MASS SPECTROMETRY (II)
MALDI-TOF AND ESI-MS
C. Electrospray Ionization Mass
Spectrometry (ESI-MS) (1980)
• John Fenn (1980)
– Nobel Prize 2002
• Production of molecular ions directly from
sample in solution
• Useful for small and large biopolymers (proteins,
peptides, carbohydrates, DNA fragments and
lipids)
• Hyphnatable (?) (HPLC-ESI-MS, CE-ESI-MS)
• Droplets electrically generated
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C.1 Ionization:
• Solution of analyte is pumped
through a stainless steel
capillary needle that is
maintained at several kVolts
relative to a cylindrical
electrode that surrounds it.
• A charged spray is generated.
If the voltage applied is
positive, the exiting spray is
positively charged.
• The spray is passed through a
desolvating capillary.
2
• Coulomb Fission :
the increased charge density, due
to solvent evaporation, causes
large droplets to divide into
smaller droplets eventually leading
to single ions.
• Ion Evaporation:
the increased charge density that
results from solvent evaporation
causes Coulombic repulsion to
overcome the liquid’s surface
tension, resulting in a release of
ions from droplet surfaces
Typical Feature of ESI
• Formation of multiply charged ions
• [M+H]+, [M+2H]2+, [M+nH]n+
• Number of peaks depends on size of the molecule and
number of acidic and basic groups
• Large proteins: series of ions of up to [M+100H]100+.
• Isotopes peaks add to the number of peaks
• Overall: complex spectra
• Relatively low m/z values
• Very high molecular weights observed
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C-3 Quadrupole Analyser
• Four parallel rod-like metal
electrodes.
• Direct (dc) and alternating
voltages (ac) are applied to
rods
• Applied potentials of
opposite rods are
¾ (U+Vcos(ωt))
¾ -(U+Vcos(ωt))
• U is a dc voltage
• Vcos(ωt) is an ac voltage.
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• Generally the frequency
of the ac field is constant
• The radius is constant
• Potential of direct current
and alternating current
are increased to allow all
ions through the
quadrupole sequentially
The Quadrupole is a “Mass Filter”.
– For given dc and ac voltages, only ions of a
certain mass-to-charge ratio pass through the
quadrupole filter and all other ions are thrown
out of their original path.
– A mass spectrum is obtained by monitoring
the ions passing through the quadrupole filter
as the voltages on the rods are varied.
– There are two methods: varying ω and
holding U and V constant, or varying U and V
(U/V) fixed for a constant ω.
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Cartesien Force Components for a singly charged
ion accelerated by the electrostatic field between
Hyperbolic Quadrupole Rods
(
)
φ = (U + V0 cos ωt ) x 2 − y 2 / r02
Potential of the
electrostatic field
(
)
(
)
Fx = −e.E x = −e
∂φ
= −e(U + V0 cos ωt ) 2 x / r02
∂x
Fy = −e.E y = −e
∂φ
= + e(U + V0 cos ωt ) 2 y / r02
∂y
Fz = −e.E z = −e
∂φ
=0
∂z
Ions Trajectories
Equation of motion of a singly charged ion of mass, m, are,
(1) : −2e
(U + V0 cos ωt )x = m ∂ 2 x
∂ x
2
( 2) : m
(3) : 2e
( 4) : m
(5) : m
∂t 2
r02
∂t 2
+ 2e
(U + V0 cos ωt )x = 0
r02
(U + V0 cos ωt )y = m ∂ 2 y
∂t 2
r02
∂2 y
∂t 2
∂2z
∂t 2
− 2e
•Axial velocity is constant
•The filtering action results from
the trajectory characteristics of
equations 2 and 4
(U + V0 cos ωt )y = 0
r02
=0
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Trajectories
• The equation of motion are obtained which
describe the trajectories, some with finite
amplitude of oscillations (stable), others with
exponentially increasing oscillations
(unstable).
• The variables in the equations are:
–
–
–
–
Mass-to-charge ratio
dc voltage
Frequency and magnitude of ac voltage
r0
C-4 Applications of ESI-MS
• Polar analytes soluble in a solvent system
suitable for spraying
– Peptides, Protein, Carbohydrates, DNA
fragments, Lipids
– Solvents: methanol, ethanol and acetonitrile
– Volatile organic solvents used to promote
cation formation: formic acid
• Mw determination
• Sequencing of peptides and DNA
fragments
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Advantages
• Can be directly coupled to separation techniques
• Soft ionization techniques: no fragmentation
observed
• Little or no fragmentation even of non covalent
complexes
• Tandem Mass Spectrometry possible: ESI-MS/MS
with three quadrupole
Disadvantages
• Complex spectra
• Requires very clean samples, low buffer and
salt concentration (0.1mM)
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