Electron ionization

Electron ionization (EI, formerly known as electron impact) is an ionization method

in which energetic electrons interact with gas phase atoms or molecules to produce ions.
This technique is widely used in mass spectrometry, particularly for volatile organic
molecules.

The following gas phase reaction describes the electron ionization process:

where M is the analyte molecule being ionized, e - is the electron and M+• is the resulting
ion.

In an EI ion source, electrons are produced

through thermionic emission by heating a wire
filament that has electric current running through
it. The electrons are accelerated to 70 eV in the
region between the filament and the entrance to
the ion source block. The accelerated electrons are
then concentrated into a beam by being attracted
to the trap electrode. A sample vapour containing
the neutral molecules is introduced to the ion
source in a perpendicular direction to the e- beam.
Upon interaction with the e- beam, the analyte
molecules ionize to radical cations which are then
directed towards the mass analyzer by a repeller
electrode. Due to the high energy electrons and
the initial thermal distribution of the neutral
molecules, the ionization process frequently
causes cleavage reactions that give rise to
fragment ions, which can convey structural
information about the analyte.

The ionization efficiency and production of fragment ions depends strongly on the
chemistry of the analyte and the energy of the electrons. At low energies (around 20
eV), the interactions between the electrons and the analyte molecules do not transfer
enough energy to cause ionization. At around 70 eV, the de Broglie wavelength of the
electrons matches the length of typical bonds in organic molecules (about 0.14 nm) and
energy transfer to organic analyte molecules is maximized, leading to the strongest
possible ionization and fragmentation. Under these conditions, about 1 in 1000 analyte
molecules in the source are ionized. At higher energies, the de Broglie wavelength of
the electrons becomes smaller than the bond lengths in typical analytes; the molecules
then become "transparent" to the electrons and ionization efficiency decreases.
Chemical ionization

Chemical ionization (CI) is an ionization technique used in mass spectrometry.

Chemical ionization is a lower energy process than electron ionization. The lower
energy yields less fragmentation, and usually a simpler spectrum. A typical CI spectra
has an easily identifiable molecular ion.

Mechanism

In a CI experiment, ions are produced through the collision of the analyte with ions of a
reagent gas that are present in the ion source. Some common reagent gases include:
methane, ammonia, and isobutane. Inside the ion source, the reagent gas is present in
large excess compared to the analyte. Electrons entering the source will preferentially
ionize the reagent gas. The resultant collisions with other reagent gas molecules will
create an ionization plasma. Positive and negative ions of the analyte are formed by
reactions with this plasma.

Primary Ion Formation:

Secondary Reagent Ions:

Product Ion Formation:

(protonation)
(H − abstraction)
(adduct formation)
(charge exchange)

Self chemical ionization occurs when the reagent ion is an ionized form of the analyte.[5]

Variations

Negative chemical ionization (NCI)

Chemical ionization for gas phase analysis is either positive or negative. Almost all
neutral analytes can form positive ions through the reactions described above.

In order to see a response by negative chemical ionization, the analyte must be capable
of producing a negative ion (stabilize a negative charge) for example by electron
capture ionization. Because not all analytes can do this, using NCI provides a certain
degree of selectivity that is not available with other, more universal ionization
techniques (EI, PCI). NCI can be used for the analysis of compounds containing acidic
groups or electronegative elements (especially halogens).[4]

Because of the high electronegativity of halogen atoms, NCI is a common choice for
their analysis. This includes many groups of compounds, such as PCBs[6], pesticides[7],
and fire retardants.[8] Most of these compounds are environmental contaminants, thus
much of the NCI analysis that takes place is done under the auspices of environmental
analysis.

Atmospheric pressure chemical ionization (APCI)

Chemical ionization in an atmospheric pressure electric discharge is called atmospheric

pressure chemical ionization. The analyte is aa gas or liquid spray and ionization is
accomplished using an atmospheric pressure corona discharge. This ionization method
is often coupled with high performance liquid chromatography where the mobile phase
containing eluting analyte sprayed with high flow rates of nitrogen and the aerosol spray
is subjected to a corona discharge to create ions.

Atmospheric pressure chemical ionization

Atmospheric pressure chemical ionization (APCI) is an ionization method used in

mass spectrometry. It is a form of chemical ionization which takes place at atmospheric
pressure.[1]

How it works

APCI allows for the high flow rates typical of standard bore HPLC to be used directly,
often without diverting the larger fraction of volume to waste. Typically the mobile
phase containing eluting analyte is heated to relatively high temperatures (above 400
degrees Celsius), sprayed with
high flow rates of nitrogen and
the entire aerosol cloud is
subjected to a corona discharge
that creates ions. Often APCI
can be performed in a modified
ESI source. This is basically a
gas phase ionisation, unlike
ESI which is a liquid phase
ionisation process. Also, we
can use nonpolar solvent for
solution making instead of polar solvent for supporting ions in solution as gaseous state
conversion of solvent before reaching to corona discharge pin is carried out here, which
well supports the ions formed. Typically, APCI is a less "soft" ionization technique than
ESI, i.e. it generates more fragment ions relative to the parent íon
Desorption atmospheric pressure photoionization

Desorption atmospheric pressure photoionization (DAPPI) is an atmospheric-

pressure ionization technique for mass spectrometry. DAPPI enables direct analysis of
solid samples without pretreatment and analysis of samples deposited on surfaces by
means of a jet of hot solvent vapour and vacuum ultraviolet light. The hot jet thermally
desorbs the sample from a surface and the vaporized sample is ionized by the vacuum
ultraviolet light and consequently sampled into a mass spectrometer.

Ionization mechanisms

The ionization mechanism depends on the analyte and solvent used and for example the
following analyte (M) ions may be formed: [M + H]+, [M - H]-, M+•, M-•.

Applications

DAPPI has the potential to analyze both polar (e.g. verapamil) and nonpolar (e.g.
anthracene) compounds. Performance of DAPPI has also been demonstrated on direct
analysis of illicit drugs

Electrospray ionization

Electrospray ionization (ESI) is a technique used in mass spectrometry to produce

ions. It is especially useful in producing ions from macromolecules because it
overcomes the propensity of these molecules to fragment when ionized. The
development of electrospray ionization for the analysis of biological macromolecules[1]
was rewarded with the attribution of the Nobel Prize in Chemistry to John Bennett Fenn
in 2002.[2]

Mass spectrometry using ESI is called electrospray ionization mass spectrometry

(ESI-MS) or, less commonly, electrospray mass spectrometry (ES-MS).

Ionization mechanism

The liquid containing the analyte(s) of interest is dispersed by electrospray into a fine
aerosol. Because the ion formation involves extensive solvent evaporation, the typical
solvents for electrospray ionization are prepared by mixing water with volatile organic
compounds (e.g. methanol, acetonitrile). To decrease the initial droplet size, compounds
that increase the conductivity (e.g. acetic acid) are customary added to the solution.
Large-flow electrosprays can benefit from additional nebulization by an inert gas such
as nitrogen. The aerosol is sampled into the first vacuum stage of a mass spectrometer
through a capillary, which can be heated to aid further solvent evaporation from the
charged droplets. The solvent evaporates from a charged droplet until it becomes
unstable upon reaching its Rayleigh limit. At this point, the droplet deforms and emits
charged jets in a process known as Rayleigh fission. During the fission, the droplet loses
a small percentage of its mass along with a relatively large percentage of its charge.[6]

There are two major theories that explain the final production of gas-phase ions:

 The Ion Evaporation Model (IEM)[7] suggests that as the droplet reaches a
certain radius the field strength at the surface of the droplet becomes large
enough to assist the field desorption of solvated ions.
 The Charged Residue Model (CRM)[8] suggests that electrospray droplets
undergo evaporation and fission cycles, eventually leading progeny droplets that
contain on average one analyte ion or less. The gas-phase ions form after the
remaining solvent molecules evaporate, leaving the analyte with the charges that
the droplet carried.

While there is no definite scientific proof, a large body of indirect evidence suggests
that small ions are liberated into the gas phase through the ion evaporation mechanism,
while larger ions form by charged residue mechanism.

The ions observed by mass spectrometry may be quasimolecular ions created by the
addition of a proton (a hydrogen ion) and denoted [M + H]+, or of another cation such as
sodium ion, [M + Na]+, or the removal of a proton, [M − H]−. Multiply-charged ions
such as [M + nH]n+ are often observed. For large macromolecules, there can be many
charge states, resulting in a characteristic charge state envelope. All these are even-
electron ion species: electrons (alone) are not added or removed, unlike in some other
ionization sources. The analytes are sometimes involved in electrochemical processes,
leading to shifts of the corresponding peaks in the mass spectrum.

Variants

The electrosprays operated a low flow rates generate much smaller initial droplets,
which ensure improved ionization efficiency. In 1994, two research groups coined the
name micro-electrospray (microspray) for electrosprays working at low flow rates.
Emmett and Caprioli demonstrated improved performance for HPLC-MS analyses
when the electrospray was operated at 300-800 nL/min.[9] Wilm and Mann demonstrated
that a capillary flow of ~ 25 nL/min can sustain an electrospray at the tip of emitters
fabricated by pulling glass capillaries to a few micrometers. [10] The latter was renamed
nano-electrospray (nanospray) in 1996.[11] Currently the name nanospray is also in use
for electrosprays fed by pumps at low flow rates, not only for self-fed electrosprays.
Unfortunately, there are no clear flow rate boundaries between electrosprays,
microsprays, and nanosprays.

Applications

Liquid chromatography–mass spectrometry (LC-MS)

Electrospray ionization is the ion source of choice to couple liquid chromatography with
mass spectrometry. The analysis can be performed online, by feeding the liquid eluting
from the LC column directly to an electrospray, or offline, by collecting fractions to be
later analyzed in a classical nanoelectrospray-mass spectrometry setup.
Noncovalent gas phase interactions

Electrospray ionization is also ideal in studying noncovalent gas phase interactions. The
electrospray process is capable of transferring liquid-phase noncovalent complexes into
the gas phase without disrupting the noncovalent interaction. This means that a cluster
of two molecules can be studied in the gas phase by other mass spectrometry
techniques. An interesting example of this is studying the interactions between enzymes
and drugs which are inhibitors of the enzyme. Because inhibitors generally work by
noncovalently binding to its target enzyme with reasonable affinity the noncovalent
complex can be studied in this way. Competition studies have been done in this way to
screen for potential new drug candidates.

Matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique

used in mass spectrometry, allowing the analysis of biomolecules (biopolymers such as
proteins, peptides and sugars) and large organic molecules (such as polymers,
dendrimers and other macromolecules), which tend to be fragile and fragment when
ionized by more conventional ionization methods. It is most similar in character to
electrospray ionization both in relative softness and the ions produced (although it
causes many fewer multiply charged ions).

The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix is used
to protect the biomolecule from being destroyed by direct laser beam and to facilitate
vaporization and ionization.

Matrix

The matrix consists of crystallized molecules, of which the three most commonly used
are 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), α-cyano-4-
hydroxycinnamic acid (alpha-cyano or alpha-matrix) and 2,5-dihydroxybenzoic acid
(DHB). A solution of one of these molecules is made, often in a mixture of highly
purified water and an organic solvent (normally acetonitrile (ACN) or ethanol).
Trifluoroacetic acid (TFA) may also be added. A good example of a matrix-solution
would be 20 mg/mL sinapinic acid in ACN:water:TFA (50:50:0.1).

The identity of suitable matrix compounds is

determined to some extent by trial and error,
but they are based on some specific molecular
design considerations:
 They are of a fairly low molecular
weight (to allow facile vaporization),
but are large enough (with a low
enough vapor pressure) not to
evaporate during sample preparation or
while standing in the spectrometer.
 They are acidic, therefore act as a proton source to encourage ionization of the
analyte.
  They have a strong optical absorption in the UV, so that they rapidly and
efficiently absorb the laser irradiation.
 They are functionalized with polar groups, allowing their use in aqueous
solutions.

The matrix solution is mixed with the analyte (e.g. protein-sample). The organic solvent
allows hydrophobic molecules to dissolve into the solution, while the water allows for
water-soluble (hydrophilic) molecules to do the same. This solution is spotted onto a
MALDI plate (usually a metal plate designed for this purpose). The solvents vaporize,
leaving only the recrystallized matrix, but now with analyte molecules spread
throughout the crystals. The matrix and the analyte are said to be co-crystallized in a
MALDI spot.

Laser

The laser is fired at the crystals in the

MALDI spot. The matrix absorbs the
laser energy and it is thought that
primarily the matrix is ionized by this
event. The matrix is then thought to
transfer part of its charge to the analyte
molecules (e.g. protein), thus ionizing
them while still protecting them from the
disruptive energy of the laser. Ions
observed after this process consist of a
neutral molecule [M] and an added or removed ion. Together, they form a
quasimolecular ion, for example [M+H]+ in the case of an added proton, [M+Na] + in the
case of an added sodium ion, or [M-H]- in the case of a removed proton. MALDI is
capable of creating singly-charged ions, but multiply charged ions ([M+nH] n+) can also
be created, as a function of the matrix, the laser intensity and/or the voltage used. Note
that these are all even-electron species. Ion signals of radical cations can be observed
eg. in case of matrix molecules and other stable molecules.

Atmospheric pressure matrix-assisted laser desorption/ionization

Atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) is an

ionization technique (ion source) that in contrast to vacuum MALDI operates at normal
atmospheric environment. The main difference between vacuum MALDI and AP-
MALDI is the pressure in which the ions are created. In vacuum MALDI, ions are
typically produced at 10 mTorr or less while in AP-MALDI ions are formed in
atmospheric pressure. Disadvantage of the AP MALDI source is the limited sensitivity
observed and the limited mass range.

AP-MALDI is used in mass spectrometry (MS) in a variety of applications ranging from

proteomics to drug discovery fields. Popular topics that are addressed by AP-MALDI
mass spectrometry include: proteomics, DNA/RNA/PNA, lipids, oligosaccharides,
phosphopeptides, bacteria, small molecules and synthetic polymers, similar applications
as available also for vacuum MALDI instruments.

The AP-MALDI ion source is easily coupled to an ion trap mass spectrometer [12] or any
other MS system equipped with ESI (electrospray ionization) or nanoESI source.

Mass spectrometer

The type of a mass spectrometer most widely used with MALDI is the TOF (time-of-
flight mass spectrometer), mainly due to its large mass range. The TOF measurement
procedure is also ideally suited to the MALDI ionization process since the pulsed laser
takes individual 'shots' rather than working in continuous operation. MALDI-TOF
instruments are typically equipped with an "ion mirror", deflecting ions with an electric
field, thereby doubling the ion flight path and increasing the resolution. Today,
commercial reflectron TOF instruments reach a resolving power m/Δm of well above
20'000 FWHM (full-width half-maximum, Δm defined as the peak width at 50% of
peak height).

History

The term matrix-assisted laser desorption ionization (MALDI) was coined in 1985 by
Franz Hillenkamp, Michael Karas and their colleagues.[13] These researchers found that
the amino acid alanine could be ionized more easily if it was mixed with the amino acid
tryptophan and irradiated with a pulsed 266 nm laser. The tryptophan was absorbing the
laser energy and helping to ionize the non-absorbing alanine. Peptides up to the 2843
Da peptide melittin could be ionized when mixed with this kind of “matrix”. [14] The
breakthrough for large molecule laser desorption ionization came in 1987 when Koichi
Tanaka of Shimadzu Corp. and his co-workers used what they called the “ultra fine
metal plus liquid matrix method” that combined 30 nm cobalt particles in glycerol with
a 337 nm nitrogen laser for ionization.[15] Using this laser and matrix combination,
Tanaka was able to ionize biomolecules as large as the 34,472 Da protein
carboxypeptidase-A. Tanaka received one-quarter of the 2002 Nobel Prize in Chemistry
for demonstrating that, with the proper combination of laser wavelength and matrix, a
protein can be ionized.[16] Karas and Hillenkamp were subsequently able to ionize the 67
kDa protein albumin using a nicotinic acid matrix and a 266 nm laser. [17] Further
improvements were realized through the use of a 355 nm laser and the cinnamic acid
derivatives ferulic acid, caffeic acid and sinapinic acid as the matrix.[18] The availability
of small and relatively inexpensive nitrogen lasers operating at 337 nm wavelength and
the first commercial instruments introduced in the early 1990s brought MALDI to an
increasing number of researchers.[19] Today, mostly organic matrices are used for
MALDI mass spectrometry.

Use

In Biochemistry
In proteomics, MALDI is used for the identification of proteins isolated through gel
electrophoresis: SDS-PAGE, size exclusion chromatography, and two-dimensional gel
electrophoresis. One method used is peptide mass fingerprinting by MALDI-MS, or
with post ionisation decay or collision-induced dissociation (further use see mass
spectrometry).

In Organic Chemistry

Some synthetic macromolecules, such as catenanes and rotaxanes, dendrimers and

hyperbranched polymers, and other assemblies, have molecular weights extending into
the thousands or tens of thousands, where most ionization techniques have difficulty
producing molecular ions. MALDI is a simple and rapid analytical method that can
allow chemists to analyze the results of such syntheses and verify their results.

In polymer chemistry

In polymer chemistry MALDI can be used to determine the molar mass distribution.[20]
Polymers with polydispersity greater than 1.2 are difficult to characterize with MALDI
due to the signal intensity discrimination against higher mass oligomers.[

Reproducibility and performance

The sample preparation for MALDI is important for the result. Inorganic salts which are
also part of protein extracts interfere with the ionization process. The salts are removed
by solid phase extraction or washing the final target spots with water. Both methods can
also remove other substances from the sample. The matrix protein mixture is not
homogenous because the polarity difference leads to a separation of the two substances
during crystallization. The spot diameter of the target is much larger than that of the
laser, which makes it necessary to do several laser shots at different places of the target,
to get the statistical average of the substance concentration within the target spot. The
matrix composition, the addition of trifluoroacetic acid and formic acid, delay between
laser pulses, delay time of the acceleration power, laser wavelength, energy density of
the laser and the impact angle of the laser on the target are among others the critical
values for the quality and reproducibility of the method.

Desorption electrospray ionization

Desorption electrospray ionization (DESI) is an ambient ionization technique that can

be used in mass spectrometry for chemical analysis. It is an atmospheric pressure ion
source that ionizes gases, liquids and solids in open air under ambient conditions. It was
developed in 2004 by Professor Graham Cooks et al. from Purdue University and is
now available commercially by Prosolia Inc. [1] DESI is a similar ionization technique to
Direct Analysis in Real Time (DART) in its versatility, applications and analysis time. It
is different in that some sample preparation is required; liquid samples have to be
deposited on a desired surface and allowed to dry and gases have to be adsorbed unto a
material.

Principle of operation

Schematic diagram of
the DESI ion source

DESI is a
combination of
electrospray (ESI) and
desorption (DI)
ionization methods.
Ionization takes place
by directing an
electrically charged
mist to the sample surface that is a few millimeters away. [2] The electrospray mist is
attracted to the surface by applying a voltage on the sample holder. After ionization, the
ions travel through air into the atmospheric pressure interface which is connected to the
mass spectrometer. DESI is a technique that allows for ambient ionization of a trace
sample at atmospheric pressure, with little sample preparation.

Ionization mechanism

In DESI there are two kinds of ionization mechanism, one that applies to low molecular
weight molecules and another to high molecular weight molecules. [2] High molecular
weight molecules, such as proteins and peptides show electrospray like spectra where
multiply charged ions are observed. This suggests desorption of the analyte, where
multiple charges in the droplet can easily be transferred to the analyte. The charged
droplet hits the sample, spreads over a diameter greater than its original diameter,
dissolves the protein and rebounces. The droplets travel to the mass spectrometer inlet
and are further desolvated. The solvent typically used for the electrospray is a
combination of methanol and water.

For the low molecular weight molecules, ionization occurs by charge transfer: an
electron or a proton. There are three possibilities for the charge transfer. First, charge
transfer between a solvent ion and an analyte on the surface. Second, charge transfer
between a gas phase ion and analyte on the surface; in this case the solvent ion is
evaporated before reaching the sample suface. This is achieved when the spray to
surface distance is large. Third, charge transfer between a gas phase ion and a gas phase
analyte molecule. This occurs when a sample has a high vapour pressure.
The ionization mechanism of low molecular weight molecules in DESI is similar to
DART’s ionization mechansim, in that there is a charge transfer that occurs in the gas
phase.

Ionization efficiency

The ionization efficiency of DESI is

complex and depends on several
parameters such as, surface effects,
electrospray parameters, chemical
parameters and geometric parameters.
[2]
Surface effects include chemical
composition, temperature and electric
potential applied. Electrospray
parameters include electrospray
voltage, gas and liquid flow rates.
Chemical parameters refers to the
sprayed solvent composition, e.g.
addition of NaCl. Geometric
parameters are α, β, d1 and d2 (see
figure on the right).

Furthermore, α and d1 affect the ionization efficiency, while β and d 2 affect the
collection efficiency. Results of a test performed on a variety of molecules to determine
optimal α and d1 values show that there are two sets of molecules: high molecular
weight (proteins, peptides, oligosaccharide etc) and low molecular weight (dizazo dye,
stereoids, caffeine, nitroaromatics etc). The optimal conditions for the high molecular
weight group are high incident angles (70-90º) and short d1 distances (1-3 mm). The
optimal conditions for the low molecular weight group are the opposite, low incident
angles (35-50º) and long d1 distances (7-10 mm). These test results indicate that each
group of molecules has a different ionization mechanism; described in detail in the
Principle of operation section.

The sprayer tip and the surface holder are both attached to a 3D moving stage which
allow to select specific values for the four geometric parameters: α, β, d1 and d2.

Sonic spray ionization

Sonic spray ionization (SSI) is method for creating ions from a liquid solution, for
example, a mixture of methanol and water. A pneumatic nebulizer is used to turn the
solution into a supersonic spray of small droplets. Ions are formed when the solvent
evaporates and the statistically unbalanced charge distribution on the droplets leads to a
net charge. Complete desolvation results in ions that can be detected using mass
spectrometry.[1]

Applications

Sonic spray ionization has been coupled with high performance liquid chromatography
for the analysis of drugs. Oligonucleotides have been studied with this method. SSI has
been used in a manner similar to desorption electrospray ionization for ambient
ionization and has been couplet with thin layer chromatography in this manner.