reverse phase column
conditions, methanol/water achieves higher
ionization efficiency than acetonitrile/water.
•
Typical APCI applications are:
pesticides, drugs, azo dyes, and
steroids
MS
analyze
r
vacuum
HPLC
N
2
gas
N
2
nebulizer
200-500°C
Atmospheric pressure
photo ionisation (APPI)
Lampe
UV
hn
APPI Ionisation
•
Allows high sensitivity analysis of compounds
that have low polarity
•
Allows high sensitivity analysis of Fusarium
toxins
•
Allows not only APPI, but also a simultaneous
APCI/APPI dual ionization mode
Theory of APPI (direct
APPI)
Molecular Weight
Non-Polar
100,000
Direct APPI
UV
(hν=10 or 10.6eV)
M+
+H
Protic solvent
Analyte
Analyte molecule M is ionized to a molecular ion M
+
.(If
analyte ionization potential is below photon energy.) In
the presence of protic solvent, M
+
may extract a
hydrogen atom to form MH
+
.
M+H
+
Theory of APPI (dopant
APPI)
Molecular Weight
Non-Polar
100,000
Dopant APPI
UV
(hν=10 or 10.6eV)
M+H
+
Analyte
Solvent
Dopant
(e.g. Toluene)
M+
+
e-
+H
M+
A photoionizable dopant is delivered in large concentration to
yield many D
+
ions. D
+
ionizes analyte M by proton or electron
transfer.
M+
APCI/APPI and ESI
APCI/APPI
ESI
mass
- ca. 1000-1500
- up to 100.000
flow
50 µl - 2 mL/min
1µL - 1mL/min
analyt
unpolar
polar
solvent
unpolare solvent possible
buffer
high tolerance
N
2
< 2.5 L/min
< 1.5 L/min
Probe
ESI
APCI
APPI
Ionization
Ion
evaporation
Chemical
ionization
Photoionization
Sample
Stability
Many sample
(Soft
ionization)
Not good for heat
unstable
compounds
Not good for heat
unstable
compounds
Sample
Polarity
Polar
compounds
Middle polar
compounds
Less polar
compounds
Typical Mass Spectra of ESI-
MS
50
100
150
200
250
300
350
400
m/z
0e3
10e3
20e3
30e3
40e3
Int.
377
399
Riboflavin C
17
H
20
N
4
O
6
Exact Mass: 376.14
50
75
100
125
150
175
200
225
250
m/z
0
250
500
750
Int.
242
213
170
156
43
256
60
185
103
77
116
198
91
130
143
(a) EI spectrum of riboflavin (vitamine
B2)
N
N
NH
N
O
O
OH
OH
HO
OH
[M+H]
+
[M+Na]
+
(b) API (ESI) spectrum of
riboflavin
Dual source (ESI/APCI)
Dual ion source
heated drying gas
Compounds Analyzed by
LC/MS
Properties of compounds: a certain polarity is
essential
With and without UV-vis absorption
Thermally stable and labile
Less and non-volatile
Less polar to ionic compound
Small to large molecules
Types of compounds:
Synthetic drugs, metabolites
Natural compounds (alkaloids, glycosides,
taxanes,
toxins, saccharides, vitamins, lipids etc)
Peptides, proteins
Non-volatile pesticides, herbicides etc
Surfactants, dyes and various organic
additives
O
HO
O
O
OH
N
O
O
O
O
O
OH
MALDI
Matrix-Assisted Laser
Desorption/Ionization
N
O
H
O
OH
4-hydroxy-picolinic acid
m = 139.05 Da
NH
2
OH
O
anthracinic acid
m = 137.05 Da
negative mode
OH
O
OH
N
-cyano-4-hydroxy-
cinnamic acid (CHCA)
m = 189.07 Da
O
H
OH
OH
O
2,5-dihydroxy-
benzoic acid (DHB)
m = 154.03 Da
positive mode
OH
O
O
H
O
O
sinapic acid (SIA)
m = 224.07 Da
small, aromatic,
organic acids:
UV-absorbing,
proton donor
Common matrices for MALDI-TOF
MS
MALDI
•Analyte is dissolved in solution with excess matrix (>10
4
).
•Sample/matrix mixture is dried on a target and placed in the MS
vacuum.
Requirements for a satisfactory matrix:
•It must co-crystallize with typical analyte molecules
•It must absorb radiation at the wavelength of the laser
(usually 337 nm)
•To transfer protons to the analyte it should be acidic
Typical successful matrices for UV MALDI are aromatic carboxylic
acids.
MALDI-TOF MS: basic principles
io n d e t e c to r
analyte molecules
incorporated in
matrix crystals
in vacuum
< 10
-7
Torr
io n d e t e c to r
+
+
laser pulse:
desorption of matrix and
analyte molecules,
ionization by charge
transfer
MALDI-TOF MS: basic principles
io n d e t e c to r
+
+
+
+
build-up of an
electromagnetic
field
acceleration of ions
MALDI-TOF MS: basic principles
io n d e t e c to r
+
+
+
+
separation of ions in a
field free drift range of
a fixed length by
velocity
(Time Of Flight)
L 1 m
no further
acceleration!
MALDI-TOF MS: basic principles
io n d e te c to r
+
+
+
+
mass-independent detection
of ions at a detector
time resolved output to
oscilloscope and computer
MALDI-TOF MS: basic principles
38
MALDI-TOF MS:
Matrix Assisted Laser Desorption/Ionization
Time-of-Flight Mass Spectrometry
ion detector
+
+
+
+
+
+
template
matrix/analyte
crystals
acceleration
zone
field-free
drift range
grid electrode
desorption
ionization
acceleration
separation
detection
m
z =
2eU
L²
t²
m: mass
z: charge
U: accelaration voltage
L: path length
t: time
e: elemetary charge
Ionization Methods for
Biomolecule Analysis
MALDI
•Very long sample lifetime;
repeated measurements
possible
•Good for mixtures
•Matrix peaks can interfere at
MW <600
•Salt tolerant
•Low maintenance
•Generate ions with few
charges
Electrospray
•Online LC/MS
possible
•Poor for mixtures
without LC
•Quantitation
possible
•Good for MW
<600
•Generate highly
charged ions
polarity
Non-Polar
Very polar
Molecul
ar
Weight
10,000
1,000
100
ESI
APCI
APPI
100,000
Mass spectrometry
MALDI
GCMS
LCMS
MALDI TOF MS
EI/ CI
MS configurations
Different types of MS
Aebersold and Mann (2003) Nature 422, 198-207
Single quadrupole MS
analyzer
Sourc
e
Detector
Nonresonant
Ion
Resonant
Ion
DC and RF
voltages
Quadrupole Mass Analyzer
Just RF applied Ion Transmission
Device
DC and RF applied Mass Analyzer
Ion Optics
A device for manipulating ion beams.
A mass spectrometer consists of many ion optical
components
Uses a combination of RF and DC voltages to operate as a mass
filter.
•
Mass analyzer.
•
Mass selection device
•
Ion transport device (RF-only collision cell).
Quadrupole Mass
Analyzer/Filter
+ U + V cos t
-U - V cos t
Mass scan and stability diagram
Working mass range: 2 to 2000 m/z
Resolution: ~ 2*m (a mass of 200 can be
analyzed with a resolution of 400 FWHM)
Linearity for quantitation: dynamic range
~ 10
5
Mass filter
• Figure A) A light ion will be dragged a large distance by the alternating field, and will find
itself in stronger and stronger regions of field. It will quickly collide with an electrode and
disappear
• Figure B) A very heavy ion will not be affected much by the alternating field, but will
gradually drift in the constant part of the field (the DC part). The alternating field is not strong
enough to drag it back as it wanders, so it also collides with an electrode, and is lost.
• Figure C) An ion that is the right weight drifts slightly in the constant part of the field, but is
always dragged back by the alternating part. The alternating part, however, is not quite strong
enough to make it spiral out of control into an electrode. Thus an ion just the right size is
stable in this quadrupole field and reaches the end, where it can be measured.
Quadrupoles theory and operation
•
By gradually increasing U and V during an experiment (i.e.
during a scan), ions with different m/z ratios can be made to
make contact with the rods or can be made to possess
stable trajectories through the quadrupole assembly
•
Only ions possessing stable trajectories through the entire
quadrupole assembly will pass through the quadrupole and
be detected
•
Generally, ions of different m/z ratios are sequentially
transmitted through the quadrupole during a scan to obtain
a mass spectrum
Quadrupoles - modes of operation
There are 2 main modes of quadrupole operation
:
1. Scanning
- the potentials applied to the quadrupole rods are
gradually increased such that ions between a
user defined m/z range sequentially possess
stable trajectories and are detected
2. Selected ion monitoring (SIM)
- The potentials applied to the quadrupole rods are
such that only ions of a specific m/z or a very
narrow range possess stable trajectories
SQ-mass analyzer
Quadrupoles - modes of operation
SIM offers increased sensitivity compared with scanning
experiments as a result of the increased (100%) ‘duty
cycle’ of a SIM experiment and reduced noise
•
in a scanning experiment, the quadrupole is
scanned across a m/z range and therefore ions of a
particular m/z only have stable trajectories for a
fraction of the total scan time
•
in SIM mode, ions of the specified m/z have stable
trajectories 100% of the scan time and therefore
more ions are detected, and hence sensitivity is
increased
Coelution
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
mi n
0.001
0.001
0.002
0.002
0.003
0.003
0.004
0.004
0.004
0.005
0.005
0.006
0.006
0.007
0.007
0.008
0.009
0.009
0.009
0.010
0.011
0.011
0.012
0.012
0.013
mAU( x1,000)
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
55.0
60.0
65.0
70.0
75.0
80.0
85.0
90.0
95.0
bar
B .Pre ss .(S ta t us)
A.Pre ss.( S t a tus)
Extr ac t -230nm,4nm ( 1.00)
Flavonoids - PDA chromatogram @230nm
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
(x1,000,000)
609.00 (1.13)
579.05 (1.11)
595.00 (1.00)
TIC
1
2
3
4
5
MS chromatogram
TIC
S
I
M
P2
MS Spectra Peak 2 - Naringin
100
150
200
250
300
350
400
450
500
550
600
650
700
750
m/z
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
Inte n.
( x1,000,000)
579.05
615.00
497.20
661.70
270.90
370.80
173.95
676.85
748.85
461.85
795.20
429.85
292.30 316.75
714.90
119.00
406.60
221.90
556.65
Naringin (MW=578)
Difficult Matrix Background
of Plasma Simplified with MS
Detection
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0.0
2.5
5.0
7.5
10.0
(x100,000)
VER:m/z 455.0 (×2)
PRO:m/z 331.9 (×6)
ALB:m/z 266.0 (×60)
PIR:m/z 260.0 (×4)
ANT:m/z 189.0 (×6)
A
LB
V
E
R
P
R
O
P
IR
A
N
T
MS chromatogram
min.
Int.
0.0
1.0
2.0
3.0
4.0
5.0
min.
-2.0
-1.0
0.0
1.0
2.0
3.0
4.0
mAU
(x10)
254nm,4nm (1.00)
6.0
Int.
PDA chromatogram
A
LB
V
E
R
P
R
O
P
IR
A
N
T
ANT:Antipyrine
PIR:Piroxicam
ALB:Albedazole
PRO:Propraolol
VER:Verapamil
High throughput
• Shorten the run time
without
sacrificing the
chromatografic
resolution
• Higher throughput
• Less solvent
UV chromatogram
MS chromatogram
0
5
10
15
20 min
0
5
10
15 min
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
mi
n
1
2
3
4
5
6
Column
: STR ODS-Ⅱ ( 2.0 × 150mm )
Mobile phase : 0.1% formic acid / methanol, gradient
elution
Flow rate : 0.2 mL / min
Ionization mode : ESI (+)
N
+
R
1
R
2
R
3
R
4
N
+
CH
3
O
H
O
CH
3
CH
3
H
O
CH
3
O
H
CH
3
M
W
R
1
R
2
R
3
R
4
M
agnoflorine M
W
341
Berberine
335
-O
-C
H
2
-O
-
O
CH
3
O
CH
3
E
piberberine
335
O
C
H
3
O
CH
3
-O
-CH
2
-O
-
C
optisine
319
-O
-C
H
2
-O
-
-O
-CH
2
-O
-
Jateorrhizine
337
O
H
O
CH
3
O
CH
3
O
CH
3
Palm
atine
351
O
C
H
3
O
CH
3
O
CH
3
O
CH
3
Qualitative Analysis of
Alkaloids
in Coptis Rhizome
Total Ion Chromatogram
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
min
Intensity
TIC
TIC
336.00
352.00
338
.00
.00
320.00
342.00
1: Magnoflorine
2:
Coptisine
3:
Epiberberine
4:
Jateorrhizine
5: Berberine
6: Palmatine
1
3
4
2
5
6
Mass Chromatograms
100
200
300
400
m/z
0e3
100e3
Int.
342.2
297.1
138.9 201.4
371.4413.6 479.6
Magnoflorine
100
200
300
400
m/z
0e3
50e3
Int.
352.2
139.1 196.3
393.4
335.6
279.6
Palmatine
100
200
300
400
m/z
0e3
100e3
200e3
Int.
338.1
323.4
196.1
465.4
117.1
283.1
Jateorrhizine
100
200
300
400
m/z
0e3
250e3
500e3
750e3
Int.
320.2
447.4
336.1
214.1
158.1
391.3
Coptisine
100
200
300
400
m/z
0e3
250e3
Int.
336.2
322.2
158.2 214.1
464.6
413.5
Epiberberine
100
200
300
400
m/z
0e3
25e3
Int.
336.2
452.2
177.0
371.4
324.6
261.0
416.9
Berberine
Mass Spectrum
Triple quad. MS analyzer
Triple Quadrupole MS
CID cell
Collision Induced Dissociation
Functions of the rod assemblies in
different scan modes
Note:
Scan
a
:
Full scan or transmission of selected ions
Pass all ions
b
or fragments:
Pass ions or fragments within a wide range of mass-
to-charge ratios
Fragment ions
c
:
Collisions with argon gas cause ions to fragment
Set
d
:
Set to pass ions of a single mass-to-charge ratio or set of mass-to-charge ratios
Product Ion Scan
Precursor Ion Scan
Neutral loss scan
SIM and SRM
MS1
Set
MS2
Set
CID
Collision Cell
RF only + Ar
M3
+
M5
+
M1
+
M2
+
M3
+
M4
+
M5
+
M1
+
M2
+
M3
+
M4
+
M3
+
m3
+
m2
+
m2
+
m1
+
SRM – Selected Reaction Monitoring
SIM – Selected Ion Monitoring
Feature of SRM
Better signal to noise ratio than SIM. However, the
absolute sensitivity of sample is poorer than SIM.
Can increase confidence by using MS/MS
fragmentation information.
Time of Flight MS analyzer
detector
reflectron
TOF
defined starting point
Reflector Time of Flight (TOF)
MS separation
+
+
+
+
+
io
n d
ete
cto
r
Linear
time-of-flight mass spectrometer
Reflector
time-of-flight mass spectrometer
m
1
= m
2
v
1
< v
2
l
mass range up to 350 kDa
l
high sensitivity
l
low resolution
l
mass range up to 5000
Da
l
low sensitivity
l
high resolution
Linear and Reflector modes
Reflector compensates for initial variation in kinetic energy,
improving resolving power and mass accuracy.
ESI-TOF
Agilent ESI MSD TOF
Bruker MicrO-TOF
ESI-TOF schematic
detector
reflectron
TOF
Pusher
electrode
Ion stream
ESI-TOF
Positive features :
•
High mass range
•
High resolution: Possible to obtain more than 10.000 FWHM resolution
•
High scan speed
•
High mass accuracy
Limitations :
•
No MS/MS
•
no structural information
•
limited suitable for quantitation: The dynamic range is limited due
to
pulsed mode of operation and small dynamic range of detector
electronics.
•
As m/z value becomes large, it becomes difficult to discriminate
between times of arrival at the detector.
Ion trap MS analyzer
Quadrupole Ion Trap
•Uses a combination of
DC and RF fields to
trap ions
•Ions are sequentially
ejected by scanning
the RF voltage
Linear Trap
• Essentially a quadrupole with end-caps
• Advantage: Larger ion storage capacity, leading to better dynamic range
Ions in
(from ESI)
3D Trap
End caps
Ions out
to detector
Ring electrode
(~V)
Insulated
spacer
He gas
1x10
-3
Torr
Trap filling
Ion traps
Ion trap
By ramping the RF voltage, or by applying supplementary
voltages on the end cap electrodes,
or by combination of both, it is possible to:
•
destabilize the ions and eject them progressively from the
trap (mass analysis)
•
keep only one ion in the trap, fragment it by inducing
vibrations, and observe the fragments (MS/MS
experiment)
•
repeat the last operation a few times to progressively
fragment the ions (MSn experiment)
Quadrupole Ion Traps
End cap electrodes
Ring electrode
Benefits
• High sensitivity
• Multi-stage mass spectrometry (MS
n
analogous to FTICR experiments)
• Compact mass analyzer
1. Low ion mass cut of
2. space-charge efects: too many ions in the trap distort
the electric fields, leading to significantly impaired
performance.
Quadrupole Ion Traps
Multistage fragmentation
MS
4
Experiment
Positive features :
• MSn experiment, multiple levels of fragmentation is possible.
• Instrument for identifying an unknown chemical from a finger print of fragments
• Suitable for structure elucidation about complicated compounds
• Suitable for the detection and quantitation of very subtle target
compounds in complicated matrix.
Limitations :
• The resolution and the performance in an ion trap are depending upon the
charge density of the ions in the trap. If too many ions at the same time in an ion
trap, the electrical fields are destorted by inductive effects. Also, collision
between the ions may occur, leading to unexpected dissociation or chemical
reactions. In this case the spectra and the quantitation will be impacted.
• Low resolution and mass accuracy
• narrow dynamic range of quantitation
Ion Trap
MSn
Q-TOF MS analyzer
Q-TOF
5x10
-7
Torr
Liner
Ion mirror
Ion
detecto
r
Pusher
Puller
Q
1
q
2
MS1 mass
selection
CID
Ion focus
&
transfer
q
0
N
2
ESI
spray tip
Roughing
pump
Turbo
pump
High
vacuum
High
vacuum
High
vacuum
Collision gas
MS2 mass
analysis
Ion
formatio
n
Q-TOF
•
The Q-TOF is a tandem mass spectrometer
(MS/MS) with two analysers
•
the first being a quadrupole analyser that is
used as an ion guide in MS mode, but as a
resolving analyser in MS/MS mode.
•
the second analyser is a reflectron time-of-flight
analyser placed orthogonally to the quadrupole.
•
the final detector is a microchannel plate
detector for high sensitivity.
Q-TOF mass analyser
Positive features :
• Proteome analysis (Mainly for qualitative analysis)
• Popular instruments for high-quality small molecule
work
• Required ions can be selected easily and efficiently
and very accurately.
• Accurate mass measurement
Limitations :
• No MSn capability
• Accurate mass mainly only in MS
• Mass calibration drifts over time
Orbitrap Technology
Image Current Detection
in Orbitrap
From Alexander Makarov’s 2008 ASMS Award Address
• 3D electric field trapping
• No need for magnet
• Easy access
• Final detection device
Orbitrap
TOF
•Simultaneous
excitation
FTICR
• Confined ion trajectory
• Image current detection
• Fourier transform data
conversion
Unique to Orbitrap
• 3D electric field trapping
• No need for magnet
• Easy access
• Final detection device
Orbitrap function
Orbitrap Discovery (Since ASMS 2007)
Orbitrap XL (Since ASMS 2007)
Orbitrap cycle time
Orbitrap Cycle time
Orbitrap Exactive
(2008)
Orbitrap mass analyser
Positive features :
• high mass accuracy
• very high resolution
• popular instruments
• easy to use software
Limitations :
• slow data acquisition or much lower performance
• Accurate mass mainly only in MS for faster analysis
• price
FT-ICR-MS
Technology
Fourier Transform Ion Cyclotron Resonance
(FT-ICR)
•Ions trapped and
measured in ultrahigh vacuum
inside a superconducting magnet.
B
0
Detect
+
+
+
++ +
+
+
+
R
C
Excite
+
+
+ + + +
+
+
+
z
m
1
Comparison
Instrument
ESI-TOF
QqQ
Ion Trap Q-q-TOF LCMS-IT-
TOF
Orbitrap
/
FT MS
MS function
MS
MS/M
S
MSn
MS/MS
MS
n
MS
n
Mass accuracy
MS
3 ppm
0,0
1
5
100
ppm
0,
1
5
100
ppm
0,
1
5
3 ppm
0,0
1
5
5 ppm
0,0
1
5
*
< 1-2
ppm
0,00
1
5
*
Mass
resolution
10,000
-
40.000
4,000
(Delta:
0.5
amu)
4,000
(Delta:
0.5
amu)
10,000
-
60.000
10,000
60,000/
>100,00
0
Quantification
+
+++
++
+
+
++
Dynamic
Range
+++
++++
+++
++
++
+++
exact number
* also in MS
n
mode
Hybrid/Tandem Instruments
•Combine (1) ion selection, (2) ion dissociation, and (3) mass analyzer
devices
•Quadrupoles and ion traps good for selective isolation of
precursor ions and for fragmentation (required for MSMS - Topic of
Lecture 2)
•Reflectron TOF, FT-ICR, and OrbiTrap have higher mass accuracy
and resolving power (high mass accuracy is good for identification
– Lecture 3)
Ion Dissociation
•Collision Induced Dissociation (CID or Collision Activated
Dissociation (CAD)
ion traps: off-resonance excitation
rf-only multi-poles: higher kinetic energy (HCD) and
cascaded CID
TOF/TOF: single collision
•Electron capture dissociation (ECD) and Electron transfer
dissociation (ETD)
ECD: FTICR, reagent: electron
ETD: ion traps, reagent: free radical anion
Other important factors to consider: how product ions are
collected and detected
Data Dependent Acquisition
•Data Dependent Scans
MSMS based on intensity ranking of precursor ions
•Dynamic Exclusion
Precursor m/z of previous MSMS are memorized and no
MSMS done on them during a defined time period
•Automatic Gain Control (AGC, unique to ion trap)
Control how many ions are scanned – to achieve signal/noise
ratio and to minimize space charge effect
Detector
Digitally recording ion
arrival
While there is an exact instant when each ion
strikes the detector, it is difficult to transfer this
perfectly into the digital world.
There are two basic approaches used to
translate a detector signal into a digital
measurement:
- the analog-to-digital converter (ADC)
- time-to-digital (TDC)
It is well known TDC has dead-time problem
TDC vs ADC
TDC Vender
Model
Waters
LCT Premier
Q-TOF
Premier
Applied Bio
systems
QSTAR XL
AD
C
Vender
Model
Agilent
LC/MSD TOF
Bruker Daltonics
microTOF
microTOF Q
Shimadzu
LCMS-IT-TOF
TDC dead time causes shift to
shorter arrival times for higher
signal levels.
Ions per transient as a function of
sample amount, showing TDC
limitations.
Questions ????
Comparison of Analyzer Types
Ion Trap/
Quadrupol
e
TOF
OrbiTrap
FT-ICR
Sensitivity
+++
++
*
to +++
++
*
+
*
Mass
Accuracy
+
**
++
+++
+++
**
Resolving
Power
+**
++
+++
++++
**
Dynamic
Range
+ to +++**
++
++
++**
Upper m/z
+
++++
+++
++
*Sensitivity lowered due to losing ions on way to analyzer, rather than
inherent sensitivity.
**Can be improved by scanning narrower mass range or slower.
