Download IONIZATION METHODS IN MASS SPECTROMETRY

IONIZATION METHODS IN MASS SPECTROMETRY
APCI and ESI Mode of ionization
In the JEOL Lcmate, components for the APCI method are standard equipment. C
omponents for the ESI method and the FAB and FritFAB ionization are optional.
In the APCI method, the sample solution is vaporized under atmospheric pressure
and ionized by corona discharge.
APCI is a chemical-ionization technique wh
ich uses gas-phase ion-molecule reactions
at atmospheric pressure. Primary ions are
produced by corona discharge on a solven
t spray. The chromatographic eluate ( from
0.1 to 1 ml/min) is directly introduced int
o a pneumatic nebulizer, where it is conve
rted into a thin fog by a high speed air or
nitrogen beam. Droplets are then displace
d by the gas flow through a carefully cont
rolled heated quartz tube , known as a de
solvation/vaporization chamber. The hot ga
s (120°C) and the compounds leaving this tube and arriving at the reaction area
of the source are chemically ionized (under atmospheric pressure) via proton tran
sfer. The evaporated mobile phase acts as the ionising gas and yields (M+H) + ion
s. The electrons needed for primary ionization result from corona discharge or ne
gatron emitters. Thermal decomposition is kept to a minimum and ionization of th
e substrate is very efficient. Differential pumping over several several stages sepa
rated by skimmers, maintains a good vacuum and allows entry of as many as io
ns as possible to the analyzer.
The APCI ionization method has the following features :
1. It is a soft ionization method generating few fragment ions.
2. The method is compatible with mobile-phase fow rates ranging from
0.1 to 1 ml/min.
3. It is suited to measurement of compounds with molecular
weights of approximately 1,500 Da or less.
4. Compounds having medium polarity can be measured with
high sensitivity.
5. The ions created under atmospheric pressure are effectively
transferred into high vacuum.
Electrospray mode of ionization
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An electrospray (ES) is produced by applying a strong electric field, under atmos
pheric pressure, to a liquid passing through a capillary tube with a weak flux (110 µl /min).
JEOL Lcmate
The electric field is obtained by applying a potential difference of 3-6 kV between
the capillary and the counter electrode, which are separated by 0.3-2.0 cm. This
field induces a charge accumulation at the liquid surface located at the end of th
e capillary, which will then break
up to form highly charged dropl
ets.
As the solvent contained in thes
e droplets evaporates, they shrin
k to a point where the repelling coulombic forces come close to their cohesion f
orces, thereby causing their " explosion". Eventually, after a cascade of further "
explosion" the highly charged ions desorb. Electrospray can also be used in the
case of molecules without any ionisable sites, through the formation of sodium, p
otassium, ammonium and other adducts. A variation of this method known as" m
icro-electrospray" uses flow rates of a few nl/min. This enables sensitivities in th
e range of a few hundred attomoles to be achieved.
The ESI mass spectra normally correspond to a statistical distribution of consecu
tive peaks characteristic of multiply charged molecular ions (M + nX)n+ where X
= mass of adduct and n = number of charges avoiding the contributions from dis
sociations or from fragmentations.
Consider an ion with charge n1 whose mass to charge ration is m1 Th, issued fr
om a molecular ion with mass M Da and an ion with charge n 2 whose mass to c
harge ration is m2 Th wit
h
Cytochro
me C + Glucagon
m2 > m1 and n1 > n2.
m= m/z of a given peak
M = molecular weight of the analyte
X = mass of adduct (eg H+, Na+ ...)
n = number of charges
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Ionspray source
TOP
Ionspray, which is another form of electrospray, creates charged droplets by usin
g a potential difference and a pneumatic nebulizer in order to accept higher flow
rates and to produce a spray that is less dependent on the nature of the liquid p
hase. The
ionspray technique consists of pu
mping the sample solution through
a pneumatic nebulizer, which is
maintained at a high voltage so as
to form a fog of charge droplets,
even if the flow rate being used i
s high. The ions are then ejected
from the droplets which evaporate,
via a low-energy process that does not produce fragments. Flow
rates are ca. 50-200 µl min-1 . A grounded screen lens acts as a
spray splitter and thus inhibits the condensation of droplets that
make the spray unstable at higher flow rates. In this way, rates
of up to 2 ml min-1 are then possible.
____________________________________
Illustration of the application ranges of different
mass spectrometric / chromatographic coupling m
ethods.
_____________________________________________
NANOSPRAY source (Jeol Lcmate ) TOP
Because the signal intensity in ESI is
concen
tration dependent,ie almost independent of the flo
w rate employed, absolute intensity ca, can be increased up to a 1000-fold when
the residence time of the analytes in the source is extended by reducing the flow
rate to 10 to 50 nL/min. Various nano-ESI sources are available commercially. S
everal analytical characteristics of nano-ESI , e.g tolerance for relatively high salt
concentration may be explained by a model which depend on the size of the initi
al droplets. A 1 to 2 µL sample will flow for 30 to 120 min providing more than
adequate time to optimize critical experimental conditions e.g. collision energy in
MS/MS analysis of selected individual peptide ions of a mixture.
Microfabricated, Silicone Chip-Based Electrospray Ion Sourc
e
TOP
Aiming to further increase the
sensitivity with concurrent re
duction of
cross-contamin
ation of analytes, multiple ESI
sources were made as parallel
etched channels on microchip
s. Electrospraying was perform
ed directly from the chip,using
flow rates of 100 to 200 nL/m
in through the channels, the s
ensitivity limit for the protein
angiotensin was 60 fmol/µL. The chip, made of glass, was designed with nine par
allel channels. Each channel was connected to two wells, which allowed for fluid
filling and sample injection into the channel. The fabrication process included th
orough cleaning of the glass with low sodium content , metal deposition (first 30
0 A° Cr and then 1000 A° Au), photolithography, chemical etching, and thermal b
onding. Smooth channels were obtained by isotropic etching with a solution of H
F/HNO3/H2O (20:14:66 v/v). For a 6 min etching time, the channel width was 60 µ
m and the depth 25 µm. The channel lengths varied from 3.5 to 5 cm depending
on their location on the glass chip. The microchip device was enclosed by therm
al bonding to a flat glass disk containing the wells. The thermal bonding was ac
complished over 3h at 620°C, after a temperature ramp of 10°C/min from room te
mperature. A comparison of scanning electron microscopic photographs before a
nd after bonding showed no noticeable alteration in channel shape.
Interface of Microchip to ESI-MS
TOP
A three - dimensional stage was constructed to hold the microchip and to align
sequentially each channel outlet with the MS inlet for optimum electrospray perfor
mance and sensitivity. The distance from the microchip channel exit to the MS in
let was between 3 and 8 mm. The surface of the outlet edge was coated with a
hydrophobic reagent, either Imunogen (Calbiochem, La Jolia, CA) or n-octyltriacet
oxysilane (Sigma, St Louis, MO), to prevent the analyzed solution from spreading
from one exit port to another. With this configuration, the channel outlet could b
e visually aligned with the MS inlet, the interface was operating at atmospheric p
ressure and room temperature.
Multiplexed Electrospray
TOP
A novel LC interface has been devised for the parallel analysis of four or eight L
C streams (single component or mixtures) with negligible crosstalk.
Another rece
ntly developed interface was described and consisting of a simple microchip-ESI
device that can be used for
rapid MS analysis. A replac
eable nanospray tip is attac
hed to the end of a microm
achined channel and the vol
tage necessary to generate t
he electrospray is applied to
a fluid reservoir on the chi
p. The system has some si
milarities to conventional na
nospray sources in that it o
perates at very low flow rates and does not require any pressure assistance for f
luid delivery. Unlike conventional nanospray sources, however, the microchip cou
pled to the nanospray tip can be used to inject discrete samples and perform se
parations prior to detection. The microchip may be used as a sample delivery de
vice enabling, for example, rapid, repetitive analysis of the same sample, to monit
or changes as a function of time, or high-throughput analysis from a chip with m
ultiple sample ports, injecting each sample individually in an automated fashio
n. One difficulty associated with a microchip device, where the spray is establish
ed directly from the planar edge of the chip, is that the fluid has a tendency to
spread over the surface prior to the onset of electrospray reducing sensitivity an
d the efficiency of separations that may be performed on the device. The hybridi
zed structure consisting of a nanospray tip coupled to a microchip eliminated the
se problems. The tips are also easy to replace extending the lifetime of the micr
ochip if desired. Fluid is continuously delivered at low flow rates ( 0.3-0.5 nL sec1
) solely by applying the electrospray voltage at one of the fluid reservoirs on th
e chip.
FAST ATOM AND ION BOMBARDMENT
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In the fast atom bombardment (FAB) ion source, ionization is accomplished by b
ombarding the sample, which is dissolved in a special liquid matrix with a high-e
nergy atom beam.
This resu
lts in molecular sputtering le
ading to the desorption of s
econdary ions of the sample,
from the liquid- vacuum inte
rface, into the gas phase. Th
e ion beam is extracted from
the ion source and mass an
alyzed, usually by quadrupol
e or magnetic sector analyze
rs in single or multistage co
mbinations. The related fast ion bombardment technique, also known as liquid se
condary-ion mass spectrometry (LSIMS), employs a stream of energetic ( ~ 30 ke
V) heavy ions (usually cesium) to accomplish the ionization. FAB sources may be
static or dynamic (continuous flow FAB). In the static mode the beam of atoms,
obtained in a special gun, bombards a conventional direct inlet probe modified t
o have a tip (ribbon of metallic or nonmetallic material ) upon which µL size dro
ps of the dissolved material
have been placed. The prim
ary atom beam of neutral ar
gon or xenon (more energeti
c) to be used for bombardin
g the target is obtained in s
pecial FAB guns. An argon
or xenon ion beam obtained
by EI is focused into a coll
ision chamber where the en
ergetic argon ions (~5 to 8
keV translational energy) be
come rapidly neutral atoms upon exchanging their charges with neutral argon or
xenon atoms. The remaining argon or xenon ions are deflected from the beam by
a potential placed on a set of electrodes, while the momentum of the neutral ar
gon or xenon atoms propels them to the probe.
Ar+. (fast) + Argon (thermal) --------mal)
Ar (fast) + Ar+. (ther
Because FAB only ejects ions into the gas phase that are already present in ionic form
in the solution or ionized by proton transfer, the selection of the proper liquid matrix is
critical. Obvious requirements are that the sample must be soluble in the matrix and
have a high diffusion rate through the matrix toward the surface.
Other chemical and
physical considerations include acid/base characteristics, purity, vapor pressure,
viscosity and surface tension.
Most FAB work has been done with glycerol,
thioglycerol and m-nitrobenzyl alcohol N-formyl- 2-aminoethanol for non polar
biomolecules and diethanolamine (negative ions) as the matrix because they dissolve
many polar compounds and have low volatility. When sample-matrix interaction occurs,
alternative matrix materials have to be explored.
Effluents
HPLC
can
from
be
analyzed by FAB
with
the
continuous - flow
FAB
technique
where the eluate
is
continuously
mixed
with
the
matrix and flows
to the FAB target
by passing through a needle where the tip is bombarded in a pulsed or continuous
manner.
MS50 CF-FAB probe design
Matrix-assisted Laser Desorption ionization
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Matrix-assisted laser desorption ionization (MALDI) is currently the most important
technique for rapidly depositing enough energy into involatile analytes in the
condensed phase to facilitate their ionization and desorption in the gas phase in one
continuous
step. MALDI
differs
from
FAB
in
four
respects :
a) The analytes are in a
matrix and co-crystallized
into solid phase rather than
being
dissolved
and
analyzed in a liquid matrix.
b) The analytes are exposed
to an intense laser light
rather than to a beam of energetic atoms or ions.
c) The laser light is applied in pulses of short duration in contrast to exposure to a
continuous beam of energetic atoms or ions.
d) The analyte is ionized by energy transfer from the matrix rather than being "sputtered
or ripped" from a liquid matrix.
In a typical MALDI analysis, a 10 µM solution is made of the analyte in water or other
appropriate solvent, and a 1 µL aliquot ( ~ 10 pmol ) is added to an ~ 10 4-fold excess of a
matrix solution that has already been placed onto the target. The solution is dried and
allowed to crystallize so that it is comprised of microcrystals of a matrix into which the
analytes are embedded. The target is placed, through a vacuum interlock, into the path
of the laser beam in the ion source. An important practical advantage is the possibility
of placing many samples in individual wells on a single target allowing for rapid
analysis of multiple samples. In commercial Maldi sources, the pulsed laser light of <
10 nsec duration is often produced by a nitrogen laser at 337 nm or a Q-switched Nd:
YAG laser ( with tripled or quadrupled fundamental wavelength at 354 or 266 nm). The
laser light is focused onto the solid sample that contains both the matrix and the
analyte. The exposure usually consists of 50 to 200 single shot pulses. Each pulse
leaves a small crater in the target region. As the fluence (irradiance) is increased, a
sharp threshold is reached at which analyte ions suddenly appear, e.g 10 mJ/cm 2 (10 ns
pulse width) for proteins.
Initially the matrix for MALDI was nicotinic or
cinnamic acid or a simple derivative of these compounds. Experimentation revealed
that the matrix material had major effects on the efficiency of ionization. In addition of
being a good absorber of energy at the wavelength of the laser , the matrix has several
distinct roles:
1. absorption of most of the laser light, thus minimizing sample damage by the
laser
irradiation.
2. vaporization when irradiated, thus causing some of the co-crystallized analytes
to
vaporize by transferring energy in a controlled manner (ablation step)
3. participation in the ionization of the vaporized analytes in the gas phase.
4. serving as a co-crystallization agent for the analyte.
5.
reducing
analyte
intermolecular
forces
and
minimizing
the
aggregation
of
the
molecules by being present in a vast excess over the sample.
Matrices such as nicotinic, cinnamic, 4-methoxy cinnamic acid or 3-hydroxypicolinic
acid, are typically low molecular weight organic acids.
In TOF mass spectrometers, the gas phase ions produced in MALDI ion sources are
subjected to controlled extraction and acceleration to a fixed energy by a strong
electrostatic field (25 to 30 KeV). The ion packets thus generated are focused by plates
at high voltages (Einzel lenses) into the field-free region of the flight tube of a TOF
analyzer for subsequent determination of the m/z values of the separated ions. TOF
anlyzers are well suited to handle the ion packets produced by the discontinuous
MALDI process. Ion traps and FTICR analyzers have also been employed.
There at least six advantages of MALDI ionization with TOF analysis:
1. very high mass range, to 1 MDa or higher
2. high sensitivity at low pmoles and often to amole levels
3. suitability for mixture analysis because the technique is not specially subject to
suppression or discrimination effects or to formation of multicharge states.
4. ability to do analysis in minutes (not including sample preparation)
5. little or no analyte fragmentation
6. reduced need for involved sample purification because a reasonable tolerance for
salts and other common biochemical additives, often to mM concentrations.
Background ions from the matrix may be a problem for analytes with MW < 1000 Da
The recent ability to conduct MALDI at atmospheric pressure ionization (AP-MALDI)
involves the pneumatically assisted transfer of ions from the region at atmospheric
pressure to high vacuum by a stream of nitrogen. Sample preparation for AP-MALDI
(including
matrix
selection) is similar to
that with conventional
MALDI. When
with
an
used
orthogonal
acceleration-TOF
analyzer,
advantages
the
of
AP-MALDI include the
possibility of using the
same instrument for
both ESI and MALDI
analyses. A
disadvantage
is
increased sample consumption with respect to vacuum MALDI.
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PHOTOIONIZATION
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Direct Laser Desorption Ionization
In this technique, analyte molecules are desorbed as intact molecules with the aid of
laser beams of 1 to 200 ns duration pulses, focused on 10 to 300 µm o.d. spots at a
variety of incident angles (30° to 75°). Ion sources have been designed with a variety of
lasers, including UV lasers, such as nitrogen (337 nm), excimer (at various wavelengths
in the 193 to 351 nm range) and IR emitting lasers. Instruments providing direct laser
desorption, such as the laser microprobe analyser ( e.g LAMMA),
have been used
to study the composition of thin (0,1 mm) biological sections, prepared in the same way
as for transmission electron microscopy.
Resonance - Enhanced Multiphoton Ionization (REMPI)
In this technique the energy for ionization is obtained by the simultaneous absorption
of two or more UV laser photons. The two-photon energy must exceed the ionization
energy of the analyte. The efficiency of the two-photon absorption , i.e, the yield of
ionization, may be increased by several orders of magnitude if the photon energy (laser
wavelength) is in resonance with the analyte's UV-spectroscopic transition. In addition
to providing high sensitivity, REMPI is also highly selective because only those
compounds can be analyzed that exhibit the required specific UV transition at the laser
wavelength applied. REMPI is a soft ionization technique, hence there is virtually no
fragmentation. Since REMPI is a pulsed type method of ionization, TOF is an ideal
mass analyzer. REMPI is a two-dimensional technique, combining both optical and
mass selectivity. Advantages are high selectivity, sensitivity and speed. Because it
takes only ~ 20 to 40 µs to obtain a spectrum, the method is well suited for
time-resolved analyses of target compounds or specific compound classes in complex
samples. An application is the real-time on-line monitoring of aromatic pollutants in
waste incinerator flue gases.
CHEMICAL IONIZATION (CI)
Chemical ionisation has become very popular for structural elucidation since it was
first developed in the mid-1960s. Here, the concept of ionisation relies on the
interaction of ions with neutral molecules and the further production of new ions. In
general, the amount of fragments ions produced is much less than in electron-impact
ionisation , since little internal energy is imparted on the ionised molecule. Also for this
reason, CI is considered as a soft ionisation technique. A schematic representation of a
chemical-ionisation
ion
source is shown in the next
figure.
If ionization occurs with
methane
at
a
pressure
between 0.1 and 1.0 torr by
electron
impact
(EI),
a
molecular ion is initially
created.
CH4 + e ------- CH4+. + 2e
Since the methane is at a
high pressure, there is a distinct possibility of the molecular ion colliding with another
methane molecule. When this happens the most likely reaction is as follows:
CH4+. + CH4 -----------
CH3. + CH5+
Therefore, if a small amount of the sample to be analyzed is introduced into the ion
source in the vapour phase, it is the species CH5+ that acts as the means of ionization
of the analyte which can react as follows:
CH5+ + M ------------
CH4 +
(MH)+
The quasi-molecular ion (MH)+ , will have an m/z value which is 1 amu greater than the
molecular ion and has a much lower internal energy than the molecular ion produced by
electron-impact ion sources. Fragmentation observed in CI are minimized compared to
fragmentation
in
electron
observed
impact
illustrated
as
for
proline. Modern
mass
spectrometers
have
combined CI and EI ion
sources
that
can
be
readily changed by the
turning of a handle. In CI
the
concentration
of
methane is at least 1000
times higher than the one
of the sample. There is a much higher probability of the sample molecule colliding with
a CH5+ ion than with an electron. In the CI spectra other quasi-molecular than (M+H)+
may be produced resulting from ion-molecule reactions leading to other reactant ions
which are able to react differently with the sample.
CH4 +.
CH3+ + H+
-----------------
The fragment ion, CH3+ , may itself undergo an ion-molecule reaction to form the C2H5+
ion :
CH3+ + CH4
----------------
C2H5+ + H2
The C2H5+ ion can also produce a quasi-molecular ion as can ethylation of the sample
molecules :
C2H5+ + M -----------
(M+H)+ + C2H4 or
M + C2H5+ ---------
(M + C2H5)+
When the proton affinity of M is lower than that of CH 4, the following reactions may take
place, resulting in a quasi-molecular ion of 1 mass unit less than the molecular
ion. This reaction is predominant in the higher alkanes.
CH5+ + M
and
C2H5+ + M
------------------------
(M - H)+ + CH4 + H2
(M - H)+ + C2H6
Other reactant gases that are used in CI are ammonia and isobutane .When ammonia is
used as a reagent gas, analytes that are more basic than NH3 yield abundant (M + H)+
ions by proton exchange while less basic analytes yield (M + NH 4)+ ions by
attachment. In the latter case, the most abundant ion in the spectrum is 18 Da higher
than M. Beneficial results may be obtained by exploring a variety of ion-molecule
reactions through consideration of the functionalities of the analyte and judicious
selection of the reagent gas.
Negative ion CI (NCI)
is, in most instances, a misnomer because it does not involve
an ion-molecule reaction but occurs when an analyte with several electronegative
atoms captures low-energy electrons present in the CI plasma. True negative chemical
ionization involving a chemical reaction is rarely used.
ELECTRON IONIZATION (EI)
Historically, electron ionization (EI, electron impact, electron bombardment) has been
the most commonly used method to ionize relatively small (< 1.2 kDa) molecules which
can be vaporized without thermal decomposition ( ~ 1 mL volume, stainless steel,
maintained at ~ 200 °C to prevent sample condensation)