B ac k t o B a sic s
Laser Desorption Ionization and MALDI
Back to Basics Section A: Ionization Processes
CHAPTER A2
LASER DESORPTION IONIZATION
AND MALDI
TABLE OF CONTENTS
Quick Guide .........................................................27
Summary ..............................................................29
The Ionization Process .........................................31
Other Considerations on
Laser Desorption Ionization ..............................33
Use of a Matrix .................................................35
Types of Laser ..................................................35
Secondary Ionization ........................................37
Uses of Lasers.................................................. 37
Conclusion ...........................................................39
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Quick Guide
Laser Desorption Ionization and MALDI
• A laser is a device for producing ultraviolet, visible or infrared light
of a definite wavelength unlike most other light sources, which give
out radiation over a range of wavelengths. The output of a single
wavelength of light is described as being coherent.
• Lasers may be tuneable, viz., although only one wavelength is
emitted at any one setting, the actual wavelength can be varied
over a small range by changing the setting of the laser.
• Other notable characteristics of the laser are concerned with the
intensity of the light emitted, its pulsed nature and the fine focusing
that is possible.
• For many lasers used in scientific work, the light is emitted in a
short pulse, lasting only a few nanoseconds but the pulses can be
repeated at very short intervals. Other lasers produce a
continuous output of light.
• The emitted beam of coherent radiation is narrow and can be
focused into a very small area. This means that the density of
radiation that can be delivered for any one pulse over a small area
is very high, much higher than can be delivered by conventional
light sources operating with similar power inputs.
• If the target at which a laser beam is directed can absorb light of
the laser wavelength then the target will absorb a large amount of
energy in a very small space in a very short time.
• The absorption of so much energy by a small number of target
molecules in such a short time means that their internal energy is
greatly increased rapidly and the normal processes of energy
dissipation (such as heat transfer) do not have time to occur. Much
of this excess of energy is converted into kinetic energy so that the
target molecules are vaporized (ablated) and leave the target zone.
• Some of the target molecules gain so much excess of internal
energy in a short space of time that they lose an electron and
become ions. These are the molecular cation-radicals found in
mass spectrometry by the direct absorption of radiation.
However, these initial ions may react with accompanying neutral
molecules, as in chemical ionization, to produce protonated
molecules.
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Laser Desorption Ionization and MALDI
• The above direct process does not produce a high yield of ions but
it does form a lot of molecules in the vapour phase. The yield of
ions can be greatly increased by applying a second ionization
method (e.g., electron ionization) to the vaporized molecules.
Therefore, laser desorption is often used in conjunction with a
second ionization step, such as electron ionization, chemical
ionization or even a second laser ionization pulse.
• Laser desorption is particularly good for producing ions from
analytically ‘difficult’ materials. For example, they may be used with
bone, ceramics, high molecular mass natural and synthetic
polymers and rock or metal specimens. Generally, few fragment
ions are formed.
• Improved ionization may be obtained in many cases by including
the sample to be investigated in a matrix formed from sinapic acid,
nicotinic acid or other materials. This variant of laser desorption
is known as matrix-assisted laser desorption ionization (MALDI).
• The laser may be used as a finely focused beam, which with each
pulse, drills deeper and deeper into the specimen giving ‘depth
profiling’. Alternatively, the beam can be defocused and moved
over an area at lower power so as to explore only surface features
of a specimen.
Summary
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Lasers are used to deliver a focused high density of monochromatic
radiation to a sample target, which is vaporized and ionized. The ions
are detected in the usual way by any suitable mass spectrometer to
produce a mass spectrum. The yield of ions is often increased by using
a secondary ion source or a matrix.
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Laser pulse
Neutral molecules and ions
begin to desorb
(a)
Sample surface
(b)
Absorbed energy starting to be converted
into kinetic energy of melted sample
Neutral molecules
pumped away
Ions drawn
into mass
spectrometer
analyser
(c)
After a few nanoseconds,
the absorbed energy has
been dissipated
Figure 1
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A laser pulse strikes the surface of a sample (a), depositing energy
which leads to melting and vaporization of neutral molecules and
ions from a small confined area (b). A few nanoseconds after the
pulse, the vaporized material is either pumped away or, if it is ionic,
it is drawn off into the analyser of a mass spectrometer (c).
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LASER DESORPTION IONIZATION
The Ionization
Process
A molecule naturally possesses rotational, vibrational and electronic
energy. If it is a liquid or a gas, it will also have kinetic energy of
motion. Under many everyday circumstances, if a molecule or group
of molecules have their internal energy increased (e.g., by heat or
radiation) over a relatively long period of time (which may only be a
few microseconds), the molecules can equilibrate the energy
individually and together so that the excess of energy is dissipated to
the surroundings without causing any change in molecular structure.
Beyond a certain point of too much energy in too short a time, the
energy cannot be dissipated fast enough so that the substance melts
and then vaporizes as internal energy of vibration and rotation is
turned into translational energy (kinetic energy or energy of motion);
simultaneous electronic excitation may be sufficient that electrons
may be ejected from molecules to give ions. Thus, putting a lot of
energy into a molecular system in a very short space of time can cause
melting, vaporization, possible destruction of material and,
importantly for mass spectrometry, ionization (Figure 1).
A laser is a device that can deliver a large density of energy into a small
space. The actual energy given out by a laser is normally relatively
small but, as it is focused into a very tiny area of material, the energy
delivered per unit area is very large. The analogy may be drawn of
sunlight which, although representing a lot of light, will not normally
cause an object to heat up so that it burns. However, if the sunlight is
focused into a small area by means of a lens, it becomes easy to set
an object on fire or to vaporize it. Thus, a low total output of light
radiation concentrated into a tiny area actually gives a high density or
flux of radiation (we could even say a high light ‘pressure’) - this is
typical of a laser. As an example, a Nd-YAG laser operating at 266 nm
can deliver a power output of about 10 Watts, somewhat like a sidelight on a motor car. However, this energy is delivered into an area
of about 10-7 cm2 so that the power focused onto the small irradiated
area is about 10/10-7 = 108 Watts/cm2 = 105 Kilowatts/cm2 (the same
effect as focusing the heat energy from 100,000 ‘one bar’ electric fires
onto the end of your finger!
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E'
Laser energy,
(a)
Absorption peak
Increasing wavelength
Increasing absorption
Increasing absorption
Laser energy,
E'
(b)
Absorption trough
Increasing wavelength
(c)
(d)
Laser beam
Laser beam
Laser beam
reflected
Sample desorbed as ions
and neutral molecules
Sample surface
Figure 2
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In (a), a pulse of laser light of a specific wavelength of energy, E’,
strikes the surface of a specimen which has a light absorption
spectrum with an absorption peak near to the laser wavelength.
The energy as absorbed, leading to the ablation of neutral molecules
and ions (c).
In (b), the laser strikes the surface of a specimen that does not have
a corresponding absorption peak in its absorption spectrum. The
energy is not absorbed but is simply reflected or scattered (d),
depending on whether the surface is smooth or rough.
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No wonder sample molecules get agitated by the laser, even if it is
only a few of them that are affected because of the small area which
is irradiated).
A molecular system exposed to a laser pulse (or beam) has its internal
energy vastly increased in a very short space of time, leading to melting
(with increased rotational and vibrational and electronic energy),
vaporization (desorption; increased kinetic or translational energy),
some ionization (electronic excitation energy leading to ejection of an
electron) and possibly some decomposition (increase in total energy
sufficient to cause bond breaking). If enough energy is deposited into
a sample in a very short space of time, it has no time to dissipate the
energy to its surroundings and it is simply blasted away from the
target area because of a large gain in kinetic energy (the material is
said to be ablated). Laser desorption ionization is the process of
beaming laser light, continuously or in pulses, onto a small area of a
sample specimen so as to desorb ions, which are examined in the
usual way by a mass spectrometer.
With continuous lasers (for example an argon ion laser), the energy
delivered is usually much less than from pulsed ones and the focusing
is not so acute. Thus, the irradiated area of the sample is more like
10-4 cm2 rather than 10-7 cm2 and the energy input is much less at
about 100 Kilowatts/cm2 rather than the 100,000 Kilowatts/cm2
described above.
Other
Considerations on
Laser Desorption
Ionization
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Consider a laser emitting radiation of energy, E’. For a substance to
absorb that energy, it must have an absorption spectrum (ultraviolet,
visible or infrared) that matches that energy. Figure 2 shows two
cases, one (a), in which a substance can absorb the energy, E’, and one
(b), in which it cannot absorb this energy. In the second case, since
energy cannot be absorbed, the laser radiation is reflected and none
of its energy is absorbed. In the second case, much or all of the
available energy can be absorbed and must then be dissipated
somehow by the system. This dissipation leads to the effects itemized
above. It follows that the capacity of a laser to desorb or ionize a
substance will depend on three factors, one the actual wavelength
(energy, E’) of the laser light, two the power of the laser and three,
the absorption spectrum of the substance being irradiated.
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Emission wavelength
of laser (energy, E ')
(a)
Increasing absorption
Absorption spectrum
of matrix
Absorption spectrum
of sample
Increasing wavelength
(b)
Laser beam
Matrix desorbed as ions
and neutral molecules
Surface of matrix material plus sample
Figure 3
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In a MALDI experiment, the sample is mixed or dissolved in a
matrix material, which has an absorption spectrum that matches the
laser wave length of energy, E’. The sample may not have a matching
absorption peak (a) but this is not important because the matrix
material absorbs the radiation, some of which is passed on to the
dissolved sample.
Neutral molecules and ions from both sample and matrix material
are desorbed (b).
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When the first and third factors match most closely and a lot of
power is available (large light flux), a lot of the laser energy can be
absorbed by the substance being examined; when the first and third
factors mismatch, whatever the power, little or none of the laser
energy is absorbed. Therefore, for any one laser wavelength, there
will be a range of responses for different substances and, for this
reason, it is often advantageous to use a tuneable laser so that various
wavelengths of irradiation can be selected to suit the substance being
examined.
Use of a Matrix
There is another way of allowing for the above variability of ionization
during laser irradiation. Suppose there is a sample substance (a matrix
material) having an absorption band that matches closely the energy
of the laser radiation. On irradiating this material. it will be rapidly
increased in energy and will desorb and ionize quickly, as described
above. Now suppose that it is not just the matrix material alone but
is a mixture or solution (a matrix) of a substance to be examined with
the matrix material. Now, at least some of the energy absorbed by the
matrix can be passed on to the sample substance causing it to desorb
and ionize (Figure 3a,b). This technique depends on the laser energy
matching an absorption band in the matrix and a match with the
sample substance is unnecessary so that the method becomes
general. It is called, matrix-assisted laser desorption ionization
(MALDI). Commonly, sinapic acid (3,5-dimethoxy-4-hydroxycinnamic
acid) or nicotinic acid are used as matrix materials for examining
organic and other compounds. The ions produced are usually
protonated molecules, [M + H]+, with few fragment ions.
Types of Laser
In theory, any laser can be used to effect desorption and ionization
provided it supplies a enough energy of the right wavelength in a short
space of time to a sample substance. In practice, for practical reasons,
the lasers, which are used tend to be restricted to a few types. The
laser radiation can be pulsed or continuous (continuous wave).
Typically, laser energies corresponding to the ultraviolet or near
visible region of the electromagnetic spectrum (e.g., 266 or 355 nm)
or the far infrared (about 20 mm) are used. The lasers are often
tuneable over a range of energies.
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Ejected (ablated)
ions and neutral
molecules
(a)
Laser pulse
A
B
C
Yield of ions at each
laser pulse
Three layers A, B, C
through the depth of a specimen
(b)
A+ ions
C+ ions
B+ ions
Number of laser pulses
Mass spectrometer recording
of ion type and yield
Figure 4a & b A laser pulse strikes the surface of a specimen (a), removing
material from the first layer, A. The mass spectrometer records the
formation of A+ ions (b). As the laser pulses ablate more material,
eventually the layer, B, is reached, at which stage, A + ions begin to
decrease in abundance and B+ ions appear instead. The process is
repeated when the B/C boundary is reached so that B+ ions
disappear from the spectrum and C+ ions appear instead. This
method is very useful for depth profiling through a specimen, very
little of which is needed.
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The so-called ‘peak’ power delivered by a pulsed laser is often far
greater than that for a continuous one. Whereas many substances
absorb radiation in the ultraviolet and infrared regions of the
electromagnetic spectrum, relatively few substances are coloured.
Therefore, a laser which emits only visible light will not be so
generally useful as ones emitting in the ultraviolet or infrared ends of
the spectrum. Further, with a ‘visible’ band laser, coloured substances
absorb more or less energy depending on the colour. Thus, two
identical polymer samples, one dyed red and one blue, would desorb
and ionize with very different efficiencies.
Secondary
Ionization
Much of the energy deposited in a sample by a laser pulse or beam
desorbs neutral material and not ions. Ordinarily, the neutral
substances are simply pumped away and the ions are analysed by the
mass spectrometer. To increase the number of ions formed, there is
often a second ion source to produce ions from the neutral materials,
thereby enhancing the total ion yield. This secondary or additional
mode of ionization may be effected by electrons (electron ionization,
EI), reagent gases (chemical ionization, CI) or even a second laser
pulse. The additional ionization is usually organized as a pulse
(electrons, reagent gas or laser), which follows very shortly after the
initial laser desorption.
Uses of Lasers
Laser desorption methods are particularly useful for substances of
high mass such as natural and synthetic polymers. Glycosides,
proteins, large peptides, enzymes, paints, ceramics, bone and large
polymers are all amenable to laser desorption mass spectrometry,
with the sample being examined either alone or as part of a prepared
matrix. Because of the large masses involved, for pulsed laser
desorption, the method is frequently used with time-of-flight or ion
trap instruments, which need pulses of ions. For MALDI, sample
preparation can be crucial, the number of ions produced varying
greatly with both the type of matrix material and with the presence
of impurities. Fragment ions are few but the true molecular mass can
be misinterpreted because of the formation of adduct ions between
the matrix material and the substance under investigation; these
adduct ions have greater mass than the true molecular mass. Some
impurities, as with common ionic detergents, may act as suppressants
to ion formation.
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B+ ions
A+ and B+ ions
Different laser
pulses
(c)
Laser pulse
A
B
Yield of ions at each
laser pulse
Surface of specimen
(d)
B+ ions
A+ ions
Number of laser pulses
Mass spectrometer recording
of ion type and yield
Figure 4c & d In (c), less power is used and the laser beam is directed at
different spots across a specimen. Where there is no surface
contamination only B+ ions appear but, where there is surface
impurity then ions A+ from the impurity also appear in the
spectrum (d).
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The laser approach without a matrix can be employed in two main
ways. Since the intensity and spot size of the laser pulse or beam can
be adjusted, the energy deposited into a sample may range from a
very large amount confined to a small area of sample to much less
spread over a larger area. Thus, in one mode, the laser can be used
to penetrate down through a sample, each pulse making the
previously ablated depression deeper and deeper. This is depth
profiling, which is useful for examining the variation in composition of
a sample with depth (Figure 4a). For example, gold plating on ceramic
would show only gold ions for the first laser shots until a hole had
been drilled right through the gold layer; there would then appear
ions such as sodium and silicon that are characteristic of the ceramic
material and the gold ions would mostly disappear.
By using a laser with less power and the beam spread over a larger
area, it is possibly to sample a surface. In this approach, after each
laser shot, the laser is directed onto a new area of surface, giving
surface profiling (Figure 4c). At the low power used, only the top few
nanometers of surface are removed and the method is suited to
investigation of surface contamination. The normal surface yields
characteristic ions but, where there are impurities on the surface,
additional ions appear.
Laser desorption is commonly used for pyrolysis/mass spectrometry,
in which small samples must be heated very rapidly to high
temperatures to vaporize them before they are ionized. In this
application of lasers, very small samples are used and the intention is
not simply to vaporize intact molecules but also to cause
‘characteristic’ degradation (the Back-to-Basics guide on pyrolysis/
mass spectrometry should be consulted).
Conclusion
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Lasers may be used in either pulsed or continuous mode to desorb
material from a sample, which may be examined as such or may be
mixed or dissolved in a matrix. The desorbed (ablated) material
contains relatively few or sometimes even no ions and a second
ionization step is frequently needed to improve the yield of ions.
Molecular or quasimolecular ions are mostly produced with few
fragment ions. By adjusting the laser focusing and power, laser
desorption can be used for either depth or surface profiling.
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