CH437 ORGANIC STRUCTURE ANALYSIS
CLASS 2: MASS SPECTROMETRY 2
Synopsis. Ionization techniques continued: atmospheric pressure chemical ionization (APCI) and
matrix assisted laser desorption ionization (MALDI). Ion selection: electromagnetic sector (BE)
analyzers, quadrupole (Q) analyzers and ion trap (QIT) analyzers.
Atmospheric Pressure Chemical Ionization (APCI)
This method (again popular in HPLC-MS) is similar to CI (it uses ion-molecule
reactions), except that it operates at atmospheric pressure and the primary ions
are produced by corona discharge on the eluent spray emerging from the HPLC
capillary, as shown below.
The high-speed nebulizer gas converts the liquid into a thin mist and the high
temperature causes desolvation of the sample before it reaches the corona
discharge electrode.
Reactions at the electrode are complex, but many involve the production of
secondary reactant gas ions via reaction between primary ions (e.g. N2+. or O2+.)
and solvent molecules (S). The secondary ions then react with analyte molecules
(M):
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Corona
discharge
N2
N2+. + SH
N2H+ + M
N2+. +
N2H+ +
eS.
MH+ + N2
As in CI, adduct formation can occur in APCI.
Collision rates are high because the reactions occur at atmospheric pressure,
hence APCI, like ESI is an efficient process, making APCIMS a very sensitive
technique. As with ESI, transition from atmospheric pressure to high vacuum
occurs using a differential pumping technique on a single (as above) or several
stages, each separated from the others by a skimmer. Declustering of ions
occurs in the intermediate pressure regions.
APCI is most useful for obtaining mass spectra of relatively polar
molecules or ionic analytes, up to about 1500 Da: it is probably the best
kind of ionization in HPLC-MS. Singly charged ions are usually formed in
preference.
Matrix-Assisted Laser Desorption Ionization (MALDI)
MALDI uses the high energy of a pulsed tunable laser (e.g. 266 nm – 20,000 nm)
to desorb and ionize sample molecules that are contained in an organic matrix,
commonly sinapic acid (3,5-dimethoxy-4-hydroxycinnamic acid) or gentisic acid
(2,5-dihydroxybenzoic acid). The matrix is needed to both absorb most of the
energy of the laser beam and to transfer protons to the sample molecules,
thereby producing ions. Hence MALDI is really a “soft ionization” technique.
Matrix molecules with carboxylic acid and phenolic hydroxyl groups are
frequently chosen, because of the ease of proton transfer.
When a narrow beam of laser light strikes the surface of the matrix/sample
mixture, the local matrix molecules absorb most of the energy, become very hot
and desorb (vaporize), along with sample molecules.
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Formation of most ions occurs via proton transfer from matrix molecules, but the
singly charged molecular species tends to dominate. Multiple-charge ions are
possible in various combinations, if the sample molecules possess several
ionizable sites (like, peptides, proteins, carbohydrates and nucleotides):
[M+H]+
H+
+
[2M+H]+
M, H
M
3H+
[M+3H]3+
M, 2H+
[2M+2H]2+
Indeed, MALDI is often the ionization technique of choice for studying
biomacromolecules and synthetic polymers. The MALDI process is illustrated
below.
Because of the pulsed nature of the production of ions, and because of the
predominant formation of ions of low charge (i.e. with large m/z values), time-offlight (TOF) analyzers are often combined with MALDI (see Class 3).
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Other Ionization Techniques
These are not discussed in this course, but include:
Fast atom bombardment (FAB)
Liquid secondary ion mass spectrometry (LSIMS)
Thermospray ionization (TSI)
Plasma desorption ionization (PDI)
Field desorption ionization (FDI)
Particle beam ionization (PBI) (this is really an HPLC-MS interface: it is
responsible for conversion of liquid phase solutes to gas phase, with ionization
occurring later)
For inorganic and organometallic compounds, there are the following ionization
methods: thermal cavity, spark source, glow discharge source and inductively
coupled plasma (ICP) source.
Mass Spectrometer Analyzers
The analyzer must be able to sort ions according to their mass-to-charge
ratios (m/z). To do this, the analyzer takes advantage of some unique feature of
each ion, as a result of the imposition of a magnetic and/or electric field.
Analyzers differ in the ways this is achieved.
Magnetic and Electromagnetic (BE) Analyzers
Along with time-of-flight (TOF) analyzers, BE analyzers are of great historical
importance and they are still used in more expensive MS instruments, especially
for high resolution and accurate mass work.
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Action of a Magnetic Field
If an ion of mass m and charge z is accelerated in the source by a potential
difference of Vs, then at the source outlet (as it enters the magnetic field B), its
kinetic energy is
mv2
=
2
zVs
(1)
In a magnetic field that is perpendicular to the ion velocity, v, the ion is subjected
to a cetrifugal force Fm, (equation (2)), according to the rule shown below.
Fm
=
(2)
zvB
Hence, the ion follows a circular trajectory with radius r, so that the centrifugal
force and magnetic force are in equilibrium,
i.e.
or,
mv2
zvB =
r
zB = mv
r
(3)
If we eliminate v from equations (1) and (3), we have:
m
z
B2r2
=
(4)
2Vs
5
Rearrangement of equation (4) and setting z = 1 (singly charged ions only), gives
r =
2mVs
(5)
B
Thus, it is possible to separate singly charged ions of different mass, as shown
below, where 1 is the least massive and 5 the most massive ion.
However, in modern magnetic sector MS instruments, it is more convenient to
collect ions that arrive sequentially at a given point (i.e. they are detected by a
point collector – see class 3/4), keeping r constant and varying B and/or Vs. In
practice, it is normal to vary B: this is called single focusing.
A problem is that the magnetic sector is essentially a momentum analyzer
and has no kinetic energy focusing ability. Since ions of the same m/z
values in practice have not just a single kinetic energy, but a range of
kinetic energies, they are brought to focus at slightly different foci at the
collector. The image is thus blurred and resolution is relatively low.
The term “resolution” is described on the accompanying hand-out.
Action of an Electrostatic Field (E)
An electrostatic analyzer (ESA) is a type of curved cylindrical condenser. The
centrifugal force acting on ions in an electric field is given by equation (6):
zE =
mv2
(6)
r
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E is the electrical potential difference between the inner and outer plates of the
ESA and r is the radius of curvature of the ion trajectory.
Rewriting equation (6), where Ek = mv2/2, gives
r =
2Ek
zE
(7)
In an electric field, the trajectory is independent of mass, so that an ESA is
not a mass analyzer, but rather a kinetic energy analyzer: it can focus ions
according to their kinetic energies. Thus, if a magnetic analyzer is used in
conjunction with an electrostatic analyzer, the defect of focusing in the former
can be minimized. Such an instrument is known as a BE or double focusing
mass spectrometer: ions of different kinetic energies, but of the same mass,
converge at one point at the collector. In practice magnetic analyzers (sectors)
have many designs and double focusing instruments depend on this and the
order in which the ions enter the magnetic and electric fields. The illustration
below is of forward geometry (electric field before magnetic field): backward
geometry is the reverse of this.
The Y-focus is an electric focusing device to compensate for the variation in field
shape with field strength, at the pole magnet tips.
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Quadrupole (Q) Analyzers.
Nowadays, the quadrupole analyzer is probably the most common type,
especially in GC-MS and HPLC-MS instruments. A quadrupole consists of four
parallel equidistantly placed circular rods (poles) that are subjected to an applied
variable electrostatic field (DC) and an alternating (AC or RF) electric field, as
shown in the diagram below.
An ion enters through the injector hole on a trajectory that is parallel to the
direction of the rods. On reaching the electric field of the rods it is subjected to
forces in orthogonal planes (x, y) to this trajectory. And undergoes a complex
oscillatory motion according to the operating conditions (especially the V:U ratio)
and its mass-to-charge (m/z) ratio. Stable and unstable trajectories are shown in
the diagram below.
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Quadrupole mass analyzers are usually operated in such a way that only ions
with a narrow range of m/z (usually within 1 Th, say M) undergo stable motion
and reach the detector. All other ions undergo unstable motion and eventually
collide with one of the rods. At a different instant of time the operating conditions
will be such that this ion takes an unstable trajectory, while an ion of different m/z
value (say M+1) acquires a stable trajectory and hence is detected.
The m/z value corresponding to stable motion is given by equation (8)
m/z (Th)
=
0.069
V
f rf r0
(8)
2
V is the amplitude
of the RF field (Volts)
f rf is the frequency of
the RF field (MHz)
r 0 is the internal radius
of the system (cm)
The DC voltage (U) does not appear in the equation, because throughout the
experiment it is kept proportional to V. The factor 0.069 is related to a pair of
dimensionless parameters, called a and q, whose ratio (a/q) is 2U/V.
A plot of a versus q results in a “stability diagram”, as shown below.
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The shaded area defines values of a and q where stable ion motion in the z
direction occurs (such ions reach the detector). The lines A, B and C of slope a/q
(=2U/V) are called scan lines). It can be seen that only one scan line (A) touches
the stability region at a single point (the apex). By passing through a unique
point, this scan line fixes unique values for a and q, rendering them constant and
relating them to one ion only, of a particular m/z value.
This ion passes to the detector under these conditions and all other ions
(under the same conditions) have unstable trajectories and collide with the
rods.
The other scan lines (B and C) are not useful, since a and q in the shaded area
correspond to a number of ions, all of which reach the detector and hence are
not resolved.
The figure below shows how the chosen scan line applies to various m/z values
during one mass spectrometry experiment. At low values of a and q (and hence
of U and V), the scan line passes through the apices of stability diagrams for low
m/z ions only, allowing them to pass through the quadrupolar system to the
detector. At the same values of a and q, the trajectories of higher m/z ions are
unstable. As values for a and q increase, the scan line passes through the apices
of the stability diagrams for ions of higher and higher m/z values (but there is a
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limit: see later). These ions have sequentially stable trajectories through the
system to the detector, whereas the lower m/z ions do not.
Thus the quadrupole is a real mass-to-charge ratio analyzer: it is
independent of the kinetic energies of ions as they leave the source.
The only requirements are that the time for ions to traverse the analyzer be short
compared with the time needed to switch from one mass to another and that the
ions remain long enough in the analyzer for a few oscillations to occur before
reaching the detector. In practice all of these requirements are normally easily
met: the rods are short (50 – 250 mm) and the m/z values are scanned very
rapidly (1000 Th/s). The small size of the quadrupole analyzer, its rapid m/z
scanning, coupled with linear mass scale make the quadrupole the most popular
and the least expensive of all analyzers. Against this, there is an upper m/z limit
of 2000 Th on most quadrupole systems, at which a resolution of 2000 (m/m)
applies, although higher m/z ions are of lower abundance. Usually quadrupole
mass spectrometers are operated at unit resolution (i.e. resolution that is just
enough to separate two peaks that are one mass unit apart). Above 2000 Th, on
most quadrupole instruments, unit masses (as in isotope clusters (see class 5)
cannot be distinguished at resolution 2000. Resolution is defined as m/m.
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The quadrupole analyzer is thus a low resolution analyzer and cannot
normally be used for accurate mass measurement.
Resolution can be increased, but only at the cost of sensitivity. In practice, most
quadrupole MS instruments are run in the 0 – 800 m/z range, using 0 – 200 V
from the DC generator and 0 – 1200 V from the AC (RF) generator.
Ion Trap (QIT) Analyzers
Strictly, these are quadrupole ion traps (hence the abbreviation QIT), since four
charged electrodes are used to trap ions. Some QIT analyzers employ only an
RF voltage of fixed frequency but of variable voltage, others allow the application
of a DC voltage as well, like quadrupole analyzers. In concept, a QIT analyzer
resembles a Q analyzer bent back on itself to form a closed loop (see below), so
that ions entering the space between the electrodes are trapped in three
dimensional trajectories which are prevented from excessive motion (ions repel)
by the presence of helium in the trap at ca. 10-3 Torr.
Ions from ion source
(e.g. from electrospray,
vi a
skimmer, accelerating lens
and gating lens
Inlet end
cap
Ring
electrode
z0
r0
Ring
electrode
z
y
x
Ion exit:
to detector
Exit end
cap
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A resonance condition exists for each m/z for a particular value of V, but a slight
increase in V will cause the trajectory to become unstable and the ion is ejected
out of the ion trap space to the detector.
Alternatively, if an RF voltage at the secular frequency of a particular ion is
applied to the end caps (i.e. in the z direction), the ion will come into resonance
and its oscillations will be so large that it will be ejected from the trap in the z
direction.
Like quadrupole analyzers, scanning is very rapid. Tandem MS experiments
(Class 4) (of the “in time” type) can be carried using QIT analyzers by collision
with helium atoms, which are always present. Fragmentation can be improved by
excitation of selected ions at their secular frequency.
The equations that govern ion motion of a QIT analyzer are similar to those for Q
analyzers and the stability diagrams are also similar.
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