IONIZATION SOURCES
Principle Scheme Advantages Disadvantages
Electrospray Ionisation • Apply strong electric field. • Practical mass limit • Low salt tolerance.
(ESI) • Redox chemistry at interface ~70,000 Da. • Di[iculty cleaning
= combined actoion of between solution and electrode • Good sensitivity. overly contaminated
repulsion and à charging of solution + • Softest ionization. instrument.
evaporation of the formation of Taylor cone. • Easily adaptable to • Low tolerance for
solvent • Repulsion of the ions à microbore LC. mixtures.
nebulisation (small droplets are • No matrix interference. • Multiple charging.
formed and there is a fast flow • Easy adaptability to • Quantitation requires
outwards. triple quadrupole internal standard.
• Counter current of (heated) gas, analysis.
• ESI source perpendicular to the mass
perpendicular to the spray à • Multiple charging.
analyser
evaporation of the solvent • Better mass accuracy.
• Multiple charging allows for calculation of
within the droplets. • Determine peptide
the MW
• Density of the surface charges modifications.
o z1*(P2-P1) = P2 – 1
increases à electrostatic • Samples can be
o M = (P1*z1) – z1
repulsion becomes higher than analysed directly.
surface tension à droplets • MS/MS capabilities.
explode into smaller droplets.
• These undergo the same
process, resulting in gradual
formation of smaller droplets.
• Happens under atmospheric
pressure.
MALDI (Matrix Assisted • Analyte is crystallized on a • Practical mass limit • Low resolution.
Laser Desorption stainless-steel plate, together with ~300,000 Da. • Matrix background.
Ionisation) an excess of matrix. • Highly sensitive. • MS/MS capability is
• Matrix is able to absorb a specific
• Soft ionization. minimal.
wavelength and is usually a weak
organic acid (proton donor) with a • Suitable to analyse • Photodegradation.
phenolic group. It can spread out complex mixtures. • Not possible to study
easily on the support. • Samples added directly noncovalent
• Interaction of laser light with the to appropriate matrix. interactions.
sample results in ionization of • Requires internal
matrix and analyte molecules. standard.
• A high potential electric field leads • Poor for determine
the ions to the mass analyser.
peptide modifications.
• Low tolerance for
various experiments.
, MASS ANALYSERS
Principle Scheme Advantages Disadvantages
Magnetic Sector • Ions are accelerated in an electric field
Analysers and pass two analysers.
• Electrostatic analyser (or ESA) functions
as a ‘kinetic energy filter’ and allows only
ions to pass with a specific kinetic
energy.
• Ions with diGerent masses and identical
speed arrive at the magnetic sector and
form a bended trajectory (deflection)
with a radius that is dependent of the
m/z value and the speed of the ion.
• Since the last one is the same for all the
selected ions, this means that only ions
of a specific m/z value form a bend that
allows them to hit the detector (m/z
filter). Adapting the magnetic field,
another set of ions will reach the
detector.
• Hence by gradually changing the
magnetic field from one extreme to the
other, ions are scanned from high MW to
low MW or vice versa.
Quadrupole • Four parallel rods. • Mass limit ±70.000 Da. • Scanning limits the time
• Combination of AC (rf region) and • High mass accuracy. to analyse ions within a
DC voltage is applied. On one pair of • Good MS/MS chromatographic peak
rods: negative voltage, on the other possibilities. (peak saturation).
pair: positive voltage. • Compatible with ESI
• Ions undergo oscillating movement sources.
when passing through the rods.
• Each pair makes up a filter: high
pass and low pass mass filters.
• Combination of these filters à
small window: only ions with a
specific m/z pass the quadrupole,
hence at a certain U, V and ω only
ions with a specific m/z value have
stabile oscillations while other ions
bend and are thrown out of the
quadrupole.
, • Scanning: this process repeats with
gradual changing U and V values
such that over time, di[erent ions
are detected.
• One scan = one duty cycle.
3D-iontrap • All ions enter the trap. • Di[erent analyses on • Space charge e[ects in
• Energetic ‘sink’ caused by RF field the same ion can be ion traps.
and helium. performed in the same
• Ions oscillate and accumulate into analyser.
the centre of the trap. • High sensitivity.
• Primary rf field is set to the ring • Possibility to study
electrode. Additional rf (or AC) is set • Adaptation of the quadrupole à 3- PTMs.
to the ‘end caps’. dimensional quadrupole built with 3
• Primary and additional rf are electrodes
gradually and simultaneously o 1 hyperbolic ring electrode
increased, with each ion (specific o 2 ‘end-cap’ electrodes which contain
m/z) resonating more and finally holes where the ions can pass.
thrown out for detection.
2D-iontrap • Based on 4 rods of a quadrupole, • More accurate.
with a positive electrode (lens) or • Volume much larger
two outside segments at each end. (more ions trapped).
Hence, radial trapping (x-axis) is • Better trapping
performed by the quadrupole, axial e[iciency (50% vs. 5%
trapping (z-axis) by the electric field with 3D-IT).
on the lenses.
• Ions move between the two outside
lens electrodes or segments (z-axis),
while they oscillate in x- and y-
direction. Ions can leave the trap in
axial or radial direction.
,FT-ICR (Fourier • Ions are injected into a small cube- • Good resolution and
Transform Ion shaped space, positioned into a very accuracy frequency
Cyclotron strong alternating fixed magnetic measurement
Resonance) field, applied on the z-axis. • MSn is possible
• Ions are introduced via the z-axis by
a trap voltage (1V) applied on the
back and front plate.
• All ions that are present in the
cyclotron are then excited (RF) after
which a fast (1 µs) scan in the
magnetic field over a broad
frequency range is applied.
• The ions rotate in the xy plane
around the z-axis so that they move
back and forward.
• Detector plates on both sides of the
cube measure the frequency by
which ions move through the tube
• Fourier transformer can convert this
complex signal into the individual
frequencies and intensities,
analogous to sound.
Orbitrap • Ions with high velocity (kinetic • Very high resolution
energy, 1600 eV) are tangentially (60.000-150.000
injected. FWHM), dependent on
• Inner electrode is adjusted to 3200V detection time.
à charged ions under influence of a • Not so expensive
centrifugal and an electrostatic • Accuracy: till ± 1 ppm.
force à ions circulate around the
inner electrode while moving along
the z-axis, also back and forth along
axis of central electrode.
• The frequency and the angular
velocity of these movements are
inverse proportional with m/z.
• Detection plates are present in the
outside electrode à similar to FT-
ICR.
,TOF (Time-Of- • Ions are accelerated by an electric Compared to quadrupoles: • Resolution.
Flight) field, set between an electrode and • No time-consuming • Accuracy.
an extraction grid. scan because of
• Ions are then introduced into a field- parallel detection of Improvements (see table
free vacuum zone. many di[erent ions underneath):
• The ion moves straight and with (µseconds), hence, • Delayed pulse
constant velocity to the detector. high throughput extraction.
• Smaller and multiple charged ions possibilities. • Reflectors.
move faster compared to heavier • Better suited for • Orthogonal
and monocharged ions. analysis of complex acceleration: ESI-TOF or
mixtures. QTOF.
• Sensitive.
• Mass limit: 300,000 Da.
• High salt tolerance.
, IMPROVEMENT OF THE RESOLUTION OF TOF
Problem Solution
Inbalanced dispersion of kinetic energy 1) Delayed pulse extraction
over ions with identical m/z • A voltage gradient is set with the highest V closest to the source (at the beginning)
• This happens during the MALDI desorption à that accelerates the fast ions less than the slow ions (which are closest to the
process. source)
o Ions expand in the field-free zone • Lower energy ions acquire more energy and catch up with the faster ones at the
and this expansion is dependent of detector.
their kinetic energy.
o For ions with identical m/z values, 2) Reflecors
the ions with higher kinetic energy • It increases the distance that the ion needs to travel.
move faster towards the detector • Consists of a gradient electric field
than ions with lower kinetic energy • Ions move inside this field according to their energy.
o Higher energy ions move faster thus deeper into the field and have to
travel a longer distance as compared to lower energy ions.
• At the end, times are focussed
3) Orthogonal acceleration: ESI-TOF or QTOF
• TOF tubes can be adapted for ESI ionization by placing them orthogonally to the ESI
source.
o ESI creates ions à focused in an ion beam that fills up the first part of an
acceleration chamber
§ This part is localized between a plate and a grid G1.
§ Initially no V is applied à ions can move freely away from the source.
o A voltage is applied on the plate causing the ions to move into a direction,
perpendicular to the ion beam.
o Ions are further accelerated towards the second grid G2 by means of a VTOF
voltage and separation can take place.
o While moving inside the TOF tube, the acceleration chamber can again filled
with new ions.
• Main advantages
o Online connection with ESI source or quadrupole
o Di[erent initial velocities are minimized after changing direction, resulting in better resolution and accuracy
Principle Scheme Advantages Disadvantages
Electrospray Ionisation • Apply strong electric field. • Practical mass limit • Low salt tolerance.
(ESI) • Redox chemistry at interface ~70,000 Da. • Di[iculty cleaning
= combined actoion of between solution and electrode • Good sensitivity. overly contaminated
repulsion and à charging of solution + • Softest ionization. instrument.
evaporation of the formation of Taylor cone. • Easily adaptable to • Low tolerance for
solvent • Repulsion of the ions à microbore LC. mixtures.
nebulisation (small droplets are • No matrix interference. • Multiple charging.
formed and there is a fast flow • Easy adaptability to • Quantitation requires
outwards. triple quadrupole internal standard.
• Counter current of (heated) gas, analysis.
• ESI source perpendicular to the mass
perpendicular to the spray à • Multiple charging.
analyser
evaporation of the solvent • Better mass accuracy.
• Multiple charging allows for calculation of
within the droplets. • Determine peptide
the MW
• Density of the surface charges modifications.
o z1*(P2-P1) = P2 – 1
increases à electrostatic • Samples can be
o M = (P1*z1) – z1
repulsion becomes higher than analysed directly.
surface tension à droplets • MS/MS capabilities.
explode into smaller droplets.
• These undergo the same
process, resulting in gradual
formation of smaller droplets.
• Happens under atmospheric
pressure.
MALDI (Matrix Assisted • Analyte is crystallized on a • Practical mass limit • Low resolution.
Laser Desorption stainless-steel plate, together with ~300,000 Da. • Matrix background.
Ionisation) an excess of matrix. • Highly sensitive. • MS/MS capability is
• Matrix is able to absorb a specific
• Soft ionization. minimal.
wavelength and is usually a weak
organic acid (proton donor) with a • Suitable to analyse • Photodegradation.
phenolic group. It can spread out complex mixtures. • Not possible to study
easily on the support. • Samples added directly noncovalent
• Interaction of laser light with the to appropriate matrix. interactions.
sample results in ionization of • Requires internal
matrix and analyte molecules. standard.
• A high potential electric field leads • Poor for determine
the ions to the mass analyser.
peptide modifications.
• Low tolerance for
various experiments.
, MASS ANALYSERS
Principle Scheme Advantages Disadvantages
Magnetic Sector • Ions are accelerated in an electric field
Analysers and pass two analysers.
• Electrostatic analyser (or ESA) functions
as a ‘kinetic energy filter’ and allows only
ions to pass with a specific kinetic
energy.
• Ions with diGerent masses and identical
speed arrive at the magnetic sector and
form a bended trajectory (deflection)
with a radius that is dependent of the
m/z value and the speed of the ion.
• Since the last one is the same for all the
selected ions, this means that only ions
of a specific m/z value form a bend that
allows them to hit the detector (m/z
filter). Adapting the magnetic field,
another set of ions will reach the
detector.
• Hence by gradually changing the
magnetic field from one extreme to the
other, ions are scanned from high MW to
low MW or vice versa.
Quadrupole • Four parallel rods. • Mass limit ±70.000 Da. • Scanning limits the time
• Combination of AC (rf region) and • High mass accuracy. to analyse ions within a
DC voltage is applied. On one pair of • Good MS/MS chromatographic peak
rods: negative voltage, on the other possibilities. (peak saturation).
pair: positive voltage. • Compatible with ESI
• Ions undergo oscillating movement sources.
when passing through the rods.
• Each pair makes up a filter: high
pass and low pass mass filters.
• Combination of these filters à
small window: only ions with a
specific m/z pass the quadrupole,
hence at a certain U, V and ω only
ions with a specific m/z value have
stabile oscillations while other ions
bend and are thrown out of the
quadrupole.
, • Scanning: this process repeats with
gradual changing U and V values
such that over time, di[erent ions
are detected.
• One scan = one duty cycle.
3D-iontrap • All ions enter the trap. • Di[erent analyses on • Space charge e[ects in
• Energetic ‘sink’ caused by RF field the same ion can be ion traps.
and helium. performed in the same
• Ions oscillate and accumulate into analyser.
the centre of the trap. • High sensitivity.
• Primary rf field is set to the ring • Possibility to study
electrode. Additional rf (or AC) is set • Adaptation of the quadrupole à 3- PTMs.
to the ‘end caps’. dimensional quadrupole built with 3
• Primary and additional rf are electrodes
gradually and simultaneously o 1 hyperbolic ring electrode
increased, with each ion (specific o 2 ‘end-cap’ electrodes which contain
m/z) resonating more and finally holes where the ions can pass.
thrown out for detection.
2D-iontrap • Based on 4 rods of a quadrupole, • More accurate.
with a positive electrode (lens) or • Volume much larger
two outside segments at each end. (more ions trapped).
Hence, radial trapping (x-axis) is • Better trapping
performed by the quadrupole, axial e[iciency (50% vs. 5%
trapping (z-axis) by the electric field with 3D-IT).
on the lenses.
• Ions move between the two outside
lens electrodes or segments (z-axis),
while they oscillate in x- and y-
direction. Ions can leave the trap in
axial or radial direction.
,FT-ICR (Fourier • Ions are injected into a small cube- • Good resolution and
Transform Ion shaped space, positioned into a very accuracy frequency
Cyclotron strong alternating fixed magnetic measurement
Resonance) field, applied on the z-axis. • MSn is possible
• Ions are introduced via the z-axis by
a trap voltage (1V) applied on the
back and front plate.
• All ions that are present in the
cyclotron are then excited (RF) after
which a fast (1 µs) scan in the
magnetic field over a broad
frequency range is applied.
• The ions rotate in the xy plane
around the z-axis so that they move
back and forward.
• Detector plates on both sides of the
cube measure the frequency by
which ions move through the tube
• Fourier transformer can convert this
complex signal into the individual
frequencies and intensities,
analogous to sound.
Orbitrap • Ions with high velocity (kinetic • Very high resolution
energy, 1600 eV) are tangentially (60.000-150.000
injected. FWHM), dependent on
• Inner electrode is adjusted to 3200V detection time.
à charged ions under influence of a • Not so expensive
centrifugal and an electrostatic • Accuracy: till ± 1 ppm.
force à ions circulate around the
inner electrode while moving along
the z-axis, also back and forth along
axis of central electrode.
• The frequency and the angular
velocity of these movements are
inverse proportional with m/z.
• Detection plates are present in the
outside electrode à similar to FT-
ICR.
,TOF (Time-Of- • Ions are accelerated by an electric Compared to quadrupoles: • Resolution.
Flight) field, set between an electrode and • No time-consuming • Accuracy.
an extraction grid. scan because of
• Ions are then introduced into a field- parallel detection of Improvements (see table
free vacuum zone. many di[erent ions underneath):
• The ion moves straight and with (µseconds), hence, • Delayed pulse
constant velocity to the detector. high throughput extraction.
• Smaller and multiple charged ions possibilities. • Reflectors.
move faster compared to heavier • Better suited for • Orthogonal
and monocharged ions. analysis of complex acceleration: ESI-TOF or
mixtures. QTOF.
• Sensitive.
• Mass limit: 300,000 Da.
• High salt tolerance.
, IMPROVEMENT OF THE RESOLUTION OF TOF
Problem Solution
Inbalanced dispersion of kinetic energy 1) Delayed pulse extraction
over ions with identical m/z • A voltage gradient is set with the highest V closest to the source (at the beginning)
• This happens during the MALDI desorption à that accelerates the fast ions less than the slow ions (which are closest to the
process. source)
o Ions expand in the field-free zone • Lower energy ions acquire more energy and catch up with the faster ones at the
and this expansion is dependent of detector.
their kinetic energy.
o For ions with identical m/z values, 2) Reflecors
the ions with higher kinetic energy • It increases the distance that the ion needs to travel.
move faster towards the detector • Consists of a gradient electric field
than ions with lower kinetic energy • Ions move inside this field according to their energy.
o Higher energy ions move faster thus deeper into the field and have to
travel a longer distance as compared to lower energy ions.
• At the end, times are focussed
3) Orthogonal acceleration: ESI-TOF or QTOF
• TOF tubes can be adapted for ESI ionization by placing them orthogonally to the ESI
source.
o ESI creates ions à focused in an ion beam that fills up the first part of an
acceleration chamber
§ This part is localized between a plate and a grid G1.
§ Initially no V is applied à ions can move freely away from the source.
o A voltage is applied on the plate causing the ions to move into a direction,
perpendicular to the ion beam.
o Ions are further accelerated towards the second grid G2 by means of a VTOF
voltage and separation can take place.
o While moving inside the TOF tube, the acceleration chamber can again filled
with new ions.
• Main advantages
o Online connection with ESI source or quadrupole
o Di[erent initial velocities are minimized after changing direction, resulting in better resolution and accuracy
