Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: LASER DESORPTION ION SOURCE
CROSS-REFERENCE TO RELATED APPLICATIONS
REFERENCES CITED
4204117 May, 1980 Aberle et at 250/287
5640010 June, 1997 Twerenbold
5663561 Sept, 1997 Franzen et at
5777324 Jul., 1998 Hillencamp
5917185 Jun., 1999 Yeung et al.
5965884 Oct., 1999 Laiko et at. 250/288
5969350 Oct., 1999 Kerley et at.
5994694 Nov., 1996 Frank et at. 250/281
6040575 Mar., 2000 Whitehouse et al
6140639 Oct., 2000 Gusev et at.
6175112 Jan., 2001 Karger et al.
6444980 Sept., 2002 Kawato et at. 250/288
2002/0175278 Nov., 2002 Whitehouse 250/281
2003/0052268 Mar., 2003 Doroshenko et at. 250/288
2003/0160165 Aug., 2003 Truche et at. 250/288
6504150 Jan., 2003 Verentchlkov 250/286
6707036 Mar., 2004 Makarov 250/
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FIELD OF INVENTION
This invention relates to the generation of gas-phase ions or charged
particles from
condensed phase sample (e.g. liquid or solid) using laser desorption
ionization and related
techniques, primarily for analysis of chemical species with mass spectrometers
or ion
mobility spectrometers.
BACKGROUND OF THE INVENTION
= Laser.desorption and ionization have been utilized to ablate and ionize a
wide variety
of surface samples for analysis with mass spectrometry.. Matrix-assisted laser
desorption/ionization (MALDI) is a desorption and ionization technique that
results in
productin of gas-phase ions from condensed-phase analyte molecules (e.g.
generally large
labilte biomolecules) by unique energy partitioning properties of absorbed
light from lasers
into target sample components. MALDI samples are generally mixtures of matrix
and
analyte, whereby the light energy from the laser is absorbed primarily by the
matrix,
facilitating both ionization and desorption of analyte. The beneficial
characteristic of these
processes is that very little of the energy is partitioned into the internal
energy of the analyte,
resulting in intact gas-phase analyte ions. Gas-phase anayte ions are
generally analyzed
by time-of-flight mass spectrometers; however, any number of gas-phase ion
analyzers
have been considered and employed for MALDI analysis.
The technique of MALDI developed primarily from research by Karas and
Hillenkamp
(1) in the late 1980. Vacuum MALDI has developed into a widely used commercial
technology for analysis of proteins and other macromolecules.
The present invention relates to the application of MALDI to desorption and
ionization in vacuum and at intermediate and higher pressures, including
atmospheric
pressure. Franzen and Koster (US 5,663,561) first described atmospheric
pressure MALDI
in reference to their atmospheric pressure desorptiOntionization technique by
stating, in
contrast to MALDI, at atmospheric pressure, the related molecules of the
decomposed
matrix material are not needed to ionize the macromolecules. The selection of
matrix
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molecules is solely dependent upon their ability to release the large
molecules." Albeit, not
explicitly claimed in this patent, the concept of atmospheric pressure MALDI
(or AP-MALDI)
was clearly first described by Franzen and Koster. Ironically, the Franzen and
Koster
patent begins by arguing that AP-MALDI Is inefficient and that augmenting
ionization
efficiency with gas phase ion-molecule reactions or desorbed neutral species
with gas
phase reagent ions at atmospheric pressure would offset some of the
transmission losses
that would occur by inefficient transport from atmospheric pressure.
Laiko and Burlingame (US 5,965,884) distinguish their AP-MALDI from Franzen
and
Koster by arguing simplicity and non-destructive matrices. This patent
dismisses the key
arguments made by Franzen and Koster that AP-MALDI is inefficient. The Laiko
patent
teaches AP-MALDI with the requirement of close coupling of a sample target to
the
conductance aperture into vacuum. The lack of efficient atmospheric pressure
optics with
this device requires precise alignment and positioning of sample and the laser
beam
relative to the vacuum inlet. In addition, Laiko provides for a sweep gas to
assist in
transport of the ions from the target surface to the vacuum inlet. The
transmission of this
device is low. The lack of time-sequenced optics with the laser pulse limit
ion extraction
and transmission efficiency.
Sheehan and Willoughby (US 6,744,041 B2) describe separation of the ionization
process [and sample target posision] from the conductance aperture using
atmospheric
pressure optics. They describe efficient atmospheric pressure transport and
compression
optics that allow relative independence of sample location from the position
of the vacuum
inlet. Components of this invention are included by reference into the present
invention.
Sheehan and Willoughby (US Patent No. 6,818,889) describe further improvement
of
transmission of MALDI generated ions at atmospheric pressure by laminating
high
transmission elements and incorporating a "back-well" geometry whereby MALDI
samples
can be placed facing away from the conductance aperture. This geometry
facilitates easier
access of the laser beam to the sample targets compared to close-coupled
designs. The
back-well geometry also provides a simplification of sample insertion and
easier access to
the ionization chamber. Components of this invention are also included by
reference into
the present invention.
Willoughby and Sheehan (US Patent No. 6,943,347) also describe improvements in
transmission of ions from atmospheric pressure sources [including AP-MALDI].
These
improvements are accomplished by precisely controlling the electric field
through the entire
conductance pathway from atmospheric pressure into vacuum. Components of this
invention are included by reference into the present invention. Willoughby and
Sheehan
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(US Patent No 6,914,243) also teach that conductance arrays and patterned
optics can further
enhance the transmission of ions from atmospheric pressure sources and improve
the
transmission of MALDI Ions from either intermediate of higher-pressure
sources.
Components of this invention are included by reference into the present
invention.
Whitehouse (US 20020175278) describes the use of a variety of RF multipole
devices and DC funnel devices to focus and entrain the flow of ions from
atmospheric and
intermediate pressure MALDI targets to detection. Components of this invention
are
included by reference into the present invention.
Truche et al. (US 6,707,039 B1) describe a wide variety of alternatives for
close-
coupling the sample target to the conductance aperture. This technology places
high
tolerance on sample position and laser position. In addition, it is envisioned
that mirrored
reflective surfaces close to the plume of the MALDI target would tend to
become
contaminated and degraded in their optical performance. In addition, the
sampling of ions
from an electric field between the target and aperture into the field-free
region of the
vacuum inlet tube would cause rim losses from field penetration and degrade
the transport
efficiency. The lack of time-sequenced optics with the laser pulse limit ion
extraction and
transmission efficiency.
Makarov and Bondarenko (US 6,707,036 02) teach of a positionally optimized
sample target device with a close-coupled conductance opening for atmospheric
pressure
and intermediate pressure MALDI. This device is still subordinate to alignment
of laser,
target, and lacks spatial or temporal optics to facilitate efficient ion
transmission to the mass
analyzer. The lack of time-sequenced optics with the laser pulse limit ion
extraction and
transmission efficiency.
1. Karas, M.; Hillenkamp, F., Anal. Chem. 1988, 60, 2299-2301.
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SUMMARY OF THE INVENTION
Dispersive sources of ions at or near atmospheric pressure; such as,
atmospheric pressure discharge ionization, chemical ionization,
photoionization, or
matrix assisted laser desorption ionization, and electrospray ionization
generally have
low sampling efficiency through conductance or transmission apertures, where
less
than 1 A [often less than 1 ion in 10,000] of the ion current emanating from
the ion
source make it into the lower pressure regions of the present commercial
interfaces
for mass spectrometry.
According to an aspect of the present invention, there is provided an
apparatus for generating gas phase ions from a sample comprising: (a) a sample
held by a sample holder; (b) an ion source for generating gas phase reagent
ions; (c)
ion optics comprising electrodes with voltages applied to direct said gas
phase
reagent ions onto said sample; (d) means for changing said voltages applied to
said
electrodes; (e) a pulsed light source for generating light pulses directed at
said
sample to produce gas phase sample related ions; and (f) means for
synchronizing
electric field changes with said pulse of said pulsed light source.
According to an aspect of the present invention, there is provided an
apparatus for analyzing chemical species comprising: (a) a sample held by a
sample
holder; (b) an ion source for generating gas phase reagent ions; (c) ion
optics
comprising electrodes with voltages applied to direct said gas phase reagent
ions
onto said sample; (d) means for changing said voltages applied to said
electrodes;
(e) a pulsed light source for generating light pulses directed at said sample
to
produce gas phase sample related ions; (f) means for synchronizing said
changing of
said voltages with said light pulses from said pulsed light source: (g) a mass
to
charge analyzer and detector; and (h) means to direct said sample related ions
to
said mass to charge analyzer and detector for mass to charge analysis.
According to an aspect of the present invention, there is provided an
apparatus for analyzing chemical species comprising: (a) a sample held by a
sample
holder; (b) an ion source for generating gas phase reagent ions; (c) ion
optics
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comprising electrodes with voltages applied to direct said gas phase reagents
ions
onto said sample; (d) a pulsed light source for generating light pulses
directed at said
sample to produce gas phase sample related ions; (e) means for changing said
voltages applied to said electrodes a first time to direct said sample related
ion away
from said sample holder; (f) a mass to charge analyzer and detector; (g) means
to
direct said sample related ions to said mass to charge analyzer and detector;
(h)
means for changing said voltages applied to said electrodes a second time to
increase transfer efficiency of said sample related ions into said mass to
charge
analyzer for mass to charge analysis; and (i) means for time synchronizing
said
changing of said voltages applied to said electrodes a first and a second time
and
said light pulses from said pulsed light source.
According to an aspect of the present invention, there is provided a
method for generating gas phase ions from a sample comprising: (a)
accumulating
charge on said sample by directing gas phase reagent ions generated from a
reagent
ion source to said sample using an electric field; (b) directing a pulse of
light at said
sample to produce gas phase sample related ions; and (c) changing said
electric field
synchronized with said pulse of light to direct gas phase ions away from said
sample
holder.
According to an aspect of the present invention, there is provided a
method for analyzing chemical species comprising: (a) accumulating charge on a
sample held by a sample holder by directing gas phase reagent ions generated
from
an ion source to said sample using an electric field; (b) directing a pulse of
light at
said sample to produce gas phase sample related ions; (c) changing said
electric
field synchronized with said pulse of light to direct said sample related ions
away from
said sample holder; (d) directing said sample related ions to a mass to charge
analyzer with detector; and (e) conducting mass to charge analysis of said
sample
related ions.
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According to another aspect of the present invention, there is provided
a method for analyzing chemical species comprising: (a) accumulating charge on
a
sample held by a sample holder by directing gas phase reagent ions generated
from
a reagent ion source to said sample using an electric field; (b) directing a
pulse of
light at said sample to produce gas phase sample related ions; (c) changing
said
electric field a first time synchronized with said pulse of light to direct
said sample
related ions away from said sample holder; (d) changing said electric field a
second
time synchronized with said pulse of light to direct said sample related Ions
into a
mass to charge analyzer with detector (e) directing said sample related ions
to a
mass to charge analyzer and detector; and (f) conducting mass to charge
analysis of
said sample related ions.
According to another aspect of the present invention, there is provided
a method for generating gas phase ions from a sample comprising: (a)
accumulating
charge on said sample held by a sample holder by directing reagent ions
generated
from a reagent ion source to said sample using an electric field; (b)
directing a pulse
of light at said sample to produce gas phase sample related ions and neutral
species;
(c) changing said electric field synchronized with said pulse of light to
direct gas
phase ions and reagent ions away from said sample holder; and (d) reacting
said
reagent ions with said sample related neutral species to generate additional
gas
phase sample related ions.
In accordance with some embodiments of the present invention,
associated methods of sample charging, laser desorption and sample ionization
are
intended to improve the collection efficiency and ionization efficiency of
atmospheric
pressure, intermediate pressure and vacuum laser desorption ionization.
Two advantages of some embodiments of the current device should be
emphasized. First, precisely timing the sequence of laser pulse with ion
extraction
under high voltage followed by reduction of the electric field in the
extraction and
focusing region before losing ions to surfaces. The field in the extraction
and
focusing region is reduced so that the ions are efficiently focused and
transmitted
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through a conductance aperture into a lower pressure region on the path to a
mass
analyzer. The second important advantage is the ability to populate the sample
surface with ions of the sample polarity as the analyte ions to be extracted.
This
condition drives the equilibrium toward product with an excess of reagent ions
compared to conventional MALDI and increases the efficiency of ionization of
analyte. One aspect of the current invention is to precharge a sample prior to
laser
desorption to enhance the yield of ions from a given sample.
Another object of this patent is to incorporate precision precharging of a
sample to predetermined spots on a sample (e.g. biopsy of suspected cancer
tissue)
in order to facilitate enhance yield of ions from a given spot. Optical
imaging can be
used to determine the precise position of sample precharging and laser pulse
impingement (e.g. dye markers or fluorescent tags visualized by microscopes
with
video recording).
Some embodiments may use specialized target surfaces with shaped
needles or electrodes behind the sample in order to control the electric field
experienced by the sample during and after laser pulse. By varying voltage in
space
and time, optimum sample precharging, ion generation and extraction of ions
can be
achieved.
The damping of motion of ions at atmospheric pressure make transport
in electric fields much slower compared to ion motion in intermediate pressure
or
vacuum. In addition, the inertial components of motion are substantially
damped at
higher pressures (above 1 Torr) and the slower ion motion is controlled by
moving
ions in the direction of optimized local electric fields. Still further
objects and
advantages will become apparent from a consideration of the ensuing
description and
drawings.
In accordance with some embodiments, atmospheric pressure,
intermediate pressure and vacuum laser desorption ion sources comprise
ionization
chambers and transmission devices encompassing targets for holding samples,
lasers to illuminate said targets resulting in desorption and ionization of
the samples,
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time-sequenced electrostatic potentials to foster efficient extraction,
focusing, and
selecting of resulting gas-phase ions. Laser desorption ion sources in
accordance
with the invention also comprise a means to accumulate charge on a sample
prior to
laser desorption of the sample and a means to conduct gas phase ionization of
laser
desorbed neutral sample molecules to increase the ionization efficiency of a
sample
during and after a desorption laser pulse.
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BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a diagram of an atmospheric pressure Laser Desorption Ionization
source,
incorporating surface charging, interfaced to a mass spectrometer.
FIG. 2A is a diagram of the atmospheric pressure Laser Desorption Ionization
source
shown in Figure 1 during the operating step of charge accumulation on the
sample surface.
FIG. 2B is a diagram of the atmospheric pressure Laser Desorption Ionization
source
shown in Figure 1 during the operating step of laser firing and charge release
from the
sample.
FIG. 2C is a diagram of the atmospheric pressure Laser Desorption Ionization
source shown in Figure 1 during the operating step of focusing the ion
population produced
into the orifice to vacuum.
FIG. 2D is a diagram of the atmospheric pressure Laser Desorption Ionization
source shown in Figure with the view turned 90 degrees showing a imaging
apparatus with
magnification.
FIG. 3 is a diagram of one embodiment of the electric fields applied during
surface
charging and ion release and focusing operation in the atmospheric pressure
Laser
Desorption Ionization source shown in Figure 1.
FIG. 4A is a timing diagram of one operating sequence embodiment used in the
atmospheric pressure Laser Desorption Ionization source shown in Figure 1.
FIG. 4B is a timing diagram of a second operating sequence embodiment used in
the
atmospheric pressure Laser Desorption Ionization source shown in Figure 1.
FIG. 5 is a diagram of an atmospheric pressure Laser Desorption Ionization
source,
incorporating surface charging, with the target surface configured in
proximity to the orifice
into vacuum.
FIG. 6 is a timing diagram of the of one operating sequence embodiment used in
the
atmospheric pressure Laser Desorption Ionization source shown in Figure 5.
FIG. 7 is diagram of an intermediate pressure Laser Desorption Ionization
source,
incorporating surface charging; interfaced to a mass spectrometer.
FIG. 8A is a diagram of the one embodiment of a Laser Desorption target
surface
configured with an insulated charging electrode.
FIG. 8B is a diagram of an alternative embodiment of a Laser Desorption target
surface configured with an insulated and shielded charging electrode.
FIG. 8C is a diagram of an alternative embodiment of a Laser Desorption target
surface configured with an array of insulated and shielded charging
electrodes.
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FIG. 9A is a diagram of one embodiment of a Laser Desorption target surface.
FIG. 9B is a diagram of an alternative embodiment of a Laser Desorption target
surface comprising an array of charging electrodes with integral fiber optics
for applying a
laser pulse to the back side of the sample.
FIG. 9C is a diagram of a renewable liquid Laser Desorption target surface
with
liquid sample delivered to the target surface through a liquid flow channel.
FIG. 9D is a diagram of a renewable liquid Laser Desorption target surface
with
integral fiber optics for applying a laser pulse to the back side of the
sample.
FIG. 10A is a diagram of an atmospheric Laser Desorption Ionization source
comprising surface charging and a annular ion focusing lens embodiment
interfaced to a
mass spectrometer during the operating step of surface charging.
FIG 10B is a diagram of the Laser Desorption Ionization source shown in Figure
10A
during the operating step of laser firing and charge release from the sample
surface.
FIG 10C is a diagram of the Laser Desorption Ionization source shown in Figure
10A
during the operating step of focusing the ion population produced into the
orifice to vacuum.
FIG 11 is a diagram of an atmospheric Laser Desorption Ionization source
comprising surface charging, a reversing annular ion focusing lens and surface
imaging.
FIG 12A is a diagram of a vacuum Laser Desorption Ionization source configured
with surface charging and a near surface potential trap configured in the
pulsing region of a
Time-Of-Flight mass spectrometer during surface charging operation.
FIG 12B is a diagram of the vacuum Laser Desorption Ionization source shown in
Figure 12A during the operating step of laser firing and charge release from
the sample
surface.
FIG 12C is a diagram of the vacuum Laser Desorption Ionization source shown in
Figure 12A during the operating step of trapping the ion population produced
on the
dynamic field trapping surface.
FIG 12D is a diagram of the vacuum Laser Desorption Ionization source shown in
Figure 12A during the operating step of pulsing the ion population produced
into the Time-
OF-Flight mass spectrometer flight tube.
DETAILED DESCRIPTION OF EMBODIMENTS
A preferred embodiment of the invention comprising an atmospheric pressure
Laser
Desorption Ionization source with sample surface charging is diagrammed in
Figure 1.
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Operating details for Laser Desorption Ionization source 1 are diagrammed in
Figures 2A
through 2D. Laser Desorption Ionization (LDI) source 1 interfaced to vacuum
system 2
comprising ion transfer optics and mass to charge analyzer with detector 3,
produces ions
from sample 4 on target plate 5. A portion of the laser desorption ion
population produced
is focused into bore 10 of capillary 11. Ions exit capillary bore 10 at
capillary exit end 12
into vacuum and are accelerated in a free jet expansion of neutral background
gas flowing
through capillary bore 10 from atmospheric pressure ion source 1. Capillary 11
may
comprise a dielectric capillary with condUCtive electrodes on the entrance and
exit faces, a
heated electrically conductive capillary, a nozzle, an orifice or an array of
orifices into
vacuum. Ions pass through skimmer 13 orifice 14 and into ion guide 15 where
their
translational energies are damped through collisions with background gas. Ions
exiting ion
guide 15 pass through exit lens 17 and are mass to charge analyzed in mass to
charge
analyzer and detector 3. Ion guide 15 may comprise a multipole ion guide, a
segmented
multipole ion guide, a sequential disk RF ion guide, an ion funnel or other
ion guides known
in the art. Ion guide 15 may extend continuously into one or more vacuum
pumping stages
or may begin and end in one vacuum stage. Mass analyzer and detector 3 may
comprise a
quadrupole, triple quadrupole, three dimensional ion trap, linear ion trap,
Time-Of-Flight
(TOE), magnetic sector, Fourier Transform Ion-Cyclotron Resonance (FTICR),
Orbitrap or
other mass to charge analyzer known in the art. Vacuum system 2 comprises
vacuum
stages 18, 19 and 20. Alternatively, embodiments of the invention may comprise
vacuum
systems with more or less vacuum stages depending on the requirements of the
vacuum
ion optics and mass to charge analyzer. Atmospheric pressure ion source 1
produces ions
from a sample deposited on or part of a surface. As will be described below,
the sample
may comprise a solid or liquid.
Sample 4 on target plate 5 is positioned in target plate chamber 22. Gas or
gas containing
ions 23 enters target surface chamber 22 through target gas controller 24.
Target gas
controller 24 comprises a gas heater and an ion source to generate reagent
ions from a gas
and/or liquid input 25. Target gas controller 25 may comprise a pneumatic
nebulization
charge droplet sprayer followed by a vaporizer producing a heated carrier gas
containing
reagent ions formed from the evaporating charged droplets. Alternatively,
target gas
controller 25 may comprise a photoionization source, a glow discharge ionizer,
a corona
discharge ionizer configured in an atmospheric pressure chemical ionization
(APCI) source
or other type of gas or liquid sample ion source. Depending on the composition
of sample 4
and the specific analysis requirements, target gas controller 24 can be
configured and
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operated to deliver unheated neutral gas, heated neutral gas or an ion and gas
mixture into
target plate chamber 22 during laser desorption ion source operation. Reagent
ion
containing gas flow 23 passes between target plate 5 and target plate counter
electrode 28
exiting target plate chamber 22 at opening 27 in target plate counter
electrode lens 28.
Electrode 28 is electrically insulated from target plate chamber 22 by
insulators 29. As will
be described below, reagent ions entrained gas flow 23 may be selectively
deposited on
sample 4, directed through opening 27 or discharged on target lens 28 during
laser
desorption ion source operation.
Target plate 5 can be moved manually or by software control in the x and y
directions using
x-y translator 26. Charging electrode assembly 8 remains fixed in position
while target
plate 5 slides over it. A more detailed diagram of charging electrode assembly
8 is shown
in Figures 2A through 2D and 8B. Charging electrode assembly 8 comprises
charging
electrode 30 and shielding electrode 32 forming an electrically conductive
cylinder around
charging electrode 30. Charging electrode 30 and shielding electrode 32 are
embedded in
dielectric block 31 to allow the application of high voltage to charging
electrode 30 without
the onset of gas phase corona discharge or arcing. Voltages are applied to
electrodes 30
and 32 through power supplies 34 and 35 respectively. Laser 7 is configured to
deliver
laser pulse 40 through lens or window 38 and reflected off mirror 39 to
impinge on sample 4
as shown in Figure 2B. Countercurrent gas 45 passes through gas heater 42 and
exits
through opening 43 of endplate electrode 44 forming countercurrent gas flow 41
in LD1
source 1. Gas 53 and desorbed ions pass through opening 52 in electrode 47 and
capillary
entrance electrode 48 into capillary 10 bore 11. Voltages applied to
electrodes 28, 44, 47
and 48 through power supplies 56, 49, 50 and 51 respectively are set to
maximize focusing
and ion transmission into capillary bore 11 as will be described below.
Charged droplet
sprayer 58 comprises liquid inlet 59, nebulization gas inlet 60, sprayer tip
61 and ring
electrode 63 as shown in Figure 2A. Voltages are applied to charged droplet
sprayer 58
and ring electrode 63 through power supplies 65 and 64 respectively. In the
preferred
embodiment shown, charged droplet sprayer 58 is configured to produce a spray
of
charged droplets oriented orthogonal to ion source centerline 68. Charged
droplets are
produced through conventional Electrospray or pneumatic nebulization in the
presence of
an electric field. Heated countercurrent drying gas 41 and target plate gas 74
aid in
evaporating the charged droplets in spray 62. In a non laser desorption
operating mode,
voltages applied to electrodes 30, 28 44, 47 and 48 are set to direct ions
generated from
evaporating the charged droplets in spray 62 into capillary bore 11. In this
Electrospray
CA 02527886 2005-12-01
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operating mode, ions produced from sample bearing solution 59 are directed
into vacuum
and mass to charge analyzed. Ion source 1 can be operated in Electrospray or
atmospheric pressure Laser Desorption ionization mode individually or both
ionization
modes can be run simultaneously. Rapid switching between Electrospray and
Laser
Desorption ionization can by achieved using the ion source embodiment shown in
Figure 1.
In an alternative embodiment of the invention, charged droplet sprayer 58 is
replaced by an
Atmospheric Pressure Chemical Ionization source comprising a pneumatic
nebulizer,
vaporizer and corona discharge needle. Alternatively, a glow discharge,
photoionization or
other type of ion source can be configured to produce ion species in region 73
between
electrodes 28 and 44. Alternatively LDI source 1 can be configured with
multiple ion
generation sources delivering ions individually or simultaneously into region
73.
In laser desorption operating mode, the voltages applied to electrodes 30, 28,
44, 47, 48,
63 and charged droplet sprayer 58 are set to direct ions 75 generated from
charged droplet
sprayer 58 to accumulate on the surface sample 4 on target plate 5 prior to
desorbing
sample 4 by laser pulse 40. Ion or charged species 75 generated from charged
droplet
sprayer 58 and ion species 71 entrained in target plate gas flow 23 are
directed to the
surface of sample 4 prior to desorbing sample 4 with laser pulse 40 as shown
in Figure 2A.
The accumulation and subsequent laser desorption of positive polarity ions is
illustrated in
Figures 2A through 2C but the same sequence of steps can be applied for
negative ion
accumulation and laser desorption with the reversal of voltage polarities
applied to
electrodes. In Figure 2A, appropriate voltages are applied to charged droplet
sprayer 58,
ring electrode 63 and electrodes 28 and 44 to produce positive polarity
charged droplet
spray 62. For illustration purposes, the potentials applied to charged droplet
sprayer tip 61,
ring electrode 63, electrode 28 and electrode 44 may be set to +4KV, +OV, -1KV
and + 1KV
respectively. The voltage applied to electrically insulated charging electrode
30 through
power supply 34 may by set to ¨ 10 to ¨ 20 KV with the shielding electrode
voltage set
close to -1KV through power supply 35. The electric field formed at the sharp
tip of
charging electrode 30 penetrates dielectric target plate 5 and extends through
opening 27
of electrode 28 into region 73 between electrodes 28 and 44 as shown in Figure
2A.
Heated target gas 74 aids in drying charged droplets produced by charged
droplet sprayer
58. Ions 75 generated from evaporating droplets produced from charged droplet
spray 62
follow electric field lines 72 and are directed to the surface of sample 4 on
dielectric target
plate 5. Either concurrently or alternatively, charged species 71 entrained in
target plate
gas flow 23 pass between target plate 5 and electrode 28 and are attracted to
the surface
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of sample 4 by the same attractive electric field formed by the electrical
potential applied to
charging electrode 30.
Charge 70 accumulates on the surface of sample 4 until the space charge limit
is reached.
When the space charge limit is reached additional positive polarity ions
turned away from
the surface of sample 4 and neutralized on electrode 28. Image charge 73, in
this case
electrons, are drawn to the tip of charging electrode 73 as positive ions
accumulate on the
surface of sample 4. Charging electrode 30 and sample 4 form a capacitor with
a charge
capacity in part determined by the electric field strength maintained between
the surface of
sample 4 and the tip of charging electrode 30. The tip sharpness of insulated
charging
electrode 30, the proximity of this tip to the surface of sample 4, the
voltage applied to
charging electrode 30 relative to the voltage applied to electrodes 28 and 44
and the
dielectric constant of target plate 5 and insulation 31 will effect the
electric field strength at
the surface of sample 4. Charge may accumulate on the surface of sample 4
until the
electric field is locally reduced and ultimately neutralized preventing
additional ions of the
same polarity from further accumulating on the surface of sample 4. Minimum
charge
migration or neutralization occurs on the surface of dielectric target plate
5. A single ion
species or a mixture of ion species can be accumulated on surface 4 depending
on the
requirements of an analytical application. For example, if sample 4 comprises
a mixture of
proteins with a matrix such as Sinapinic acid typically used in Matrix
Assisted Laser
Desorption Ionization (MALDI), protons may be an optimal choice of charged
species to
accumulate on the surface of sample 4. Protons can be directed to the surface
as
protonated water or protonated methanol ions generated from charged droplet
sprayer 58
or a charged droplet sprayer or APCI ion generator configured in target gas
controller 24.
Proteins form ions generally as protonated species so the protons accumulated
on the
surface of sample 4 will supply a source of protons to increase ionization
efficiency during
laser desorption of sample 4. Alternatively, metal ions such as sodium can be
accumulated
on the surface of sample 4 if carbohydrate analysis is required to enhance
ionization
efficiency. If sample 4 comprises a liquid such as water or a low volatility
surface such as
glycerol, accumulating ions can react with or attach to sample species in
solution prior to
laser desorption. Infrared lasers can be used to desorb aqueous sample
solutions at
atmospheric pressure. Sample 4 may include no matrix and laser desorption may
occur
directly from the sample as is used with Direct Ionization Off Surfaces (DIOS)
techniques.
Accumulating charged charged species may be in direct contact with sample
molecules
when no matrix is used on target plate 5. This direct charge species and
sample species
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association can improve ionization efficiency for select sample types when
compared with
charge accumulation in the case where the sample is associated with a matrix.
Different
ion species may be supplied by charged droplet sprayer 58 and target' gas
controller 24.
Ions species may be generated from charged droplet sprayer 58 and target gas
controller
24 simultaneously or individually. Charged species production by either device
may be
rapidly switched off or on, if required during laser desorption ionization
operation. Charged
droplet sprayer 58 can be rapidly turned off and on by adjusting the relative
potentials
applied sprayer tip 61 and ring electrode 63.
When sufficient positive charge has accumulated on thezsurface of sample 4,
laser pulse 40
is applied to the surface of sample 4 from laser 7 to desorb sample from
target plate 5. The
voltage applied to charging electrode 30 is rapidly reversed just prior to,
during or just after
laser pulse 40 to release the charge from the surface of sample 4. This
effectively reverses
the potential across the capacitor formed by the charge accumulated on the
surface of
sample 4 and the image charge accumulated ,near the tip of charging electrode
30. The
laser pulse step is illustrated in Figure 2B where the attracting electric
field 72 is gone and
electric field 77 attracts ions desorbed from sample 4 toward entrance orifice
78 of capillary
10. Figure 3 is a diagram of one set of electrical potentials that may be
applied during the
ion accumulation and ion desorption steps. Curve 80 shows one example of the
relative
potentials applied to Electrodes 30, 28 44, 47 and 48 during accumulation of
positive
charge on the surface of sample 4. Curve 82 represents the relative but off
axis electrical
potentials applied to charged droplet sprayer tip 61 and ring electrode 63
during production
of positive polarity charged droplets from sprayer tip 61 and accumulation of
positive
polarity ions on the surface of sample 4. Curve 81 shows the reversal of
voltage polarity
applied to charging electrode 30 and 28 to facilitate desorption and
ionization of sample
components from sample 4 when laser pulse 40 is applied. The voltage applied
to ring
electrode 63 as shown by curve 83 is set to minimize distortion of the
centerline focusing
electric field directing desorbed ions into capillary entrance 78. Charged
droplet sprayer
nebulizing gas flow is switched off during the laser desorption and ion
focusing steps.
When charged droplet sprayer 58 is operated in non nebulizing Electrospray
mode, the
charged droplet spray turns off when the voltages on ring electrode 83 are set
approximately equal to the voltage applied to sprayer tip 61 as shown in curve
83 or Figure
3. The timing diagram of the voltage transitions illustrated in Figure 3 is
shown in Figure 4A.
The surface charging time period is followed by laser pulse 85 and a rapid
change in
voltage 86 applied to charging electrolle 30. The voltage changes applied to
Electrodes 30,
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28 and 63 are maintained during the ion focusing period to allow time for
desorbed ions
from sample 4 time to reach capillary entrance 78 where they are swept through
capillary
bore 11 into vacuum by gas flow 53. In the example described, voltages applied
to
Electrodes 44, 47 and 48 remain constant during the sample charging, ion
desorption and
ion focusing steps illustrated in Figures 2A, 2B and 2C.
When positive reagent ions are generated from target gas controller 24,
relative voltages
can be set between electrodes 30 and 28 to allow these reagent ions to pass
through
opening 27 in electrode 28 and mix with neutral molecules 75 and ions 88
desorbed from
sample 4. Through exchange or attachment of charge from the reagent ions to
desorbed
neutral species, the ionization efficiency of the desorption process is
improved increasing
mass to charge analysis sensitivity. As diagrammed in Figures 2B and 2C,
reagent ions 90
mix with desorbed neutral species when the appropriate voltages are applied to
electrodes
30 and 28 to direct reagent ions 71 through opening 27 and along centerline 68
moving as
gas phase ions 90 toward capillary entrance 78. Before countercurrent gas flow
41 sweeps
desorbed neutrals away from opening 43 in endplate electrode 44, reagent ions
90 have a
the opportunity to collide with and exchange charge or attach to a neutral
desorbed sample
molecule. Target plate gas flow 74 meeting countercurrent gas flow 41 in
region 73 form a
stagnation and mixing area in region 71 that promotes charge exchange or
attachment
between reagent ions 90 and desorbed neutral species 75. Once a neutral sample
molecule has been ionized in the gas phase, focusing fields 77 direct the ions
towards
capillary entrance 78. Reagent ions species may also be selected to promote
desired gas
phase reactions with desorbed analyte sample molecules. Reagent ion flow
through
opening 27 in electrode 28 can be stopped during the ion focusing step by
applying the
appropriate relative voltages between electrodes 30 and 28 to direct reagent
ions to
neutralize on electrode 28 before entering opening 27.
An alternative sequence of surface charging step 92, sample desorption,
extraction and ion
focusing step 93 and gas focusing step 94 is shown in timing diagram 4B. The
charging
and desorption steps illustrated by Figures 2A and 2B are similar to the two
step sequence
of Figure 4A as shown in the timing diagram shown in Figure 4B. However, as
the
desorbed ions 88 approach capillary entrance orifice 78, the potentials
applied to electrodes
30, 28, 44, 47 and 48 are set approximately equal, as shown in step 94 of
timing diagram
4B, to allow gas dynamics forces to dominate ion motion, sweeping ions into
and through
capillary bore 11. The application of steep electric fields near capillary
entrance 78 serve to
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focus ions toward the centerline but can also drive ions into the edge of
capillary entrance
electrode 48 where they are neutralized. Reducing the electric field just
before the ions
reach capillary entrance orifice 78 allows initial ion focusing as desorbed
ions traverse from
sample 4 to capillary entrance orifice 78 but reduces the amount of ion
impingement
occurring on capillary entrance electrode 78 as the ions enter capillary bore
11. This
additional gas dynamic ion focusing step improves ion transport efficiency
into vacuum
increasing sensitivity in mass to charge analysis. The timing of the voltage
switch to the
gas focusing step can be optimized for any set of focusing voltages applied by
using a
calibration procedure in which the duration of ion desorption, extraction and
focusing step
93 is varied to find the maximum mass spectrometer signal response. The
diagrams of
timing sequences and steps shown in Figures 2A through C, Figure 3 and Figures
4A and
4B are given to illustrate examples of operating sequences, however other
switching
patterns or variations on switching patterns can be employed to optimize
performance for
different applications. Voltages can be applied to maximize ionization and
sampling
efficiency of negative ions. Variations of step sequences and additional steps
may be
added to sequences to maximize performance and to optimize for differences in
samples,
applications, and ion source lens geometries, gas composition, temperature and
flow rates.
For example multiple laser shots can be conducted on the same spot or on
different spots
while the voltage applied to charging electrode 30 is transitioned from charge
accumulation
to charge rejection potentials. Laser beam 40 spot can be moved or target
plate 5 can be
moved between each laser shot in a series.
An alternative embodiment, or addition to the embodiment of the invention, is
diagrammed
in Figure 2D. Figure 2D is a diagram of laser desorption ion source 1 viewed
from an angle
rotated 90 degrees to the view shown in Figures 2A through 2C. Charged droplet
sprayer
58 is with sprayer tip 61 is pointing orthogonal to the viewing plane.
Configured 90 degrees
rotated from charged droplet sprayer' 58 and Laser 7 is optical imaging device
95 with
image magnifiers 96 and mirror 97. Imaging device 95 may comprise a video
camera for
digital imaging or a microscope for manual viewing of the sample surface.
Imaging device
95 is used to provide and image sample surface 4 allowing optimization of the
target plate 5
position relative to the tip of charging electrode 30 and laser pulse 40.
Positioning the tip of
charging electrode 30 under a sample feature will maximize charge accumulation
at that
location. Laser desoption ionization efficiency can be improved with sample
mixed in
MALDI matrices when a laser pulse is applied to a MALDI crystal located using
optical
imaging with feedback to the target plate x-y translator stage 26. Less ion
yield results
CA 02527886 2005-12-01
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when a laser pulse impinges on a MALDI matrix in a location where no matrix
crystals are
present. Imaging device 95 can be used to located the position of MALDI matrix
crystals in
sample 4. Based on the image information and sample coordinates provided,
target plate 5
is moved to line up the tip of charging electrode 30 and laser pulse 40 with
the MALDI
matrix crystal position in sample 4. The position of laser beam 40 hitting
sample 4 can be
adjusted independent of target plate 5 movement or the location of the tip of
charging
electrode 30. Mirror 39 can be configured with a fine resolution movement
device such as
a galvanometer to allow rapid steering of laser beam 40 impinging on sample 4.
Alternatively, the position of charging electrode 30 can be positioned using a
separate x-y
translator stage to provide movement of charging electrode 30 independent of
target plate 5
x-y movement. Additional illuminating devices such as lower power lasers can
be
incorporated into imaging device 95 to enhance the image from florescent dyes
used to
stain sample 4. For example, if sample 4 is a tissue slice and laser
desorption source 1 is
used to conduct molecular imaging of stained tissue samples, individual cells
can be
optically imaged using imaging device 95 to allow laser charge accumulation on
and laser
desorption from selected cells in tissue sample 4. Laser beam 40 can be
focused down to
small spot dimensions and target plate 5 can be fabricated as a very thin
dielectric sheet
allowing the insulated sharp tip of charging electrode 30 to rest just under
but very close to
an imaged and selected cell. Laser desorption ionization from individual cells
or from a
small group of cells in a tissue can be performed with an appropriately
focused laser spot
and a small local charge accumulation area. Imaging device 95 can also be used
determine when a sample has been depleted or damaged after several laser
shots.
Target plate 5 and charging electrode 30 may be configured in alternative
embodiments.
Target plate 5 may be configured as a moving dielectric belt. The eluant from
a liquid
chromatography (LC) run can be deposited on the moving belt as a continuous
track or
spots with a MALDI matrix added on line. A second track of calibration sample
can be
added along side the LC sample track. Two charging electrodes can be
positioned under
each track or spot train to provide simultaneous charging of both LC and
calibration
samples. Laser beam 40 can be rastered across both tracks or spots during the
desorption
step to generate ions from both the LC and calibration samples as the
dielectric belt target
moves past opening 27 of electrode 28. The charging and laser desorption steps
can occur
rapidly with multiple step cycles conducted per second to maximize sample
throughput.
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An alternative embodiment of the invention is diagrammed in Figure 5 where
electrodes 44
and 47 are removed and target plate 100 is positioned closer to the capillary
bore entrance
102. Charged droplet sprayer 105 produces charged droplet spray 108 as
described in
Figure 2A above. Evaporating charged droplets generate ions that can be
directed to
accumulate on the surface of sample 101 to enhance the ionization efficiency
of laser
desorption or directed toward capillary bore entrance 102 when conducting
Electrospray or
pneumatic nebulization ionization of a sample substance. Alternatively,
charged droplet
sprayer 105 may be configured as an APCI, a photoionization, glow discharge,
corona
discharge or other ionization source to generate of charged species for charge
accumulation on sample 101 prior to laser pulse 108. Multiple alternative
ionization probes
can be configured in one ion source with laser desorption producing ions in
region 113 of
ion source 114 shown in Figure 5 or region 73 of ion source 1 shown in Figure
1 and 2A
through 2D. Different ionization methods can be separately controlled to
provide ion
accumulation on sample 101 and 4 prior to laser desorption or to generate ions
that are
directed into vacuum through capillary bore 104 and 11 for mass to charge
analysis.
Combinations of multiple probes can be run simultaneously or independently in
one ion
source without the need to change hardware.
The operating sequence of laser desorption ion source 114 shown in Figure 5 is
analogous
to that illustrated in timing diagram 4B described above. In positive ion
operating mode, a
negative voltage is applied to charging electrode 112 through power supply 123
relative to
the voltages applied to target plate counter electrode 111, capillary entrance
electrode 115,
capillary nosepiece electrode 117, charged droplet sprayer 105 and ring
electrode 106
through power supplies 118, 119, 120, 122 and 121 respectively. Charged
species
generated by charged droplet sprayer 105 and/or target gas controller 124 are
directed to
the surface of sample 101 on dielectric or semiconductor target plate 100.
Charge is
accumulated on the surface of sample 101 until the space charge limit is
reached for the
relative electrode voltages applied. The time period 128 of this sample
charging step is
illustrated in the timing diagram shown in Figure 6. Laser pulse 108 is fired
from laser 110
to desorb material from sample 101 as the voltages on electrodes 112, 106 and
117 are
changed to facilitate extraction of desorbed ions from the surface of sample
101 and
focusing of the ion population produced into capillary bore entrance 102. The
ion
desorption, extraction and focusing step 129 is shown to occur simultaneously
with laser
pulse 108. Alternatively, the electrode voltage transitions can occur before
or after the laser =
pulse and additional laser pulses can occur during or after such electrode
voltage transition.
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Prior to the desorbed ion population reaching capillary bore entrance 102, the
relative
voltages applied to electrodes 112, 111, 106, 115 and 117 are set to be
approximately
equal to reduceithe electric field in region 113 between target plate 100 and
capillary
entrance electrode 115. As illustrated in the timing diagram shown in Figure
6, shortly after
the ion extraction and focusing voltages are applied, the relative voltages of
electrodes are
set to be approximately equal to initiate gas focusing step 130. With a
minimum electric
field in region 113, the desorbed ions are swept into capillary bore by gas
flow 131. The
reduction of the electric field in region 113 prior to the desorbed ions
reaching capillary
entrance electrode 115 reduces neutralization of ions on electrode 115 and
improves ion
transmission efficiency into vacuum through capillary bore 104. The duration
of the gas
focusing step 130 time period is sufficient to allow the desorbed ion
population to enter
capillary bore 104 prior to switching the electrode potentials back to ion
accumulation step
132. Heated countercurrent gas flow 127 sweeps neutral species away from
capillary bore
entrance 102 during ion extraction and focusing step 129 and provides the
carrier gas for
sweeping ions into vacuum. As described for laser desorption ion source 1, gas
phase ion
species may generated in target gas controller 124 and carried in target gas
133 to charge
the surface of sample 101 and provide subsequent gas phase ionization of
desorbed
neutral molecules traversing region 113. The charging, desorption and gas
focusing steps
can be conducted in rapid succession cycling multiple times per second to
minimize sample
analysis time. As described above the laser pulse 108 spot, target plate 100,
and charging
electrode 112 positions can be positioned independently with or without
optical imaging to
optimize analytical performance for a given application.
An alternative embodiment of the invention is diagrammed in Figure 7 where
target plate
140 and target plate chamber 142 are positioned in vacuum stage 160. The
pressure
maintained in vacuum stage 160 may range from above 4 torr to below 10'4 torr
depending
on the analytical application, total gas flow through target plate gas
controller 143 and ion
generator 147 and vacuum stage 160 pumping speed. Ion or charged species
generator
147 with ion focusing electrodes 148 and target gas controller 143 may
comprise a
chemical ionization, glow discharge, electron bombardment, photoionization or
other
vacuum compatible ion source to generate charged species. Similar to the
operation of the
atmospheric pressure ion sources described above, charging of the surface of
sample 141
occurs in intermediate pressure laser desorption ion source 164 prior to
applying laser
pulse 165 from laser 151 to desorb sample components and ions from sample 141.
Charged species in either positive or negative ion operating mode are
accumulated on the
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surface of sample 141 by applying the appropriate potentials as described
above to
charging electrode 166, target plate counter electrode 146, skimmer electrode
149, ion
generator 147 and focusing electrodes 148. Ion species are supplied from
target gas
controller 143 and ion generator 147 individually or simultaneously during the
sample
charging step. The voltages applied to electrodes 166, 146, 149 and 148 and
ion generator
147 are rapidly changed while laser pulse 165 is applied to aid in desorbing,
extracting and
ionizing sample components from sample 141. After ion and neutral sample
components
have been desorbed and extracted from sample 141, voltages applied to these
electrodes
are then changed to optimize transmission efficiency of the desorbed ion
population
through skimmer opening 150 into ion guide 154. Timing sequence similar to
that shown in
Figures 4A, 4B and 6, can be applied in the operation of intermediate pressure
laser
desorption ion source 160. Additional gas phase ionization of neutral desorbed
sample
molecules can occur through charge exchange or ion attachment with ion species
supplied
in target gas 144 as the desorbed sample plume expands in region 167 between
the target
plate and skimmer 149. Ion guide 154 can be operated as an ion trap to allow
additional
reaction time between reagent ions supplied from target plate gas controller
143 trapped in
ion guide 154 to react with desorbed neutral species flowing through skimmer
opening 150
and into ion guide 154. The accumulation of charge on the sample prior to
desorption and
addition of further gas phase ionization increases the ionization efficiency
and sensitivity of
intermediate pressure laser desorption ionization and allows for ion molecule
reactions with
sample components prior to, during or after laser desorption of sample 141.
Target plate gas flow 144 aids in directing reagent ions to the surface of
sample 141 during
the sample charging step. Target plate gas flow 145 exiting target plate
chamber 142
through opening 168 in electrode 146 provides a gas load in vacuum stage 160
and,
passing through skimmer 149 opening 150 into vacuum stage 161, provides a
local
increase in background gas pressure at the entrance of ion guide 154. The flow
of target
plate gas 145 through electrode 146 serves to collisionally damp translational
energy
spread of ions generated in the desorption process. The translational energy
spread of the
desorbed ion population continues to be reduced through collisional cooling in
ion guide
154. Desorbed ions can be focused in region 167 by applying the appropriate
relative
voltages to electrode 146 and skimmer electrode 149. Ions accelerated and
focused
between electrode 146 and skimmer opening 150 experience collisions with
background
gas that may increase or decrease internal energy of the ions depending on the
rate of
acceleration imposed by the applied voltages. If required, ion internal energy
can be
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increased in region 167 to decluster or fragment of ions prior to conducting
mass to charge
analysis in mass to charge analyzer 158. Intermediate pressure laser
desorption ion
source mass spectrometer 157 comprises vacuum stages 160, 161 and 162.
Sufficient
vacuum pumping is provided in each vacuum stage to allow optimal performance
of
elements within each vacuum stage. Less than three or more than three vacuum
stages
may be configured in alternative embodiments of the invention to provide
optimal
performance for specific mass analyzer types. Ion guide 154 as shown in Figure
7 extends
into multiple vacuum stages and serves as the gas conductance orifice between
vacuum
stages 161 and 162. Ions traversing ion guide 154 pass through exit electrode
155 into
mass to charge analyzer and detector 158. Voltage applied to exit electrode
155 may be
increased relative to the offset potential applied to ion guide 154 to trap
ions in ion guide
154. Trapped ions can be released from ion guide 154 by lowering the voltage
applied to
exit electrode 155. The release of trapped ions from ion guide 154 need not be
sychronized with laser pulses in ion source 160 allowing.decoupling of mass
spectrometer
analysis timing with the pulsed production of ions in ion source 160. Ions
from multiple
laser desorption shots may be stored in ion guide 154 before releasing trapped
ions into
mass to charge analyzer 158.
Alternative embodiments of sample target plates, charging electrodes and laser
optics
assemblies are diagrammed in Figures 8 and 9. Figure 8A shows charging
electrode 170
insulated by dielectric insulator 171 in contact with the opposite side of
dielectric target
plate or belt 172 from sample spots or lines 173. Voltage is applied to
charging electrode
170 through Power supply 174. In the embodiment shown in Figure 8A, charging
electrode
170 is not surrounded by a shielding electrode. This allows the attractive
electric field to
extend over a broader region on target plate 172 during the charging of sample
173 prior to
applying a laser pulse. The additional ions collected during sample charging
are available
for gas phase ionization of sample molecules after the laser pulse desorption
and ion
extraction step improving ionization efficiency. Charging electrode 170 can be
fixed in
position with target plate or belt 172 moving over it or both charging
electrode 170 and
target plate 172 can be translated independently to optimize performance.
Cylindrical
shielding electrode 174 is added to the charging electrode assembly 179 in
Figure 8B to
constrain the electric field formed by charging electrode 175 during the
sample charging
and desorption and ion extraction steps. Shielding electrode 174 prevents ions
in the target
gas from being attracted to the back side of target plate or belt 176 during
the sample
charging step. Charging electrode 175 with shielding electrode 174 insulated
by dielectric
CA 02527886 2005-12-01
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insulator 177 can be fabricated with very small dimensions. A small diameter
charging and
shielding electrode assembly contacting a thin target plate or belt allows
charging of a small
sample area when desorbing sample from specific spatial regions of sample 178.
The
smaller dimensions of these elements coupled with a small laser spot size
allows improved
spatial resolution when desorbing sample from surfaces. This is advantageous,
for
example, when selectively desorbing material from specific cells or groups of
cells in a
tissue sample. Target plate or belt 176 is moved along the surface of charging
electrode
assembly 179 while remaining in contact with dielectric material 177 or the
tip of charging
electrode 175. 'Higher relative electrical potentials can be applied to
charging electrode 179
if it is entirely insulated in dielectric 177. Shielding electrode 174 maybe
incased in or
surrounding insulator 177. Multiple charging electrodes 180 with common
shielding
electrode 181 are insulated in dielectric 182 that also serves as the sample
target surface in
target plate assembly 185 shown in Figure 8C. As charging electrode and target
plate
assembly 185 are translated to align laser pulse 186 with each sample spot
187, electrical
contact is made with aligned charging electrode 188 and power supply 183
through spring
contact 184. Integrated assemblies 185 have the advantage that shorter
distances and
more reproducible tolerances can be maintained between sample spots 187 and
the tip of
charging electrodes 188. This allows more reproducible and higher charging of
sample
surfaces to be achieved for different sample spots and for different target
plates.
Figure 9A shows a conventional laser desorption target plate 190 typically
used for MALDI
applications where laser beam 191 impinges on the front side of target plate
190 with no
prior charging of sample. Typically target plate or the surface of target
plate 190 comprises
a conductive material to prevent the buildup of charge during laser desorption
operation.
The invention comprises elements and configurations that provide improved
performance
but depart from configurations employed conventional laser desorption ion
sources that
utilized target plates as shown in Figure 9A. Embodiments of laser desorption
target plates
shown in Figures 8A through 8C and 9B through 9D contain elements and
configurations
not employed in laser desorption ion sources found in the prior art. A diagram
of laser
desorption target plate assembly 194 comprising fiber optic bundles 195
surrounded by
charging electrodes 196 configured in dielectric block 198 is shown in Figure
9B. Sample
202 is deposited on the end of each fiber optic bundle 195 on target plate
surface 203.
Laser pulse 204 from laser 200 is focused through optical lens assembly 201
and sent
through a portion of fiber optic bundle 207 to impinge on the back side of
sample spot 208.
Laser pulse 204 can be directed to different areas of sample spot 208 by
sending laser
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pulse 204 through different areas of fiber optic bundle 207. This can be
achieved by
steering laser beam 204 or by moving target plate assembly 194 using x, y and
z axis
translation. Voltages are applied to charging electrode 197 from power supply
205 through
spring contact 206 to allow charging of the surface of sample spot 208 prior
to applying
laser pulse 204. The embodiment of the invention shown in Figure 9B allows
close
positioning between a sample and an orifice into vacuum or an adjacent pumping
stage.
The laser optics are simplified and the laser beam is oriented perpendicular
to the sample
surface allowing a smaller laser beam spot size. Alternatively, sample spots
or lines 208
may be mounted on an optically transparent plate and the plate can be slid
over the exit
end of fiber bundle 207. This would allow more rapid loading and running of
sample plates
without the need to clean the exit end of fiber optics bundle 207 between
sample runs. A
lens may be added to the exit end of fiber optic bundle 207 or incorporated in
to a glass
target plate to allow tighter focusing of laser beam 204 as it exits fiber
optic bundle 207.
A liquid sample 210 is introduce through bore 215 of dielectric element 211 of
liquid surface
laser desorption probe 212 diagrammed in Figure 9C. Charging electrode 213 is
electrically insulated from solution 210 in dielectric element 211. If
solution 210 has low
conductivity or is electrically floating, charge can be accumulated at surface
214 and in bore
215 when a high potential of opposite polarity is applied to insulated
charging electrode 213
through power supply 218. Charge species accumulating on the surface of and in
liquid
210 are delivered to liquid surface 214 prior to laser pulse 217 as described
above for the
solid surface laser desorption samples. Liquid 210 can flow through channel
215 or be
loaded as a static sample during laser desorption ionization. Desorbed ions
can be formed
by laser desorption of sample components from water using infared lasers.
Glycerol can be
used as a liquid surface with low volatility in atmospheric pressure and
intermediate
pressure laser desorption ion sources. Precharging the liquid surface prior to
applying a
laser pulse can improve the ionization efficiency of such samples during laser
desorption.
In an alternative embodiment of liquid sample laser desorption probe 226,
laser pulse 220
is applied to the underside of liquid sample surface 224 as diagrammed in
Figure 9D. Fiber
optic bundle 221 passed through dielectric block 227. Liquid sample 225 is
introduced
through annulus 228 forming sample surface 224 as it exits annulus 228.
Charging
electrode 229 is electrically insulated in dielectric block 227 with voltage
applied through
power supply 230. Precharging of electrically floating surface 224 and
solution 225 can
occur when an opposite polarity electrical potential is applied to charging
electrode 226
attracting gas phase charged species to surface 224. When saturation of
charging in
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electrically isolated solution 225 is achieved, laser 222 delivers laser pulse
220 through
optical focusing elements 223 and fiber optic bundle 221 to laser desorb
sample liquid 225
from surface 224. Liquid sample solution 225 may contain matrix components
that absorb
the wavelength of laser light used to enhance laser desorption efficiency.
Increased flexibility in target plate design and laser desorption source
operation can be
achieved while improving performance by separating the laser desorption region
from the
ion focusing region into a vacuum orifice in atmospheric pressure laser
desorption ion
sources. An alternative embodiment of the invention in which the ion
generation and
sampling regions are separated is diagrammed in Figures 10A through 10C. Laser
desorption ion source 240 comprising target plate chamber 241 with target
plate 270 and
charging electrode 244 is interfaced to three stage vacuum system 288 with
mass to charge
analyzer and detector 267. Target plate chamber 241 is separated from endplate
electrode
255, focusing electrode 256 and capillary entrance electrode 271 by annular
electrode
assembly 252. No line of sight exists between sample 245 and capillary
entrance 259
reducing the transport of contamination neutrals and charged particles into
vacuum
minimizing contamination vacuum ion optics and decreasing chemical noise in
acquired
mass spectrum. Ion focusing region 272 where ions are focused into vacuum
orifice 259 is
separated from ion generation region 251 allowing independent optimization of
both
functions. Charge droplet sprayer 274, employing pneumatic nebulization, is
positioned in
center section 275 of annular electrode assembly 252 with face electrode 253
serving .as
the ring electrode for charged droplet sprayer 274. Alternative ion generation
means as
described above for alternative ion source embodiments, can be can be
configured in laser
desorption ion source 240 replacing pneumatic nebulization charged droplet
sprayer 274.
In the embodiment shown, charged droplet sprayer 274 is positioned on the
centerline 285
of ion source 240 spraying toward sample 245. Target plate gas controller 242,
with similar
configurations and functions as described above, supplies heated target gas
243. If
required, ions 247 can be generated in target plate gas controller 242 and
delivered to
target plate chamber 241 entrained in target gas flow 243. Target plate gas
flow 243 exits
target plate chamber 241 through opening 287 in target plate counter electrode
250. Target (
plate gas flow 288 entering region 251 directly opposes nebulization gas flow
280 from
charged droplet sprayer 274 forming a gas stagnation and mixing region in
region 251.
In Figure 10A, the relative voltages applied to charging electrode 244, target
plate counter
electrode 250, annular electrode 253 and charged droplet sprayer 274 are set
to
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accumulate charge 246 on the surface of sample 245. Target plate gas flow 288
facilitates
drying of charged droplets produced from charged droplet sprayer 274. Ions 248
generated
from the evaporating charged droplets are directed toward sample 245 by the
electric field
applied in region 251. Charged species 247 entrained in target plate gas flow
243 are also
directed toward the surface of sample 245 by the applied electric field. When
sufficient
charge has been accumulated on the surface of sample 245, laser pulse 281 is
fired at
sample 245 from laser 282 through lens 283 and reflected off mirror 284 as
shown in Figure
10B. As described for alternative embodiments above, relative voltages applied
to
electrodes 244, 250 and 253 and charged droplet sprayer 274 are changed just
before,
concurrent with or just after laser pulse 281 is fired to facilitate the
release of charged
species from sample 245. The timing of the voltage change relative the laser
pulse event is
optimized to maximize sample ionization efficiency. In the example shown, the
voltage
applied to electrode 253 remains constant during the sample charging, ion
desorption and
ion focusing steps. Nebulization gas flow 280 from charged droplet sprayer 274
and target
plate gas flow 280 remains on during the sample charging, ion desorption and
extraction
and ion focusing steps providing a gas phase stagnation and mixing region in
region 251
during each operating step. This mixing region facilitates gas phase
ionization of desorbed
neutral sample molecules by ions 247 entrained in target plate gas flow 288
during the
desorption, extraction and ion focusing steps. Following a short delay after
laser pulse 281
to allow desorbed ions and neutral species to move into region 251, the
relative voltages
applied to electrodes 244 and 250 and charged droplet sprayer 274 are changed
to
optimize ion transmission and focusing into bore 258 of capillary 257 through
annulus 292
of annular electrode assembly 252 as illustrated in Figure 10C.
Countercurrent drying gas 262 traverses gas heater 261 and flows through the
center
aperture of endplate,eleCtrode 255. Heated drying gas flow 260 is directed
along endplate
electrode 255 and through annulus 292 of annular electrode assembly 252.
Heated
countercurrent gas flow 260 becoming gas flow 277, moves in the bpposite
direction to ion
movement through annulus 292 of annular electrode assembly 252 as ions are
directed
from region 251 to capillary bore entrance 259 as shown in Figure 10C. Heated
countercurrent gas flows 260 and 277 sweep any neutral contamination species
away from
annulus 292 of annular electrode assembly 252 preventing neutral contamination
species
from entering vacuum through capillary bore 258. Voltages are applied to the
electrodes in
electrode assembly 252 to focus and direct ions from region 251 to region 295
and into
capillary bore entrance 259. Voltages applied to electrodes 294, 254, 255, 258
and
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capillary entrance electrode 271 are set to direct desorbed and gas phase
generated ions
290 leaving electrode assembly annulus 292 through the center opening in
endplate
electrode 255 and focus ions 291 into capillary bore entrance 259 as shown in
Figure 10C.
Annular electrode assembly 252 decouples ion formation region 251 from the
capillary
entrance region allowing the performance in both regions to be optimized
independently.
Gas flows and gas temperatures, surface charging, ionization efficiency and
the transport of
ions into annular lens assembly 252 can be optimized in region 251. Ion
focusing into
capillary bore 258 in region 291 is decoupled from variable settings and step
sequences
occurring in region 251 allowing optimization of ion transport and focusing
separate from
performance optimization in region 251. Optimization of variables in focusing
region 295
increases sensitivity of mass to charge analysis by increasing the efficiency
of ion transport
into capillary bore 258. Desorbed or gas phase generated ions entering
capillary bore 258
pass into vacuum, pass through skimmer 297 and ion guide 266 and are analyzed
in mass
to charge analyzer and detector 267. Target gas flow 243, pneumatic nebulizer
gas flow
280 and countercurrent gas flow 260 and 277 exit laser desorption ion source
at gas outlet
298. Laser desorption ion source 240 may be operated at near atmospheric
pressure.
Alternatively laser desorption ion source 240 can be operated at pressures
above one
atmosphere to prevent outside contamination from backstreaming into the ion
source
chamber or at pressures below one atmosphere to accommodate negative pressure
venting systems.
An alternative embodiment of the invention is diagrammed in Figure 11 where
combination Electrospray and laser desorption ion source 300 comprises annular
electrode
assembly 301. Charged droplet sprayer 302 with or without pneumatic
nebulization
generates charged species that are directed to the surface of sample 303
during the charge
accumulation step. Sample is desorbed and ionized by laser pulse 317 'fired
from laser 310.
The ions generated are directed into and through annular electrode assembly
301 by
applying the appropriate voltages to back electrode 308, charged droplet
sprayer 302,
charging electrode 305 and annular electrode assembly 301. Ions exiting
annular electrode
assembly 301 are focused into bore 313 of capillary 314 moving against
countercurrent
drying gas 315. Optical imager 309 can be used to image the surface of sample
303.
Based on this image, the position of laser pulse 317 and the tip of charging
electrode 305
can be adjusted to provide optimal performance. Alternatively, sample ions can
be
generated from charged droplet sprayer 302. Target plate gas flow 307 aids in
drying
charged droples 307 produced by charged droplet sprayer 302. Ions generated
from the
CA 02527886 2005-12-01
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evaporating charged droplets produced by charged droplet sprayer 302 are
directed and
focused into annulus 318 of annular electrode assembly 301. The charged
droplet spray
generated ions are directed through annulus 318 and focused into bore 313 of
capillary 314.
Alternatively, charged droplet sprayer 312 positioned orthogonal to target
plate 304 can
generate ions for charging sample 303 prior to laser desorption or can
generate sample
ions directly for mass to charged analysis. Annular lens assembly 301
configured in
multiple ionization type ion source 300 decouples the ion production region
from the ion
focusing region into bore 313 of capillary 314 allowing decoupled optimization
of each
region and reducing mass spectrum noise from neutral contamination components
entering
vacuum. The sensitivity of mass to charged analysis is increased by the
improved focusing
of ions passing though regions 319 and 320 into capillary bore 313. Laser
desorption of
sample 304 and Electrospray ionization of a sample solution can occur
simultaneously or
independently in ion source 300. Running Electrospray simultaneously with
laser
desorption ionization allows gas phase ion-molecules reactions or the addition
of known
internal calibration peaks during mass spectrum acquisition.
The charging of a sample surface prior to conducting laser desorption can
improve
the efficiency of ion production in vacuum. Time-Of-Flight mass to charge
analysis of ions
generated from laser desorption or matrix assisted laser desorption in vacuum
is well
known in the art. Charging of sample surfaces prior to laser desorption can
reduce mass
measurement accuracies and resolving power in conventional MALDI TOF mass to
charge
analysis. When the steps of ion desorption and acceleration into the TOF
flight tube are
coupled, the kinetic energy of the desorbed ion species can effect the ion
flight time.
Charging of the ion surface can change the desorbed ion energy from laser shot
to laser
shot modifying the flight time of the desorbed ion species. Time delay
acceleration of ions
into the TOF pulsing region after a laser pulse can reduce the effects of
initial ion energy
spr?ad and neutral gas interference but cannot compensate entirely for shot to
shot
differences in surface charging. Charging of a 'sample prior to a laser pulse
in vacuum can
be used in TOF mass to charge analysis if the laser desorption step and
subsequent
acceleration of ions into the TOF flight tube are decoupled. US Patent Number
US
6,683,301 B2, (US patent '301) incorporated herein by reference, describes the
apparatus
and method for decoupling the steps of laser desorption of a sample in vacuum
and
subsequent pulsing of the ions generated into a TOF flight tube (for mass to
charged
analysis. As described in US patent '301, ions generated in the laser
desorption step are
directed to and trapped above a surface in near field potential wells formed
by a high
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frequency electric field. The trapped ion population is subsequently
accelerated into the
TOF flight tube. Charging of the sample surface prior to the laser desorption
step can be
incorporated into such an apparatus and method to improve ionization
efficiency or to
conduct ion molecule reactions prior to laser desorption as diagrammed in
Figures 12A
through 12D.
An alternative embodiment of the invention is diagrammed in Figure 12A through
12D mounted in vacuum chamber 340. Figure 12A illustrates the step of charge
accumulation on the surface of sample 341 positioned on dielectric target
plate 342.
Charge is accumulated on the surface of sample 341 by directing ion beam 345
to the
surface of sample 341 by applying the appropriate focusing and accelerating
potentials to
charging electrode 346, focusing electrode set 344, target plate counter
electrode 347, TOF
pulsing region entrance electrode 348, trapping surface 350, trapping
electrode 349, and
ion accelerating electrodes 351, 352 and 353. Ion beam 345 is generated by ion
'source
343 operating in vacuum. Ion source 343 may be an electron bombardment,
chemical
ionization, glow discharge, or other vacuum ion source known in the art. When
the
maximum charging of the surface of sample 341 has been achieved, laser pulse
358 is
directed to sample 341 from laser 359 through optical lens 360 and reflected
off mirror 361
as shown in Figure 12B. The voltages applied to electrodes 346, 347, 344, 348,
349, 351
and trapping surface 350 are changed to direct the population of desorbed ion
species 362
toward trapping surface 350 and trap desorbed ions 362 above trapping surface
350 as
shown in Figures 12B and 12C. As described in US patent '301, the reduction of
kinetic
energies of ions 365 trapped above dynamic electric field trapping surface 350
may be
achieved by ion collisions with neutral background, gas or by laser cooling of
ions.
Sufficient neutral background gas may be locally present in TOF pulsing region
364 to
reduce trapped ion kinetic energy or neutral gas may be added to TOF pulsing
region 364
through a pulsed gas valve. Alternatively, laser cooling may be applied to
reduce the
trapped ion kinetic energy. Redirected laser pulse 358 aimed at or along
trapping surface
350 may be used for laser cooling of trapped ion 365 kinetic energy although a
reduction in
power may be required compared with laser desorption pulse 358. Laser pulse or
beam
358 can be redirected toward trapping surface 350 by moving the angle of
mirror 361 and
the laser power can be reduced by defocusing laser pulse 358 using lens 360 or
reducing
the power output of laser 359. After the kinetic energy spread of trapped ions
365 has been
reduced, voltages are changed on trapping surface 350, electrode 349 and grid
electrodes
351 and 352 to accelerate or push-pull trapped ions 365 into TOF flight tube
355 through
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60412-4219
grid electrodes 351, 352 and 353. Accelerated ions 368 may be steered in TOF
flight tube
355 using steering electrode set 354. Ion 368 are accelerated from trapping
surface 350
into TOF flight tube 355 to maximize TOF performance by changing voltages
applied to
trapping surface 350 and electrodes 349, 351 and 352 as more fully described
in US patent
'301. In the embodiment shown in Figures 12A through 12D grid electrode 353
forms part
of the TOF flight tube and the voltage applied to electrode 353 and remains
constant during
the sample charging, laser desorption, ion trapping and ion acceleration steps
described
above.
Ions accelerated from trapping surface 350 Into TOF flight tube 355 are mass
to charge
analyzed and detected. TOF flight tube may comprise a linear flight path or be
configured
with one or more ion reflectors to increase mass to charge analysis resolving
power.
Multiple sample charging and laser desorption steps may be conducted for each
step of
accelerating ions Into TOF Flight tube 355. This will increase analytical
speed if the trapped
ion kinetic energy cooling step is the longest step in the ion charging,
desorption, extraction
and analysis sequence. Target plate 342 can be rotated or translated to move
different
samples into position or to optimize the sample position relative to the tip
of charging
electrode 346 and laser pulse 358. Optical imaging of the sample may be
performed to
direct adjustment of the sample surface for optimal performance. Target plates
are
removed and replaced by the changing of flange 370. Flange 370 may be replaced
with an
automatic target plate loading and pumpdown system that allows removal and
loading of
target plate 342 without venting TOF flight tube vacuum chamber 340. Unlike
conventional
vacuum laser desorption, the flatness tolerance, dimensional reproducibility
and material
selection of target plate 342 are relaxed in the embodiment of the invention
shown. This
reduces cost and improves selection of materials that may be more compatible
with specific
samples.
Sample charging prior to laser desorption can be configured with ion guides in
atmospheric
pressure, intermediate pressure and vacuum laser desorption ion sources. US
Patent
Number US 6,707,037 B2 (US patent '037) describes
laser desorption ion sources comprising multipole ion guides configured in
atmospheric
pressure, intermediate pressure and vacuum regions. The step of charge
accumulation on
or near the sample surface prior to applying a laser desorption pulse can be
added to
embodiments described in US patent '037. Separately generated reagent ions can
be
introduced axially through the ends of multipole ion guides or radially
through the gaps
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between rods in multipole ion guides prior to applying a laser desorption
pulse to a sample.
The added reagent ion charge can accumulate on the sample surface or be
trapped in the
multipole ion guides to enhance ion-molecule reaction gas phase ionization of
neutral
desorbed components through ion-molecule reactions. Reagent ions of the
opposite
polarity can be added to the multipole ion guide volume to promote gas phase
ion-ion
reactions. For desorbed positive multiply charged ions, the addition of an
electron to
multiply charged positive polarity ions through ion-ion gas phase reactions
may lead to
positive ion fragmentation through electron capture or electron transfer
fragmentation
mechanisms. Ions generated through laser desorption or gas phase ion-molecule
reactions
are directed through the ion guide to a mass to charge analyzer for mass to
charge analysis
employing methods and apparatus as described in US patent '037. Other ion
guides such
as sequential disk RF ion guides or other ion guide types known in the art may
be used as
an alternative to the multipole ion guide embodiments.
Ions generated in the laser desorption ion sources described above
alternatively be
analyzed using ion mobility analyzers or combinations of ion mobility
analyzers with mass
spectrometers. Although the present invention has been described in accordance
with the
embodiments shown, one of ordinary skill in the art will recognize that there
could be
variations to the embodiments, and those variations would be within the spirit
and scope of
the present invention.
Configuration and operation of the embodiments of laser desorption ion source
as
described above provide performance improvements as described above and as
listed
below:
a) By precisely timing and positioning the laser desorption process to
coincide with a potential pulse to the sample, the sample can be desorbed and
ionized from the target in optimum electric fields and flow leading to
efficient
extraction of ions from the target, and by subsequently cycling the electric
potential
to more appropriate focusing fields the ions can be more efficiently focused
and
transmitted to and through the conductance opening to lower pressures.
b) By charging the sample surface with reagent ions or electrons prior to
the laser desorption process, the ionization process can occur more
efficiently.
c) By charging the sample with selected reagent ions the selectivity of
ionization process can be improved and analyte can be chemically labeled or
tagged.
d) By charging the sample with selected reagent ions at a predetermined
collection point and matching the collection point with the laser pulse, a
specific point
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on a sample (e.g. stained spot of 2D gel or organelle in tissue sample) can be
selectively desorbed and ionized.
e) By laser desorbing and ionizing samples at higher pressures,
such as
at atmospheric pressure, the motion of the gas-phase ions is more controllable
than
performing desorption and ionization at lower pressures because the ions tend
to
follow the electric field in absence of flow or other forces. The addition of
flow as a
ion focusing parameter gives the device more degrees of freedom to control
motion
and enhance focusing (e.g. counterflow in focusing field can enhance
focusing).
By introducing sample from a liquid stream such as capillary
electrophoresis or liquid chromatography, the device can operate as a
continuous
interface for LC/MS or CE/MS.
g) By controlling the extraction and focusing fields in a time-sequence to
optimize both processes, the alignment and position of the sample relative to
the
conductance opening is less critical.
h) By using optical alignment instead of positional alignment of sample
and conductance opening, the loading of the sample into the source becomes
much
easier and the nature of the sample (e.g. direct tissue samples, direct 2D
gels or
western blots, flowing sample) can be far more diverse than conventional MALDI
spots.
