Note: Descriptions are shown in the official language in which they were submitted.
CA 02590656 2007-06-04
MULTIPLE SAMPLL INTROllUCTION NIASS SPECTROMETRY
FIELI) OF THE INVENTION
The presetit iiiventioii relates to nlass spectronietry, and more particularly
to a niultiple
saiuple introcluctiozl mass spectrometry.
BACKGROUND OF THE INVLN'I'ION
Atmosplleric Pressuce lonization (API) Sourees irlcluding Eleetrospray (ES),
Atmosplleric Pressure Clienlical lonization (APCI) ancl Incluctively Coupled
P1asnla
(ICP) iol1 sources interfaced to mass aualyzeis ai=e typically opei'ated with
a siilgle sample
introduction probe. In mass spectrometric applications where iiiternal standai-
ds are
required, additional cotzlponents can be added to the primary sample solution
wl7ere the
resulting niixttu=e is delivered through one probe itito the API source. The
mixture of
compounds in a single solution iiitroduced through the sanle probe are ionized
and mass
aualyzed. A ktiown saiilple wlien iiiixec[ with ati utilulowai sample can
serve as an inteenal
mass scale or quantltatlotl calibration staticlard for the unknown
cojzlponeiits peaks
appearing in the mass spectrLUii actluit-ed in this inaiuler. Ilowever, mixing
a lcnown
comPoGU-id calibration solution with an tinlulowiz satnple solution can have
utidesired
analytical consequences. 7'he lctiown aiid unknown solution coniponents inay
a1:I'ect one
aiiolher in an unpredictable manuec dw=ing the solution transport or
ioilizatioa process.
One component i-tiay react witli
CA 02590656 2007-06-04
another in solution or one or more components may suppress the ionization
efficiency of
other components during the ionization process. A solution with a known
component
mixture may be difficult to eliminate as a source of chemical contamination in
a probe
which is running a series of unknown samples at the trace component level. If
it is
desirable to deliver a known solution as a mixture through the sample
introduction probe
on an intermittent basis, the occasional sample introduction will be subject
to the
constraints of solution flow rates through the probe, efficiency of mixing
solutions, dead
volume losses.and flushing of the probe to eliminate the known solution prior
to the next
analysis. The invention avoids performance and sample introduction problems
encountered
when mixing liquid samples prior to ionization in an API source, by conducting
simultaneous mass analysis of two different solutions without th need to mix
solutions in
the same probe prior to analysis. One aspect of the invention is the
configuration and
simultaneous operation of multiple probes or multiple sprayers or nebulizers
within a
probe assembly through which different sample solutions can be introduced
simultaneously
into an API source during operation.
In one embodiment of the invention, multiple sample introduction means have
been
configured in Electrospray Atmospheric Pressure Ion sources which are
interfaced to mass
analyzers. At least two sample introduction Electrospray probes are operated
simultaneously in an Electrospray ion source. At least one ES probe is
supplied a sample
which is different from the sample solution supplied to additional ES probes
operating
within the same ES source chamber. In this manner a calibration solution can
be
introduced through one ES probe while an unknown sample is introduced through
another
ES probe or second channel within the same ES probe assembly. Ions produced
from both
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CA 02590656 2007-06-04
solutions via the simultaneous spraying of both ES probes blend or mix in the
atmospheric
pressure ES chamber background gas prior to entering the orifice into vacuum.
The
mixture of ions resulting from the solutions delivered from at least two ES
probes is
simultaneously mass to charge (m/z) analyzed resulting in a mass spectrum
containing an
internal standard for calibrating or tuning the mass analyzer. The internal
calibration
standard contained within the acquired mass spectrum is achieved without
mixing known
and unknown samples in solution. Simultaneous introduction of different
samples through
multiple ES probes also enables the study of mixed ion and molecule reactions
at
atmospheric pressure in the ES source chamber prior to introduction into
vacuum. Each
ES sample introduction probe assembly can be configured with nebulization gas
and liquid
layered flow. An internal calibration solution can be included in the layered
flow or the
primary flow of any given ES probe configured in the ES source chamber. The
individual
sample solution flows or nebulization gas flows to any combination of ES
probes can be
switched on or off during an analytical run without the need to reposition
probes. In
another aspect of the invention, an Atmospheric Pressure Chemical Ionization
(APCI)
source assembly can be configured with multiple inlet channels o probes. These
multiple
APCI inlet probes can include pneumatic nebulization and the solution and gas
flow
supplied to each inlet probe can be individually or simultaneously turned on
or off. In
both the ES and APCI sources, multiple probe sample solution ionization can be
controlled
without the need to reposition probes by switching voltages, controlling the
nebulization
gas flows or controlling the sample solution flows. Configurations of multiple
sample
introduction inlet probes can also be extended to a system that has a
combination of both
Electrospray and APCI ion production means in the same API chamber. Each ES or
APCI sample inlet probe can include pneumatic or ultrasonic nebulization.
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CA 02590656 2007-06-04
Configurations of Electrospray ion sources which include more than one sample
introduction needle or nebulizer have been described in the literature.
Kostianinen and
Bruins, Proceedings of the 41st ASMS Conference on Mass Spectrometry, 744a,
1993,
described the configuration and use of an assembly of multiple Electrospray
inlet tips with
and without pneumatic nebulization mounted in an Electrospray ion source. Each
ES tip
was supplied the same sample solution delivered from a single pump with a
single solution
source. The sample solution, delivered from a liquid chromatography pump,
flowed into
an assembly or array of one, two or four ES or pneumatic nebulization assisted
ES sprayer
tips in an attempt to improve ion signal intensity at higher liquid flow
rates. In the
arrangement reported, the solution flow to individual sprayer tips could not
be turned on
and off independently and different solutions could not be introduced
selectively to
individual sprayer tips in the assembly of multiple ES sprayer tips.
Rachel R. Ogorzalek Loo, Harold R. Udseth, and Richard D. Smith, Proceedings
of the
39th ASMS Conference on Mass Spectrometry and Allied Topics, 266-267, 1991 and
J.
Phys. Chem., 6412-6415, 1991 and Richard D. Smith, Joseph A. Loo, Rachel R.
Ogorzalek Loo, Mark Busman, and Harold R. Udseth, Mass Spectrometry Reviews,
10,
359-451,1991 describe the configuration of an Electrospray ion source
interfaced to a
quadruple mass analyzer apparatus which included dual Electrospray ion sources
delivering
ions to two separate entrance apertures of a Y shaped capillary. Positive ions
created in
one Electrospray source were introduced into one inlet branch of the Y shaped
capillary
and negative ions created from the second Electrospray ion source were
introduced into the
second inlet branch of the Y shaped capillary. The positive and negative ions
swept into
the two entrance orifices of the capillary tube began mixing where the two
inlet branches
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CA 02590656 2007-06-04
of the capillary tube met well downstream of the capillary entrances located
in the two ES
atmospheric pressure source chambers. Dual Electrospray ionization sources or
a separate
ES source and a gas phase corona discharge source individually delivered ions
into two
entrance orifices of a Y shaped capillary. For all experiments reported, the
first ES source
produced ions of opposite polarity to the second ES or gas phase corona
discharge source.
The opposite polarity ions produced in separate ion sources were not mixed in
the
atmospheric pressure ion source but entered a split capillary tube at two
separate entrance
orifices and mixed in partial vacuum downstream in the capillary tube.
Bordoli, Woolfit and Bateman, Proceedings of the 43th ASMS Conference on Mass
Spectrometry and Allied Topics, 98, 1995 described an Electrospray ion source
which
included a calibration ES probe configured with a second microtip (50 nl/min
flow rate)
sample probe interfaced to a magnetic sector mass analyzer. The sample probe
included a
microtip attached directly to a syringe needle. The syringe was mounted on an
X-Y-Z
positioning stage to optimize the position of the microtip sprayer. The
calibration ES
probe was configured such that it could be moved into a position when a
calibration
solution was sprayed at 500 nl/min while no sample flowed through the primary
ES
sample probe. After acquisition of a calibration mass spectrum, the
calibration ES probe
was retracted and the calibration solution flow turned off. The sample flow
through the
microtip sample ES probe was then turned on and a separate mass spectrum was
acquired
from the Electrosprayed ions produced. In this manner, an external calibration
mass
spectrum was acquired prior to acquisition of a mass spectrum of the primary
sample. The
calibration mass spectrum and the sample mass spectrum were then added
together in the
data system prior to calculating the mass assignment of the sample related
peaks. For the
CA 02590656 2007-06-04
ES source configuration reported, the two ES probes were not operated
simultaneously and
no gas phase mixture of calibration and sample ions was created at atmospheric
pressure
and no mass spectrum was acquired from a mixture of calibration and sample
ions. No
single mass spectrum was acquired which included sample related peaks and
calibration
compound related peaks with the apparatus described. Neither ES probe
described was
configured to operate with pneumatic nebulization assisted Electrospray. The
ES
calibration probe position required adjustment prior to acquiring a
calibration spectrum to
enable effective spaying near the orifice into vacuum. After acquisition of a
calibration
mass spectrum, the ES calibration probe was retracted to avoid interference
prior to the
mass spectrum acquisition from the sample solution delivered through the
primary ES
probe.
In one embodiment of the invention described, multiple samples are introduced
into an
API source simultaneously where ions are produced from all samples and mixed
in the
atmospheric pressure ion source chamber. A portion of the gas phase ion
mixture is then
swept into vacuum through an orifice or capillary where the ions are mass
analyzed. In
this manner a solution containing calibration compounds can be ionized
simultaneously
with a sample solution resulting in an acquired mass spectrum containing an
internal
standard without mixing calibration components and sample components in
solution.
Higher mass accuracy's can be achieved with an internal standard when m/z
assignm.ents
are calculated for sample ion related peaks in an acquired mass spectrum. In
addition to
independently introducing calibration compounds in an API source, multiple
sample inlet
probes can be used to introduce multiple samples individually or
simultaneously into an
API source. Mounting multiple probes in an API chamber such as ES and APCI
probes,
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CA 02590656 2007-06-04
allows multiple ionization techniques to be run individually or simultaneously
in a single
API source assembly. Multiple Electrospray probes can be configured to
collectively
provide optimal performance over a wide range of sample flow rates and
solution
chemistries. ES probe positions can be configured to fall directly on the
vacuum orifice
centerline to a position angled to well over 100 degrees off the centerline.
Different liquid
flow rates can be delivered to separate ES or APCI probes within the same API
source.
ES and/or APCI probes mounted at different positions in the ES source chamber,
can
operate simultaneously, in pairs or in groups at different flow rates and
introducing
different sample solutions. The multiple ES probes may be operated with or
without
nebulization assist.
Summary of the Invention
One embodiment of the invention is the configuration of an API source with
multiple
sample solution inlets, connected to different sample delivery systems,
interfaced to a mass
analyzer. Individual sample inlet probes can be operated independently or
simultaneously
in the same API source chamber. The composition and flow rate of solution
introduced
through each individual API probe can be controlled independently from other
sample
introduction ES, APCI or ICP probes. Multiple samples are introduced into the
API
source through multiple API probes without mixing separate sample components
in
solution prior to solution spraying and ionization. Ionization of components
from multiple
sample solutions occurs in the gas phase at or near atmospheric pressure. The
API source
may include but is not limited to Electrospray, APCI or ICP ionization means
or
combinations of each ionization technique. Another aspect of the invention is
the
technique of introducing a calibration solution into at least one API source
inlet probe and
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CA 02590656 2007-06-04
the sample of interest through another API source inlet probe. Both
calibration and
sample solutions are introduced through separate inlet probes but are sprayed
and ionized
simultaneously in the API source resulting in a mixture of gas phase
calibration and
sample related ions. A portion of the resulting ion mixture is mass analyzed
producing a
mass spectrum which includes known component ion peaks that can serve as an
internal
standard to improve m/z measurement and even quantitation accuracy.
Alternatively,
multiple sample solutions can be introduced separately but simultaneously
creating a
mixture of ions at or near atmospheric pressure to study gas phase ion and
molecule
interactions and reactions. Multiple inlet probe API sources can be interfaced
to any MS
or MS/MS" mass analyzer type including but not limited to, Time-Of-Flight
(TOF),
Quadrupole, Fourier Transform (FTMS), Ion Trap, Magnetic Sector or a Hybrid
mass
analyzer.
In one embodiment of the invention, an Electrospray ion source is configured
with
multiple Electrospray probes. Each probe may or may not be configured with
pneumatic
or ultrasonic nebulization assist and/or a second liquid layer. The multiple
ES probes and
each liquid layer of each ES probe may be connected to different liquid
delivery systems.
In this manner, different samples, mixture of samples and/or solvents can be
sprayed
simultaneously or individually in a variety of combinations. The liquid
delivery systems
include but are not limited to liquid chromatography pumps, syringe pumps,
gravity feed
vessels, pressurized vessels, and or aspiration feed vessels. Samples may also
be
introduced using auto injectors, separation systems such as liquid
chromatography (LC) or
capillary electrophoresis (CE), capillary electrophoresis chromatography (CEC)
and/or
manual injection values connected to any or all ES probes. Multiple and
independent
8
CA 02590656 2007-06-04
solution introduction allows multiple samples to be analyzed simultaneously
with
Electrospray ionization without changing ES probe positions. The ability to
introduce
sample solution through one ES probe and have the option to selectively and
simultaneously introduce additional secondary samples into the ES chamber
through other
ES probes can be used to generate mass spectra, even on-line during LC or CE
separations, with internal or external calibration standards. Different sample
mixtures
which span a range of m/z values or sample types can be introduced through
different ES
probes. Depending on the unknown sample being analyzed, an optimal calibration
solution
can be chosen from another ES probe. For example one m/z range calibration
solution can
be chosen which produces singly charged ES ions when analyzing singly charged
compounds and likewise multiple charged ES generated calibration ions can be
produced
when analyzing compounds which form multiply charged ions in Electrospray
ionization.
The solution flow for any secondary ES probe can be controlled independent of
the
solution flow to a primary ES sample solution probe without having to change
or adjust
any probe position, change the ES source voltages, shut off the primary sample
solution
flow or contaminate the solution being introduced through the primary sample
solution
probe. Multiple probe sets can be operated simultaneously or in sequence with
other
probe sets in the same API chamber. The configuration and operation of
multiple ES
probes can facilitate API MS detection from multiple sample sources. In
particular,
multiple sample probes facilitates and improves the analytical throughput of
unattended
automated operation of a single mass analyzer as a detector for multiple
Liquid
Chromatography separations systems.
In another embodiment of the invention, multiple nebulizers are configured in
an
9
CA 02590656 2007-06-04
Atmospheric Pressure Chemical Ionization source. Similar to ES, multiple
sample
solutions can be introduced into the gas phase and ionized without mixing
solutions. In
this APCI source embodiment, multiple nebulizers spray individual sample
bearing
solutions into a vaporizer where the mixture of nebulized droplets is
evaporated prior to
ionization in the corona discharge region. Calibration solutions can be
introduced through
one or more sample inlet probes independently and simultaneously with sample
solution
introduction through yet another inlet probe. No adjustment to probe position,
applied
voltages or vaporizer temperature may be required when controlling the
solution flow to
multiple inlet probes. This independent sample flow control with little or no
mechanical
adjustment in an APCI source increases the system level analytical flexibility
and sample
throughput with manual or automated operation while minimizing multiple
solution cross
contamination. Multiple APCI and ES probes can be configured in one API source
in
another embodiment of the invention. The combination ES and APCI source
expands the
range of analytical capability of an API-MS instrument interfaced to a variety
of
separation systems particularly for automated operation with a variety of
samples.
The use of multiple probes with API sources, including ES, APCI or ICP
ionization
techniques allows a more rapid introduction of samples particularly when a
fast mass
analyzer such as Time-Of-Flight is used. Rapid sample introduction can be
limited by the
cycle time of an LC, CE or CEC separation system or auto injector. Sample
introduction
cycle time can also be limited by the time it takes for an injected sample to
travel from
the injector valve to the ES or APCI probe outlet. Multiple LC, CE or CEC,
auto
injectors, injector valves and API probes can be conftgured to decrease the
cycle time of
sample introduction and analysis time of an API MS system.
CA 02590656 2007-06-04
Description of the Figures
Figure 1 is a diagram of an Electrospray ion source configured with multiple
independent
Electrospray probes installed.
Figure 2 is a diagram of the Electrospray ion source of Figure 1 showing a
cross section
top view of the ES dual probe assembly positioned near the ES source
centerline.
Figure 3 is a diagram of the Electrospray ion source of Figure 1 showing a
cross section
side view of a dual ES probe assembly configured off axis from the ES source
centerline
and an ES dual probe assembly positioned near the centerline.
Figure 4a is a mass spectrum of a sample solution containing the doubly
charged peak of
Gramicidin S Electrosprayed from one tip of a dual tip off axis ES probe
operating with
pneumatic nebulization assist.
Figure 4b is a mass spectrum of a calibration solution Electrosprayed with
pneumatic
nebulization assist from the second ES tip two of a dual tip off axis ES
probe.
Figure 4c is a mass spectrum of a sample solution Electrosprayed from tip one
and a
calibration solution Electrosprayed from tip two simultaneously from a dual
tip off axis
probe.
Figure 5 is a diagram of a six tip ES probe array with pneumatic nebulization
assist
mounted near the axis to the ES source chamber centerline.
11
CA 02590656 2007-06-04
Figure 6 is a cross section diagram of two ES probe assemblies with
independent x-y-z tip
position adjustment configured in an ES source.
Figure 7a is a mass spectrum of a sample solution containing Leucine
Enkephalin
Electrosprayed with pneumatic nebulization assist through an off-axis ES probe
assembly
into the ES chamber.
Figure 7b is a mass spectrum of a calibration solution containing Tri-Tyrosine
and Hexa-
Tyrosine
Electrosprayed with pneumatic nebulization assist from a second ES probe
positioned near
the ES source centerline.
Figure 7c is a mass spectrum of the sample and calibration solutions
Electrosprayed
simultaneously into the ES chamber from an off-axis ES probe and an ES probe
positioned near the ES source centerline respectively.
Figure 8 is a diagram of an Electrospray source configured with three
independent
Electrospray probes with two off-axis ES probes connected to two LC separation
systems.
Figure 9 is a diagram of an Atmospheric Pressure Chemical Ionization source
with two
independent sample inlet probes configured with one probe angled off-axis to
the APCI
source centerline and one probe aligned with the APCI source centerline.
Figure 10 contains mass spectra of sample and calibration solutions sprayed
separately
12
CA 02590656 2007-06-04
from individual APCI inlet probes and a mass spectrum of sample and
calibration solutions
sprayed simultaneously in a dual inlet probe APCI source configured as shown
in Figure
9.
Figure 11 is a diagram of an Atmospheric Pressure Chemical Ionization source
configured
with two APCI sample inlet pneumatic nebulization tips oriented to spray in a
substantially
parallel direction.
Figure 12 is a cross section diagram of a two layer Electrospray probe tip.
Figure 13 is a cross section diagram of a three layer Electrospray tip.
Figure 14 is a diagram of an Atmospheric Pressure Ion Source configured with
Electrospray probe assembly and an Atmospheric Pressure Chemical Ionization
probe
assembly.
Figure 15 is a series of mass spectrum acquired separately and simultaneously
from
different sample solutions delivered to the Electrospray and APCI probes
configured as
shown in Figure 14.
Figure 16 is a diagram of an Electrospray ion source comprising two
Electrospray probes
which are configured to produce Electrospray ions of opposite polarity.
Figure 17 is a diagram of an APCI source comprising two APCI probe and
vaporizer
13
CA 02590656 2007-06-04
assemblies which are configured to produce ions of opposite polarity.
Figure 18 is a diagram of an APCI source comprising three APCI probe and
vaporizer
assemblies which are configured to produce a mixture of positive and negative
ions
simultaneously.
Description of the Invention
One embodiment of the invention, as diagrammed in Figure 1, comprises an
Electrospray
ion source which includes multiple Electrospray solution inlet probes. The
Electrospray
ion source is interfaced to a mass spectrometer which is configured in vacuum
chamber
31. Individual Electrospray probe assemblies can be configured in the
Electrospray ion
source atmospheric pressure chamber 30 where solution is sprayed from
individual probe
tips at flow rates ranging from below 25 nL/min to above 1 mL/min. The
spraying of a
solution from an Electrospray tip may or may not include nebulization assist.
Electrospray
source assembly 1 includes two ES probe sets 2 and 5 each configured with dual
ES tips.
ES dual probe assembly 2 comprises two Electrospray tips 3 and 4 configured
with
pneumatic nebulization assist. Each ES tip 3 and 4 is supplied solution
independently
through delivery lines 9 and 10 respectively. ES sprayer tips 3 and 4 are
located off
center line or axis 24 of ES source 1 as defined by the centerline of
capillary 21 orifice 23
into vacuum. A second ES dual probe assembly 5 is comprises two parallel ES
tips 6 and
7 which are configured with pneumatic nebulization assist. Solution is
independently
supplied to ES tips 6 and 7 through solution delivery lines 14 and 15
respectively during
ES operation. ES probe tips 6 and 7 are positioned near centerline 24 of ES
source 1.
Each ES dual probe assembly is configured to provide gas flow concentrically
at tips 3, 4,
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CA 02590656 2007-06-04
6 and 7 through gas supply lines 11, 8, 12 and 13 respectively. The gas flow
to each ES
probe tip can be controlled individually or collectively to allow ES operation
with
pneumatic nebulization assist or to provide gas such as oxygen or sulfur
hexaflouride
(SF6) at an ES tip to suppress corona discharge during positive or negative
Electrospray
ion production. In the embodiment shown, solutions can be Electrosprayed from
ES tips
3, 4, 5 and 6 individually or simultaneously or with combinations of
simultaneous spraying
from individual ES probe tips during Electrospray operation. A portion of the
ions
produced from the solutions Electrosprayed into ES chamber 30 are transported
into
vacuum through bore 23 in capillary 21 where they are mass to charge analyzed
by a mass
spectrometer and detector.
In the embodiment shown in Figure 1, the axis of ES tips 3 and 4 are
positioned to be
approximately parallel in dual tip ES probe assembly 2. The position of ES
probe
assembly 2 can be adjusted in the x direction and rotationally, effectively
moving ES tips
3 and 4 in the y direction. The position of ES probe tips 3 and 4 can be
locked in place
after adjustment with locking screw 16. The x and y ES tip position adjustment
sets
location and direction of the spray produced from probe tips 3 and 4 relative
to centerline
24 of ES source 1. As will be explained in more detail below, the position
adjustment
allows optimization of the ion mixture delivered to vacuum when
Electrospraying
simultaneously from ES probe tips 2 and 3 over a wide range of liquid flow
rates and
solution chemistries. Similarly, the x and rotational or y positions ES tips 6
and 7 can be
adjusted by moving ES probe assembly 5 and locking the position in place with
locking
screw 19. The x and y ES probe tip position adjustment, relative to ES source
axis 24 and
capillary orifice 23, allows optimization of performance when sprayiiig sample
solutions
CA 02590656 2007-06-04
from ES probe tips 6 and 7 individually or simultaneously. As is diagrammed in
Figure 6,
ES probe assemblies 2 and 5 may alternatively be configured to include full x-
y-z tip
position adjustment. Depending on the initial ES dual probe assembly mounting
position
and the range of tip position adjustment, the orientation of the ES probe tip
axis may be -
configured to extend over a range of angles from 0 to greater than 90 degrees
relative to
the x-z ES source plane. Zero degrees is defined as the z axis pointing into
bore 23 of
capillary 21. An ES probe tip axis, and consequently the centerline of an
Electrospray
plume produced, can be oriented maximize the production of ions near nose
piece 25
opening 28 to optimize performance. Charged liquid droplets produced in the
Electrospray
or pneumatic nebulization assisted Electrospray process evaporate to form ions
in
Electrospray chamber 30 aided by heated countercurrent drying gas 27 flowing
through
endplate nosepiece opening 28. A portion of the ions formed in ES chamber 30
are
directed into capillary bore 23 where they are swept into vacuum by the gas
flow through
capillary bore 23. Charged droplet evaporation can also occur during the
transfer of
partially evaporated Electrosprayed charged droplets into vacuum through
capillary bore
23. Capillary 21 can be heated to aid in the charged droplet evaporation
process. A
detailed description of the invention is given below using the cross sections
diagrams
shown in Figures 2 and 3.
Figure 2 is a top view diagram of an Electrospray ion source 1 showing dual
tip ES probe
assembly 5. Figure 3 is a side view of ES source I shown in Figure 1
configured with
dual off axis probe assembly 2 and 5. ES source 1, is operated by applying
electrical
potentials to cylindrical electrode 20, endplate electrode 26 and capillary
entrance electrode
40 while maintaining all ES electrode tips at ground potential. Heated counter
current
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CA 02590656 2007-06-04
drying gas flow 41 is directed to flow through endplate heater 42 and into ES
source
chamber 30 through endplate nosepiece 25 opening 28. The orifice into vacuum
as shown
in Figures 1 and 2 is a dielectric capillary tube 24 with entrance orifice 48.
The potential
of an ion being swept through dielectric capillary tube inner bore 23 into
vacuum is
described in U.S. patent number 4,542,293. To produce positive ions, negative
kilovolt
potentials are applied to cylindrical electrode 20, endplate electrode 26 with
attached
electrode nosepiece 25 and capillary entrance electrode 40. Typically, for
generating
positive ions, -4,000, -3,500 and -3,000 Volts are applied to capillary
entrance 40, endplate
26 and cylindrical electrode 20 respectively during Electrospray operation and
ES probe
assemblies 2 and 5 with ES tips 3, 4, 6 and 7 remain at ground potential. To
produce
negative ions, the polarity of the electrical potentials applied to electrodes
20, 26 and 40
are reversed while ES probe tips 3, 4, 6 and 7 remain at ground potential.
Alternatively,
if a nozzle, thin plate orifice or conductive metal capillaries are used as
orifices into
vacuum, kilovolt potentials can be applied to ES probe tips 3, 4, 6 and 7 with
lower
potentials applied to cylindrical electrode 20, endplate electrode 26 and the
orifice into
vacuum during operation. Alternatively, heated capillaries, nozzles or thin
plate orifices
can be configured as the orifice into vacuum operating with or without counter
current
drying gas during ES or APCI ionization.
Referring to Figure 2, when the appropriate potentials are applied to elements
6, 7, 20, 26
and 40 in ES source chamber 30, charged liquid droplets are produced from the
unassisted
Electrospraying or Electrospraying with pneumatic nebulization assist of
separate solutions
delivered to ES tips 6 and 7. In the embodiment shown in Figure 2, the
position of ES
tips 6 and 7 are fixed relative to each other during Electrospray operation.
Alternatively,
17
CA 02590656 2007-06-04
ES probe assembly can be configured to allow adjustment of the relative
positions of tips
6 and 7. The charged droplets Electrosprayed from each solution exiting from
ES tips 6
and 7 are driven by the electric field against the counter current drying gas
flow 27. As
the charged droplets evaporate, ions are formed from the components originally
in the
solutions delivered through tips 6 and 7, and mix in region 43. A portion of
the mixture
of ions in region 43 is swept into vacuum through the capillary bore 23 are
directed into
mass analyzer and detector 45, located in vacuum region 46, where they are
mass
analyzed. If a heated capillary is configured as an orifice into vacuum with
or without
counter current drying gas, a mixture of partially evaporated charged droplets
sprayed
from ES tips 6 and 7 are swept into the heated capillary orifice. Charged
droplet
evaporation and the production of a mixture of ions can occur in the capillary
when
Electrosprayed charged droplets are not completely evaporated in atmospheric
pressure
chamber 30 prior to being swept into the capillary orifice. The resulting ions
produced
from a mixture of charged droplets produced from two Electrosprayed solutions
in the
heated capillary will form an ion mixture in the capillary and in vacuum. Ions
formed
from multiple solutions can also be mixed and stored in ion traps in vacuum.
Three
dimensional ion traps and multipole ion guides operated in two dimensional
trapping mode
can hold mixtures of ions which are trapped simultaneously or sequentially
from multiple
solutions sprayed in one APIsource. Mass analysis of the ion mixtures is then
conducted
using mass analyzer and detector assembly 45.
For example, the multiple ES probe API source embodiment shown in Figure 1 can
be
interfaced to a multipole ion guide Time-Of-Flight mass analyzer where the
multipole ion
guide is operated in two dimensional trapping mode as described in U.S. Patent
Number
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CA 02590656 2007-06-04
5,689,111. lons fornied (i-om spraying a solution 1i=om ES probe 7 can
initially be trapped
by a multipole ion guicle operated in two climensioilal trapping iiiocle. T'he
solution flow
to ES probe 7 can tlieii be turnecl off aad a clitlerent solution flow
througlz ES probe 6
turned oli forming ions which are also trapped in the sanle nlultipole ioti
guide operating
as a two dimensional trap. T'he ioii mixture lorinecl in this ma.nuer cail be
trapped fot- a
period of titne to promote iozl-ion interactions or ion-molecule interactions
and/or
reactions with addecl 1ieutral baclcgrottncl gas. The restilting trappecl ion
mixture can t[leri
be released from the multipole ion guide trap at-icl tnass analyzed in the
7'iii1e-Of=Fliglit
mass analyzer. Alternatively, MS/MS" experiments can be eoncluetecl on the
trappeci ion
populatiozi as is described in U.S. Paleiit No. 6,011,259.
Two ctifferent sample solutions can be sprayed from ES ln=obe tips 6 and 7
indepencleiitly
or siniul.taneously cluriilg ES source operatioli. As described above, wlien
two solutions
are Electrosprayed, witli or without pneuniatic nebulization assist,
sintultaneously .froin
ES probe tips 6 and 7, iotls resttlting froiii the two separate sprays mix in
regiot) 43. A
portion of the ion mixtttre is swept into vacuuM through capillary bore 23
atid
subsequently mass to cllarge analyzed. Using this etnboclinlent of tlie
inven.tioii, the
sarziple solution ftom ES probe tip 11as a minir unl elfect oii the ions pi-
oducecl froni the
sauaple solution sprayed fronl ES probe tip 7. Cheniical components in the
saniple
solutions cleliveted li=oin inclependent solutioia soucces througli ES probe
tips 6 and 7 clo
not naiY in solution prior to spraying. Charged droplets azxt ioias of tlie
sarne polarity are
produced wlieii 1-.;lectrosprayin.g :Fro.m ES probe tips 6 atid 7. Charged
droplets aiicl ions of
like polarity have minimal cheiiiical iriteractim) durijig evaporation in ES
cl.ianiber 30 due
to charge repulsion so miliiiaial disto.ctioti of the illdividual ion
pohulation produced froai)
19
CA 02590656 2007-06-04
each solution occurs prior to entry into vacuum. Compounds of known molecular
weight,
referred to as calibration compounds, can be added to the solution sprayed
from ES probe
tip 6 while a sample solution is sprayed from ES probe tip 7. If the
calibration and
sample solutions are sprayed simultaneously from ES probe tips 6 and 7
respectively, the mass spectrum acquired from the resulting ion mixture
contains a set of internal calibration
peaks corresponding to the known molecular weight compounds included in the
calibration
solution. Using this embodiment of the invention a mass spectrum can be
acquired
containing an internal standard set of peaks without having mixed the
calibration and
sample compounds in solution. Known component and sample component ion mixing
occurs in the gas phase prior to mass analysis. Alternatively, the solution
flow through ES
probe tips 6 and 7 can be turned on sequentially. If oneES probe contains a
calibration
solution, sequential spraying of ES probes 6 and 7 allows acquisition of a
mass spectrum
which can be used as an external standard close in time to the acquisition of
the
subsequent sample mass spectrum. The probe positions remain fixed during
Electrospraying with MS acquisition while spraying simultaneously or
separately in time.
Including internal standards in an acquired mass spectrum allows increased
accuracy in
assignment of the molecular weights of sample related peaks contained in the
spectrum.
Internal standards in a mass spectrum can also serve to improve quantitative
accuracy.
Conventionally, to acquire a mass spectrum which includes an internal
standard, calibration
compounds are mixed with sample bearing solution prior to Electrospraying.
Typically
when acquiring a external calibration mass spectrum, the calibration solution
is delivered
through the same ES probe that the following sample solutions will flow
through.
CA 02590656 2007-06-04
Calibration compounds contaminant the transfer lines and ES probe tip internal
bore and
can result in unwanted peaks in a mass spectrum acquired from a sample
solution. Mixing
calibration compounds in solution, directly or through a layered flow
Electrospray probe
configuration, to create an internal standard in the resulting acquired mass
spectrum, can
also cause suppression of sample ion signal during the Electrospray ionization
process.
Mass calibration compounds contaminate sample delivery lines and are often
difficult to
eliminate when switching between applications that require internal standards,
external
standards or no calibration peaks in the acquired mass spectrum. Long flushing
time may
be required to remove calibration compounds from transfer lines and ES probe
assemblies,
adding to analysis time. Due to this contamination problem, mixing calibration
solutions
with sample solutions in the liquid phase does not allow rapid application and
removal of
calibration compounds during API source operation. The invention overcomes the
analytical disadvantages of mixing calibration and sample solutions to acquire
mass spectra
containing internal standards. Simultaneous operation of multiple ES probes
produces ions
from independently spraying solutions that mix in the gas phase prior to mass
analysis.
Each independent ES probe spray can be rapidly turned on and off with no
residual
unwanted compound contamination appearing in subsequently acquired mass
spectrum.
The Electrospray generated ions are produced from charged droplets produced
from
separate sprayers. Any sample or calibration ion interaction is limited to
those processes
occurring in the gas phase. As the ions produced are of the same polarity,
chemical
interference through interaction in the gas phase is minimal. By varying
relative solution
component concentrations and compositions, the invention allows independent
control of
the intensities and m/z locations between the calibration and sample component
peaks in
an acquired mass spectrum.
21
CA 02590656 2007-06-04
Adjusting the location of the ion mixing region 43 relative to nose piece
opening 28 and
capillary entrance orifice 28, varies the ratio of ions from each spray which
enter capillary
bore 23. For a given calibration solution concentration, the calibration peak
intensities
relative to the sample peak intensities can be changed by moving probe
assembly 5 in the
x direction and locking with locking knob 19. Depending on the relative liquid
flow rates
and nebulization gas flow rates through probe ES tips 6 and 7 rotational
adjustment of ES
probe assembly 5 can also be used to change the placement of ion mixing region
43
relative to capillary entrance orifice 48 to optimize performance. For many
analytical
applications, it is desirable to maximize sample ion signal even while adding
calibration
component related peaks to the acquired mass spectrum. Adjustment of the
position of ES
probe assembly 5 with fixed relative ES probe tip positions allows
introduction of
calibration peaks in an acquired spectrum with minimum sample signal loss. The
parallel
ES tip configuration allows a wide range of liquid flow rates to be sprayed
independently
from each tip with efficient mixing of ions produced. Consequently, optimal
performance
over a wide range of analytical applications can be achieved using a parallel
sprayer
configuration without the need to re-adjust the position probe assembly 5. An
example of
a mass spectrum acquired while simultaneously Electrospraying solutions
delivered at two
different liquid flow rates through two ES tips is shown in Figures 4.
An Electrospray probe assembly, similar to ES probe assembly 2, configured
with two ES
tips oriented to spray approximately in a parallel direction as diagram.med
Figures 1 and 3,
was used during acquisition of the mass spectra shown in Figures 4a through
4c.
Electrospray ion source 1 was interfaced to a quadrupole mass spectrometer for
the data
acquired in Figures 4a through 4c. Figure 4a shows mass spectrum 60 acquired
from a 10
22
CA 02590656 2007-06-04
ng/ul gramicidin S, in a 1:1 methanol: water sample solution, continuously
infused through
delivery line 9. The solution containing the gramicidin S sample was
Electrosprayed with
pneumatic nebulization assist from ES tip 3 at a liquid flow rate of 50
ul/min. The doubly
charge peak 61 of Gramicidin S is the dominant peak in the spectrum with a
relative
abundance of 3,100 as shown by ordinate 62. The orientation of the axis of ES
probe tips
3 and 4 was approximately 60 degrees angled up from the horizontal (z-x) plane
which
intersects ES source centerline 24. For the data acquired in Figure 4 82 = 60
degrees
where 92 is the angle formed by the ES probe tip axis relative to the z axis
and is axially
symmetric around the z axis. The axis of ES tips 3 and 4 were positioned
approximately
parallel and each tip was positioned an equal distance from the z-x plane
during spraying.
ES tips 3 and 4 were separated by fixed distance of approximately 8 mm during
acquisition of mass spectra 60, 64 and 68. ES tips 3 and 4 were positioned
approximately
1.5 cm along the z axis and up approximately 1.0 cm along the y axis as shown
by
dimensions Z and r respectively in Figure 3. The position of ES tips 3 and 4
along the x
axis was adjusted to optimize performance after which the dual ES tip
positions were
locked in position during acquisition of the mass spectra series shown in
Figures 4a
through 4c. A mixture of calibration compounds valine (50 ng/ul), tri-tyrosine
(25 ng/ul)
and hexa-tyrosine (50 ng/ul) in a 79% water, 19% iso-propanol and 2% propionic
acid
solution was delivered to ES probe tip 4 at a flow rate of 500 ullmin. The
calibration
solution was Electrosprayed from probe tip 4 with pneumatic nebulization
assist. Mass
spectra 64 acquired while Electrospraying the calibration solution from ES
probe tip 4 is
shown in Figure 4b. Peaks 65, 66 and 67 with mass to charge values of 118, 508
and 998
respectively were formed from the singly charged protonated molecular ions of
the
23
CA 02590656 2007-06-04
calibration components of known molecular weight. Other peaks present were
from
contamination compounds present in solution. The abundance of peak 65 (118 mlz
) is
approximately 4,300. Mass spectrum 68 in Figure 4c was acquired while
simultaneously
spraying sample and calibration solutions from ES tips 3 (50 ul/min) and 4
(500 ul/min)
respectively. Sample or gramicidin S peak 71 abundance of approximately 2,600
has been
reduced by less than 15 % when compared to the gramicidin S peak 61 acquired
when
independently sprayed. The calibration peak heights have changed less than 15%
comparing mass spectra 64 and 68 acquired with single and simultaneous
solution
spraying.
The nebulization gas flow and the calibration solution flow through ES tip 4
was turned
off during the acquisition of mass spectrum 60 shown in Figure 4a. Conversely,
the
nebulization gas flow and the sample solution flow through ES tip 3 was turned
off during
the acquisition of mass spectrum 64 shown in Figure 4b. Both calibration and
sample
solution flows and nebulization gas flows to ES tips 3 and 4 were turned on
during
acquisition of mass spectrum 68 shown in Figure 4c. Ions formed from the two
independent simultaneous Electrosprays mixed in the gas phase prior mass
analysis
allowing acquisition of a mass spectrum with an internal standard. A
quadrupole mass
analyzer was used to acquire the data shown in Figures 4a through 4c.
Alternatively, other
types of mass analyzers could be used such as Time-Of-Flight, three
dimensional
quadrupole ion traps, magnetic sector, Fourier Transform Mass Spectrometers
and triple
quadrupoles. Internal standards within a mass spectrum can be used to improve
the
accuracy of mass to charge assignments of sample peaks, particularly for mass
spectra
acquired with higher resolution. The sequence of mass spectra shown in Figures
4a
24
CA 02590656 2007-06-04
through 4c can be acquired in under one minute limited only by the mass
spectrum
accumulation time and the speed with which individual liquid flow rates can be
turned on
or off. The invention allows the efficient mixing of gas phase ions produced
from
multiple solutions Electrosprayed simultaneously over a wide range of liquid
flow rates.
Sample and calibration solutions can be introduced through multiple ES probe
tips with no
need to adjust probe tip position after initial optimization. The invention
increases the
versatility of an analytical mass analysis system that can accept multiple
solution inputs
with unattended operation. An Electrospray ion source comprising multiple
inlet probes,
configured for independent or simultaneous spraying, minimizes system
downtime,
maximizes sample throughput, allows selective acquisition of mass spectra with
internal
standards without contacninating sample solutions. As will be described below,
a multiple
inlet probe API source an also be used to study ion-ion gas phase interactions
at
atmospheric pressure.
In the example shown in Figures 4a through 4c, the solution flow to ES tips 3
and 4 was
supplied through delivery lines 9 and 10 respectively by liquid pumps which
could be
turned on or off independently with or without nebulization gas flow.
Alternatively,
solution 44 can be supplied to ES tip 7 from solution reservoir 45 as shown in
Figure 2.
Solution 45 is drawn to ES tip 7 through delivery line 15 by the venturi force
induced
from the nebulization gas supplied to ES tip 7 through line gas delivery line
13. With
solution reservoir 45 positioned below ES probe tip 7, solution flow to ES tip
7 stops
when the nebulization gas is turned off. If no nebulization assist is used
when
Electrospraying from ES tip 7, a gas pressure head can be applied to solution
45 in
reservoir 44 to aid in initially forcing liquid to ES tip 7. The electrostatic
forces from the
CA 02590656 2007-06-04
electric field applied during unassisted Electrospraying can also maintain
solution flow
through ES tip 7. Liquid flow to ES tip 78 can then be turned off by removing
the gas
pressure head on solution 45 in reservoir 45 and reducing the electric field
at ES tip 7.
Unassisted Electrospray can be turned on or off by applying the appropriate
relative
potentials to an individual ES tip and then removing the potential from the
tip. For
example if two independent ES probes are configured in an ES source and 6,000
volts is
applied to each probe independently during ES operation then the spraying from
a given
probe can be switched on or off by applying kilovolt potentials to the ES
probe or lowing
the probe voltage to stop the Electrospray. Each ES tip 3, 4, 5 and 7 can be
individually
configured to optimize performance for a specific set of applications with a
range of liquid
flow rates and solution chemistries. ES tips can be configured with single,
double and
triple tube layers to accommodate various gas and liquid layers at the ES tip
connected to
specific solution and gas delivery lines. Single layer tips such as
replaceable microtips
which allow low ES low rates may be pre=loaded prior to installation in an ES
source and
do not require solution delivery lines. Multiple microtips can be configured
to spray
simultaneously if is desirable to acquire mass spectra with an internal
standard while
Electrospraying at liquid flow rates in the 25 to 500 nanoliter per second
range. For
higher liquid flow rates, layered ES tip configurations are typically used.
Figure 12 is a diagram of a two layer Electrospray tip. With a two layered ES
tip
configuration, nebulization gas 74 can be supplied through annulus 71 between
a second
layer tube 70 surrounding liquid sample introduction tube 72 to assist the in
the formation
of charged liquid droplets during Electrospray operation. Sample bearing
solution is
delivered to exit end 73 of inner tube 72 through bore 75. A second liquid
layer can be
26
CA 02590656 2007-06-04
delivered through annulus 71 replacing the gas flow if liquid layering is
desired during
operation at the ES probe tip. Alternatively, ES probe tips may be configured
with three
concentric layers as diagrammed in Figure 13. Typically with a three layer ES
probe,
sample solution is introduced through bore 88 of inner tube 80, a second
solution can be
introduced through annulus 84 between tubes 80 and 81 and, if required, a gas
flow 85
can be delivered through annulus 83 between tubes 81 and 82. The solutions
delivered
through bore 88 and annulus 84 mix at the first layer tube exit 86 in region
87 during ES
operation. The second solution delivered through annulus 84 may contain known
calibration compounds which mix with the sample solution delivered through
bore 88 in
region 87 during ES operation. Conventionally, calibration compounds are mixed
with
sample bearing solution prior to the solution being delivered through bore 88.
One ES probe tip or combinations of ES probe tips 3, 4, 6 and 7 can be
configured as two
or three layer assemblies similar to that shown in Figures 12 and 13.
Depending on the
analytical application, solution introduction tube 72 or 80 can be configured
as a Capillary
Electrophoresis column, a microbore packed capillary column, or an open bore
tube of
either dielectric or conductive material. Single, two and three layer ES probe
tips which
are configured in off-axis positions or positioned near the API source
centerline are
conunercially available. An off-axis probe position is typically used for
higher liquid flow
rate applications in Electrospray ion sources. The present invention embodies
the
configuration of multiple ES probes with single, double or triple layer tips
in an API
source with the ability to conduct individual or simultaneous spraying of
solution from two
or more probe tips with or without nebulization assist. Multiple probe tip
positions can be
fixed during API operation allowing sequential or simultaneous spraying from
multiple tips
27
CA 02590656 2007-06-04
without the need to adjust probe location and allowing rapid, efficient and
unattended
switching of solution spraying from variety of inlet probes.
Figure 5 shows an alternative embodiment of the invention. Electrospray source
114 is
configured with ES probe assembly 90 comprised of six ES tips 91 through 96
with
individual liquid supply lines 101 through 106 respectively. Position adjuster
97 can be
used to move ES probe assembly 90 such that any ES tip can be located near ES
source
centerline 115. Gas line 98 supplies nebulization gas to ES probe tips 91
through 96.
Alternatively, ES probe assembly 90 can be configured such that each ES tip 91
through
96 is configured with an individual nebulization gas supply each of which can
be
independently turned on and off. In the embodiment diagrammed in Figure 5 ES
tips 95
and 92 can be supplied with individual calibration solutions while separate
sample
solutions are supplied to ES tips 91, 93, 94 and 96. With this arrangement,
mass spectra
acquired from the Electrospraying of any sample solution can have internal
standard peaks
added by turning on the nearest adjacent ES tip supplied with calibration
solution. In the
embodiment shown in Figure 5, several sample solutions can be rapidly analyzed
with
little or no cross contamination which can occur when multiple samples are
delivered to
the ES source sequentially through the same ES probe tip. After acquiring MS
data from
a sample solution spraying from ES tip 96 simultaneously with a calibration
solution
spraying from ES probe tip 95, ES probe assembly 98 can be translated using
adjuster 97
such that ES tip 94 is positioned near ES source centerline 115. ES tip 95 can
be used to
spray calibration solution simultaneously with the Electrospraying of a sample
solution
from ES tip 94 to provide internal standard peaks in the acquired sample
solution mass
spectrum. Further ES probe assembly translation can be used to position ES tip
92 near
28
CA 02590656 2007-06-04
ES source centerline 115 to selectively spray calibration solution during
sample solution
Electrospraying from either tips 91 or 93. The linear ES tip configuration of
ES probe 90
can be extrapolated into a two dimensioni array of tips with automatic x and y
position
translators. Also, flow-through ES tips can be replaced by pre-loaded
microtips.
Alternatively, all tips of ES probe assembly 90 can be used to spray sample
solutions and
a single off axis ES probe can used to Electrospray calibration solution when
it is
desirable acquire a external standard calibration mass spectrum or to add an
internal
standard to the acquired sample solution mass spectra. Kilovolt potentials can
be applied
to ES source elements 110, 111 and 112 to initiate Electrospray with ES probe
assembly
90 operated at ground potential. Alternatively, kilovolt electrical potentials
can be applied
to ES probe tips 91 through 96 during Electrospray operation. ES source 114
can be
configured with heated counter current drying gas to aid in the evaporation of
the
Electrospray produced charged droplets sprayed sequentially or simultaneously
from one,
two or more ES tips.
The ES probe tip positions can either be fixed with respect to each other and
the ES
source capillary entrance or the tip positions can be adjustable. As is shown
in Figure 1,
ES tip positions 3 and 4 are fixed relative to each other but, as a set,
movable in the x
direction and rotationally around the ES probe 2 mounting block rotational
axis. An
alternative to the invention is shown in Figure 6 where ES probe assemblies
120 and 122
include full x, y and z position adjustments for ES tips 121 and 123
respectively. ES
probe assembly 122 is positioned parallel to ES source 130 centerline 131. The
angle of
ES probe tip 123 axis 124 relative to ES source centerline 130 is equal to
zero degrees, 0,
= 00. Sample bearing solution can be introduced into liquid delivery tube 129
of ES probe
29
CA 02590656 2007-06-04
assembly 122 or into entrance tube 132 of ES probe assembly 120 with
independent liquid
delivery systems. In this manner, different samples or mixture of samples andl
or solvents
can be sprayed simultaneously or individually. Liquid delivery systems may
include but
are not limited to, liquid pumps with or without auto injectors, separation
systems such as
liquid chromatography or capillary electrophoresis, syringe pumps, pressure
vessels,
gravity feed vessels or solution reservoirs. During ES source operation, the
spray
produced from each ES probe can be initiated by turning on the liquid flow
using a
solution delivery system. With the appropriate solution reservoir
configuration, pneumatic
nebulization gas flow can also be used to initiate Electrospray. When
nebulization assist is
not used, the Electrospray from either ES tip 121 or 123 can be turned on by
increasing
the voltage applied to an ES tip relative to the voltage applied to ES source
electrodes
140, 141 and 142. For example, if the voltages applied to capillary entrance
electrode
140, endplate and nosepiece 141 and cylindrical electrode 142 are set at -500,
0 and +500
V respectively, the Electrospray from ES tip 121 can be initiated by
increasing the voltage
applied to ES tip 121 to + 5,000 V. The Electrospray from ES tip 121 can be
stopped by
setting the potential applied to ES tip 121 to 0 V. Electrospray from ES tip
123 would
remain off with an appropriate voltage (approximately OV) applied to ES tip
123 such that
the electric field at ES tip 123 is effectively neutral. Electrospray from ES
tip 123 can be
turned on by applying +5,000 V to ES tip 123. Nebulization gas supplied to ES
tips 121
and 123 through gas delivery lines 136 and 128 respectively can be turned on
when
kilovolt potentials are applied to the ES tips to aid in the Electrospray
charged droplet
formation process. The nebulization gas flow to an individual ES tip can be
turned off
when the appropriate voltage is applied to the ES tip to shut off the
Electrospray.
Switching voltage and nebulization gas would allow rapid turning on and off of
the
CA 02590656 2007-06-04
Electrospray at an ES tip even if the sample bearing solution continued to
flow through
the tip for a period of time. Alternatively, as was shown in Figure 2 where a
reservoir is
used as a solution source, the liquid flow to ES probe tip 123 or 121 can be
controlled by
turning the nebulization gas flow on or off. When the nebulization gas flow is
turned on,
the venturi effect at the ES probe tip pulls solution from the reservoir to
the ES probe tip
where it is nebulized. In the case where Electrospray is sustained by
supplying pneumatic
nebulization gas flow to the ES probe, a simple and inexpensive solvent
delivery system
can be employed.
ES probe assembly 120 axis 137 shown in Figure 6 is positioned at an angle of
70
degrees, 0120 = 70 , from ES source centerline 131. ES probe assembly 120 is
configured
with three layer ES probe tip 121 having sample solution inlet 132, layered
flow solution
inlet 138 and nebulization gas inlet 136. A diagram cross section of ES probe
tip 121 is
shown in Figure 13. Liquid sample enters bore 88 of first layer tube 80
through transfer
line 132. A second solution can be added through transfer line 138 into
annulus 84
between tubes 80 and 81 and this solution forms a sheath liquid surrounding
and mixing
with the sample solution at exit end 86 in region 87. Nebulizer or corona
suppression gas
can be introduced to ES probe tip 121 through gas delivery or transfer line
136 into
annulus 83 between tubes 81 and 82. Liquid layering of solutions in region 87
at the tip
of three layer ES probes has been used to interface LC, CE or CEC separation
systems to
ES sources. When interfacing to CE, CEC or microbore LC columns, sample
introduction
tube 80 may actually be the CE, CEC or LC column itself. The second layer
solution
flow may also be used to add a calibration compounds to the sample solution
exiting from
tube 86 of ES probe tip 121. The resulting mass spectrum acquired from such a
mixed
31
CA 02590656 2007-06-04
solution spray would contain an internal standard. The calibration solution
could be
started or stopped by turning on or off the liquid delivery system supplying
solution
through transfer line 138. The introduction of a calibration solution in this
manner avoids
contaminating the original sample solution source but still necessitates
mixing of solutions.
in region 87 prior to spraying. The calibration components in the resulting
mixture may
effect the Electrospray ionization efficiency of the sample compounds present
thus causing
peak height distortion in the acquired mass spectrum. The relative positioning
of the exit
ends of tubes 80 and 81 can effect the relative ititensity of ion populations
layered from
the two solutions produced in the Electrospraying process. The layered liquid
flow can
also be used to introduce a diferent solvent system to study ion-neutral
interactions in a
multiple probe spray mixture. A range of solution compositions can combined in
the
liquid phase using the three layer probe tip assembly shown in Figure 13 if
required in an
analytical application. A four layer variation of the three layer probe shown
in Figure 13
can be operated such that no liquid mixing occurs by separating the liquid
solution layers
with nebulizer or corona suppression gas. For example, a four layer probe tip
embodiment
can have liquid solution delivered through the innermost tube one, nebulizer
gas supplied
through the annulus between tubes one and two, a second liquid solution
delivered through
the annulus between tubes two and three and a nebulizer gas supplied through
the annulus
between tubes 3 and 4. Alternatively, gas can be supplied through the
innermost tube one
with a liquid, gas and liquid layering. Three or more liquid solutions can be
layered
where some of the solutions delivered through separate layers are mixed in the
liquid state
as they emerge from the layered tip similar to the solution mixing shown in
Figure 13.
Layered liquid flow allows the introduction of additional solutions through
one or more
Electrospray probes, and can serve as a means of interfacing ES with one or
more
32
CA 02590656 2007-06-04
separation systems such as CE, CEC and LC.
ES probe tip 123 is configured as a two layer probe, shown in Figure 12, with
calibration
solution 145 supplied from reservoir 144. With little or no pressure head or
gravity feed
applied, calibration solution 145 can be pulled from reservoir 144 using the
venturi suction
effect of the nebulizing gas applied at ES probe tip 123. Calibration solution
144 can be
sprayed from ES tip 123 when nebulization gas flow is applied through gas
delivery tube
128. Solution delivery tube 139 can be initially filled with solution by
applying head
pressure to reservoir 144, by gravity feed from reservoir 144 or by turning on
the
nebulizing gas ES probe tip 123. Once solution delivery tube 129 and the inner
tube of
ES tip 123 are filled with calibration solution, any head pressure in the
attached reservoir
is relieved and, with no gravity feed applied, the liquid flow through
solution delivery tube
129 can be started and stopped by turning the nebulizing gas flow to ES tip
123 on and
off. Calibration solution can be selectively sprayed from ES probe tip 123
individually or
simultaneously with a sample solution Electrosprayed from ES probe tip 121.
Alternatively, solution can be delivered to ES probe tip 123 using a syringe
pump, liquid
chromatography system or other liquid delivery system. Solution flow to ES
probe tip 123
can then be turned on or off by turning the solvent delivery system flow on or
off.
The x-y-z and angular positions of ES probe tips 121 and 123 relative ES
source axis 131
and capillary entrance 148 as shown in Figure 6 may be adjusted to optimize ES
performance while spraying from single ES probes individually or from two ES
probes
simultaneously. The rotational position of ES tip 121 around ES probe assembly
axis 137
is adjusted with positioning knobs 133 and 134. The position of ES tip 121
along the axis
33
CA 02590656 2007-06-04
of ES tip 121 is adjusted by turning knob 135. Similarly, the rotational and
axial position
of ES tip 123 is adjusted with positioning knobs 125, 126 and 127
respectively. ES probe
tip positions may require adjustment to optimize ES performance for given
liquid flow
rates and solution or sample types. Once optimized for individual or
simultaneous
spraying, the probe positions can remain fixed during ES operation. For the
embodiments
shown in Figures 1 and 6, a portion of each ES probe assembly is located
outside the ES
source chamber housing. This allows full adjustment of x-y-z and angular
position while
operating the ES source to achieve optimal performance. ES probe assemblies
120 and
122 as diagrammed in Figure 6 also allow adjustment of the relative layered
tube exit tip
positions. For example, adjustment of nut 149 will move the inner tube 80 exit
86
position, as shown in Figure 13, along the axis of ES probe tip 121 relative
to the second
and third layer tube exit positions. The relative position of innermost tube
exit end 73 as
shown in Figure 12 can be adjusted using nut 150 for optimizing the nebulizing
gas
performance at ES tip 123. These ES tip adjustments allow for optimization of
layered
liquid flow and/or gas nebulization tube tip positions while operating the ES
source.
Different liquid flow rates can be delivered through ES probe tips 121 and 123
during
simultaneous Electrospraying from both ES probe tips. The solution flow rate
range used
for ES applications extends from below 25 nanoliters per minute to over 2
milliliters per
miniute. For a 25 to 1,000 nanoliters per minute range of liquid flow rates, a
single layer
flow through or replaceable micro Electrospray probe tip can be configured to
replace two
layer ES probe tip 123 in ES source 130. Unassisted Electrospray operation can
be
conducted from ES probe tips individually or simultaneously with pneumatically
assisted
ES probes. Two or more pneumatic nebulization assisted ES probes configured
with full
tip position adjustment can be operated simultaneously in one ES chamber.
Combinations
34
CA 02590656 2007-06-04
of single, two layer and three layer ES probes can also be configured and
operated
simultaneously in a single ES chamber.
ES source 130, as diagrammed in Figure 6, is configured with two ES probes
with
independent adjustable ES tip positions. Axis 124 of ES probe assembly 122 is
positioned
along ES source centerline 131 with ES probe tip 123 spaced a distance Z1
along ES
source centerline 131 from endplate nosepiece face 149. Axis 137 of ES probe
assembly
120 is positioned at an angle of 0120 = 70 degrees relative to ES source
centerline 131.
Tip 121 of ES probe assembly 120 is shown located a distance Z2 axially from
end plate
nosepiece face 149 and a distance r2 radially from ES source centerline 131
with a radial
angle 6,20 = 0 degrees. Angle B; is defined as the radial angle around
centerline 131
looking in the direction that the gas flows through the capillary or the
positive z axis
direction as shown in Figure 1. The 12 o'clock position above centerline 131
is defined as
0 degrees with the angle increasing clockwise to 360 degrees. Setting Z, = 2
cm, Z2
=
1.5 cm and r2 = 1.5 cm, higher liquid flow rates can be introduced through ES
probe tip
121 and lower liquid flow rates, with a solution containing calibration
compounds, can be
introduced through ES probe tip 123. Both ES probe tips 121 and 123 can be
operated
with pneumatic nebulization assist, for the tip positions and angles given.
When higher
liquid flow rates are sprayed from ES probe tip 123 the probe tip axis angle,
di122, relative
to ES source centerline 131 can be increased by turning adjustment knobs 125
and/or 126,
Alternatively ES probe assembly 122 can be positioned off ES source centerline
131 but
spraying approximately in a direction parallel to centerline 131. Depending on
the specific
analytical problem requiring ES MS analysis or ES MS/MS analysis, multiple ES
probes
CA 02590656 2007-06-04
can be positioned in the ES source to optimize performance for individual or
simultaneous
spraying operation.
Mass spectra acquired from a dual probe ES source configured similar to that
shown in
Figure 6 are shown in Figures 7a through 7c. Figure 7a shows a mass spectrum
of a
sample solution of 1:1 methanol:water containing Leucine Enkephalin
Electrosprayed with
pneumatic nebulization assist at a liquid flow rate of 100 ul/min from ES
probe tip 123 in
dual probe ES source 130. Protonated m/z peak 153 of Leucine Enkephalin is the
dominate peak in acquired mass spectrum 150. No solution was flowing through
off axis
probe ES probe tip 121 during acquisition of mass spectrum 150 shown in Figure
7a.
Mass spectrum 151 shown in Figure 7b was acquired while a Electrospraying,
with
pneumatic nebulization assist, a calibration solution from ES probe tip 121
configured in
dual probe ES source 130. The calibration solution contained containing known
molecular
weight compounds Tri-Tyrosine (50 pmoUul) and Hexa-Tyrosine (50 pmol/ul) in an
80:20
solution of water:isopropanol with 2% propionic acid delivered from a sample
reservoir at
a flow rate of 5uUmin. Calibration solution flow was driven primarily by the
venturi force
of the pneumatic nebulization gas flow at ES tip 121. Protonated molecular
ions of Tri-
Tyrosine 154 and Hexa-Tyrosine 155 are the primary peaks in mass spectrum 151.
No
solution was flowing through the ES probe tip 123 during acquisition of
calibration
spectrum 151 shown in Figure 7b. Figure 7c shows mass spectrum 152 acquired
while
simultaneously spraying calibration and sample solutions from ES probe tips
121 and 123
respectively. Protonated molecular ion peaks 156 and 158 resulting from
Electrospraying
of the calibration solution can be used as an internal standard to improve the
accuracy of
the calculated mass assignment of the sample Leucine Enkephalin peak 157 or
another
36
CA 02590656 2007-06-04
unknown compound molecular weight. As was shown in Figures 4a through 4c,
little
signal loss is observed when comparing single and dual probe spraying. ES
probe tip 121
and 123 positions were not changed during acquisition of the mass spectra 150,
151 and
152 shown in Figure 7.
It is obvious to one skilled in the art that any number of combinations of
multiple
Electrospray probe tip positions may be configured in an Atmospheric Pressure
Ion Source
where:
the Electrospray tip angles (0l, 02, .... ON) can range from ~t = 0 to 180 ,
the Electrospray tip locations (ri, 6,, zl,), (r2, 02, zZ), ... (rN, BN, ZN)
can have values
where r; may equal any distance within the ES chamber, 8; = 0 to 3600
measured clockwise, and z; may equal any distance within the ES chamber,
and
the relative Electrospray tip radial angle of separation (9 1- 9 2), ... (8 1-
0 N) for
any two ES probe tips i and k can range from Bi- 0 k= 0 to 360 ,
Electrospray probe assemblies may be configured with two or more parallel tips
or with
individual tips. ES probe tip positions may be adjustable or fixed in the ES
chamber.
Although Figures I and 6 show Electrospray sources configured with one off
axis ES
probe assembly, several off axis ES probe assemblies with different angles 0t
can be
configured into an ES source chamber which may also include an ES probe
assembly
located near the ES source centerline. In addition, individual Electrospray
probe tips may
be configured with but not limited to any of the following ES tip types: a
single layer
37
CA 02590656 2007-06-04
Electrospray probe tip, a replaceable micro Electrospray tip, a flow through
micro
Electrospray tip, a pneumatic nebulization assisted Electrospray tip with or
without liquid
layer flow, an ultrasonic nebulizer assisted Electrospray tip or a heated
Electrospray tip.
Any combination of ES probe tip types can be configured into an ES source and
operated
individually or simultaneously. ES probes can be configured to extend through
the wall of
the ES chamber or be mounted entirely within the ES chamber.
Figure 8 is a diagram of an alternative embodiment of the invention where
three ES
probes are configured within ES source 160. Electrospray source 160 includes
cylindrical
electrode 162 dielectric capillary 163, counter current drying gas 167, gas
heater 168,
endplate electrode 165 and attached endplate nosepiece 166. Alternatively, a
non dielectric
capillary, a heated capillary, a flat plate orifice or a nozzle can be
configured as an orifice
into vacuum replacing dielectric capillary 163. Multiple ES source probes can
be
configured with different arrangements of drying gas flow direction relative
to the ES
source axis and the axis of the orifice into vacuum such as those arrangements
used with
"z spray" or "pepperpot" Electrospray source geometries. ES probe assemblies
170, 171
and 172 are mounted in ES source chamber 161 each with x-y-z and angular
position
adjustment of ES probe tips 173, 174 and 175 respectively as was previously
described for
the ES probe assemblies 120 and 122 in Figure 6. In the embodiment shown in
Figure 8,
the x-y-z and angular position of ES probe tips 173, 174 and 175 can be
adjusted during
tuning of Electrospray source performance. Each ES probe tip position can be
adjusted to
optimize ES-MS or ES-MS/MS" performance during single or simultaneous multiple
probe
operation for a wide range of combinations of liquid flow rates and solution
compositions.
Once the positions of ES probe tips 173, 174 and 175 are optimized during ES-
MS
38
CA 02590656 2007-06-04
operation tuning, no further adjustment is required during ES source operation
and MS
data acquisition. ES probe assemblies 170 and 172 are each configured with
three layer
ES probe tips 173 and 175 respectively as is shown in Figure 13. ES probe
assembly 171
is configured with two layer ES tip 174 as is shown in Figure 12. Solution can
be
Electrosprayed from ES probe assemblies 173 and 175 with or without pneumatic
nebulization assist and/or liquid layer flow. The positions of ES tips 173,
174 and 175
are, Z173, R1731 Zi75, R175 and Z174 respectively with ES tips 173 and 175 set
spray
angles of (P173 and 0175, and radial angles 0 173 and 0 175, respectively. As
examples shown
in Figure 8, ES probe tip 173 is set at an angle of + 60 degrees (0173 = + 60
) and ES
probe tip 175 is set at an angle of -60 degrees (0I75 =- 60 or +300 degrees)
relative to
ES source centerline 177. The included angle, (0i73 -'0175), between ES probe
tips 173
and 175 in the embodiment shown is 120 degrees, however, this included angle
can vary
from zero degrees to 180 degrees. The relative radial angle of separation
between ES
probe tips 173 and 175 (0173 - 9175 ) equals 180 degrees. ES probe tip 174 is
positioned
with its axis falling on ES source centerline 177. The relative angle between
either ES
probe tip 173 or 175 and ES probe tip 174 is 60 degrees. The relative angles
between all
ES tips probes mounted simultaneously in ES source chamber 161 can vary from
close to
zero to over 180 degrees depending on the analytical application being run.
The radial
probe separation can range from 0 to 360 degrees. Multiple ES probes can
alternatively
be mounted on ES source back plate 179 as is shown in Figure 1 or through the
side walls
of ES chamber 161 as shown in Figure 8, each with fixed positions or
individual position
adjusters. One or more ES probes can be mounted on the back plate as shown in
Figure 1
or ES probe assemblies mounted on back plate 178 may be configured with one or
more
39
CA 02590656 2007-06-04
ES probe assemblies which extend through a side wall or walls of ES chamber
161 as
shown in Figure 8.
A portion of the ions produced from the simultaneous Electrospraying of
solutions from at
least two of ES probes tips 173, 174 and / or 175 are swept into vacuum,
through capillary
orifice 164, where they are mass analyzed. With the appropriate liquid
delivery systems,
the solution flow to ES probe tips 173, 174 or 175 can be turned on or off
independent of
the layered liquid flow or nebulizer gas flow supplied to any given ES probe
tip. For
example, Electrospray from ES probe tip 173 can be turned off if the sample
liquid flow
through line 179 to ES probe assembly 170 were tuned off independent of
whether the
sample liquid flow through line 180 to ES probe assembly 172 remains on. The
nebulizer
gas flow to ES probe assembly 170 supplies through line 180 can remain on
independent
of the sample solution flow status through line 178. Leaving the nebulizer gas
flow on,
even with solution flow through ES probe 170 turned off, retains the optimal
drying gas
flow characteristics in ion mixing region 182 where the nebulization gas from
ES probes
and ES source counter current gas flow 183 meet. After the gas flow balance
into region
182 has been optimized, the gas flow into this region can remain constant even
when
sample flow is introduced through one or more ES probes individually or
simultaneously.
Optimal ES-MS performance can be achieved when multiple nebulization gas flows
remain
on even with combinations of sample flows being turned on an off independently
through
multiple ES probe tips. Alternatively, the gas and liquid flow supplied to ES
probe tip
175 can be alternately switched on when the gas and liquid flow supplied to ES
probe tip
173 is turned off. The liquid and gas flow through ES tip 174 can remain ion
while
spraying sample solution from either ES probe tips 173 or 175. In the
embodiment
CA 02590656 2007-06-04
diagrammed in Figure 8, ES probe tips 173 and 175 are located in a positions
that are
radially symmetric relative to the position of ES probe tip 174. Gas flow
through ES
probe tips 173 and 175 can be adjusted to be symmetric and equal in mixing
region 182
when the liquid and gas flows to ES probe tips 173 and 175 are switched on and
off in an
alternating manner. The relative positions of each probe can also be adjusted
so that
performance is optimized different liquid flow rates are delivered through ES
probe tips
173 and 175. In the case of alternating Electrospraying through ES probe tips
173 and
175, calibration solution can be delivered through ES probe 174 to provide an
internal
standard in the acquired mass spectrum when spraying individually or
simultaneously from
ES probe tips 173 and 175. When a heated capillary is configured in API
source, heated
counter current gas flow 183 may or may not be required. Partially evaporated
charged
liquid droplets swept into a heated capillary evaporate further on the way to
vacuum. Ions
produced from multiple solution sources, mix in partial vacuum or in vacuum
prior to
mass analysis. Ion mixtures may be formed by trapping ions produced from
different
Electrospray probes in three dimensional ion traps or multipole ion guides
operated as two
dimensional ion traps in vacuum as well. Mixtures of ions in three and two
dimensional
ion can be formed by trapping ions formed from simultaneous or individual
sequential
Electrospraying from multiple ES probes.
Individual separation systems such as LC, CE or CEC can serve as the solution
delivery
systems to different ES probes configured in an ES chamber. Multiple ES probes
configured in an Electrospray ion source allow a single ES mass spectrometer
system to
serve as a detector for multiple separation systems without the need to switch
eluting
samples through a common probe. A common ES probe may not be optimally
configured
41
CA 02590656 2007-06-04
or even compatible for each separation system configured with the ES source.
Multiple
ES probes avoids cross contamination from one sample injection to the next
delivered
from individual separate systems. The separation of compounds spatially in
solution is
generally the slow step of an LC, CE or CEC MS analytical analysis,
particularly when a
mass spectrometer capable of rapid data acquisition, such as Time-Of-Flight,
is used. The
use of multiple ES probes combined with efficient manual or automated sample
introduction increases analytical throughput with no risk of performance loss
due sample
cross contamination. The mass spectrometer, configured to operate in MS or
MS/MSn
mode with multiple separation systems, can serve as a detector for a wide
range of
chemical analysis run in a manual or automated mode without the need to change
or adjust
component hardware. One embodiment of multiple separation systems interfaced
to a
single ES source is diagrammed in Figure 8. A first gradient liquid
chromatography
system 184 comprises LC gradient pump 185, injector valve 186, manual or auto
injector
187, liquid chromatography column 188, switching valve 191, and connecting
line 180 to
ES probe assembly 172. Similarly, a second gradient LC system 194 comprises LC
gradient pump 195, injector valve 196, manual or auto injector 197, liquid
chromatography
column 198, switching valve 199, and connecting line 179 to ES probe assembly
170.
Sheath liquid flow can be delivered through transfer line 192 to ES probe
assembly 172
and through connecting line 201 to ES probe assembly 170. Nebulizing gas is
delivered
through lines 193 and 181 to ES probe assemblies 172 and 170 respectively. In
the
configuration shown, the following sequence could be used to double the sample
throughput with LC-MS analysis using one Electrospray mass spectrometer
detector.
Assume that during each LC-MS run, calibration solution is sprayed
continuously from ES
42
CA 02590656 2007-06-04
probe tip 174 while MS data is being acquired. The LC-MS analytical sequence
begins
with valve 191 switched so that solution delivered from LC gradient pump 185
is directed
to flow through line 189 with no sample solution flow directed to ES probe
inlet line 180.
With valve 191 switched to this position, column 188 can be flushed or
reconditioned after
an LC gradient run without introducing contamination into ES source 160. The
pneumatic
nebulization gas flow to ES probe tip 175 may or may not be turned on
depending on how
the gas flows in mixing region 182 are initially balanced. Valve 199 is
switched so that
solution delivered from LC gradient pump 195 flows into transfer line 179 to
ES probe
assembly 170 exiting at ES probe tip 173. LC column 198 has been reconditioned
or
flushed and the solution composition being delivered from LC pump 195 is the
solution
required for initiation of an LC gradient run. Sample is injected from manual
or
autoinjector 197 into valve 196 and an LC separation is initiated when
injector valve 196
is switched from load to run placing the injected sample on line with column
198.
Nebulization gas and, if required, liquid layered flow is delivered to ES
probe tip 173 in
addition to the sample solution. As the LC gradient separation through column
198
proceeds, components eluting from column 198, travel through valve 199 and
line 179
where they are Electrosprayed from tip 173. A portion of the ions produced the
sample
solution during the Electrospray ionization process are subsequently mass
analyzed.
During and prior to the completion of the analytical gradient LC run which is
occurring in
LC column 198, column 188 is being flushed, reconditioned, or re-equilibrated
and the
solution gradient reset for another LC gradient separation. When the LC
gradient run
through column 198 is complete, valve 199 is switched so that the eluate from
LC column
198 flows through line 202 and not through line 179. Alternatively, an
additional solvent
flow can be supplied through line 200 into line 179 through valve 199 in this
switch
43
CA 02590656 2007-06-04
position to flush line 179 prior to the start of the LC gradient run through
ES probe
assembly 172. When valve 199 is switched to divert the flow through column 198
to line
202, valve 191 is switched to connect the flow exiting column 188 to line 180
and ES
probe assembly 172. If the pneumatic nebulization gas flow to ES probe 172 was
turned.
off while the gradient LC run through column 198 was occurring, it is turned
back on at
this point. Nebulization gas supplied through line 181 to ES probe assembly
170 may
remain on or be turned off depending on how the spray gas balance in region
182 has
been optimized. A sample is injected into injector valve 186 with manual or
auto injector
187 and an LC gradient separation begins with LC system 184 when valve 186 is
switched
from inject to run. Sample bearing solution eluting from column 188 is
delivered to ES
probe tip 175 through line 180 and is Electrospray into ES chamber 161. A
portion of the
sample ions resulting from the Electrospray process are drawn into vacuum
through orifice
164 where they are mass analyzed. When the gradient LC run through LC column
188 is
complete, valve 191 is once again switched so that solution flow from LC
column 188 is
directed to flow through line 189 and the cycle described above begins again.
Solution
flow can be delivered through line 190 to ES probe assembly 172 to flush line
180 prior
to initiating the next gradient run through LC column 198.
The analytical sequence example described above includes switching between two
LC
separation systems using one ES-MS detector to increase sample throughput .
While one
LC column is being flushed after an LC run, an analytical separation is being
conducted
using a second LC separation system. Sample solution from LC system 194 is
delivered
to ES source 160 through ES probe assembly 170 and sample solution from LC
separation
system 184 is delivered to ES source 160 through ES probe assembly 172. A
calibration
44
CA 02590656 2007-06-04
solution can be delivered to ES source 160 through ES probe assembly 171
simultaneously
with the Electrospraying of either LC separation solutions to create an ion
mixture. A
mass spectrum acquired from the resulting ion mixture contains an internal
standard peaks
which can be used for mass calibration and/or quantitative analysis
calculations.
Several variations to the multiple ES probe embodiment diagrammed in Figure 8
can be
configured. One variation would be to eliminate switching valves 191 and 199
and send
the solution flow from columns 188 and 198 directly into ES probe assemblies
170 and
172. This would reduce dead volume and even allow the incorporation of fused
silica
packed columns as the first layer sample delivery tube configured in ES probe
assemblies
170 and 172 exiting at ES tips 173 and 175 respectively. During the column
flushing
period prior to an LC analytical run, say for ES probe assembly 170, the
position of ES
probe tip 173 can be moved so that any spray from tip 173, from flow through
column
198, would be directed away from mixing region 182 when ES probes 171 and 172
are
spraying. Probe tip 173 would then be moved back into position when the
analytical
separation through column 198 was reinitiated. ES probe tip 175 would then be
moved to
a position during flushing of LC column 188 such that any spray from tip 175
would not
be directed into mixing region 182. In this second position, any spray from
tip 175 during
flushing through colunm 188 would not contribute chemical noise to acquired
mass spectra
during the LC-MS analysis of samples flowing through LC column 198. The
positions of
ES probe assemblies 170 and 172 can be changed with automated adjustment means
during programmed multiple LC column analysis sequences.
An alternative and simpler method to recondition or flush LC columns between
LC runs
CA 02590656 2007-06-04
through an ES probe assembly without the need to move the ES probe position,
is to turn
off the nebulizing gas through the appropriate ES probe tip and change the
electrical
potentials applied to the ES probe tip during LC column reconditioning. The
electrical
potential should be switched or changed to a value which prevents unassisted
Electrospray
from occurring from the ES probe tip during LC column reconditioning. Solution
exiting
the ES probe tip from the LC column being reconditioned would then drip off
and flow
out the ES source chamber drain. As an example of this method, consider an LC
gradient
run Electrosprayed with nebulization assist through ES probe tip 175 while LC
column
198 is being reconditioned with solution flowing through ES probe tip 173. In
this
example, switching valves 191 and 199 have been eliminated and LC columns 198
and
188 are connected directly to or are incorporated into ES probe assemblies 172
and 170
respectively. Nebulization gas flow to ES probe tip 173 is turned off during
the LC
column reconditioning and any ions produced from unassisted Electrospray of
the liquid
emerging from ES probe tip 173 may be prevented from effectively entering
mixing
region 182 by the opposing nebulizing gas flow from ES probe assembly 172.
Unassisted
Electrospray from ES probe tip 173 can be prevented by applying a potential to
ES probe
tip 173 which is effectively equal to the local electric field potential
collectively formed by
the electrical potentials applied to ES source cylindrical lens 162, endplate
165 and
capillary entrance electrode 204. Liquid flowing through LC column 198 which
emerges
at ES probe tip 173 will drip off into ES source chamber 161 without
contributing ions
into mixing region 182. Similarly, the nebulizing gas flow can be turned off
and the
electrical potential applied to ES probe tip 175 can be changed to prevent
unassisted
Electrospray when liquid is flowing from LC column 188 though ES probe tip 175
during
reconditioning.
46
CA 02590656 2007-06-04
Additional analytical apparatus configurations are possible with combinations
of multiple
LC, CEC and / or CE separation systems configured in series or in parallel
supplying
solution to multiple ES probes. As an example, a capillary column or micro
bore column
can be configured in LC system 194 while and LC system 184 is configured with
a
standard 4.6 mm inner diameter LC column. ES probe assembly 175 can be
configured
with the capillary LC column incorporated as part of the ES probe assembly to
minimize
dead volume while ES probe assembly 170 is configured to accommodate the
higher liquid
flow rates delivered from larger bore column 198. The location of probe tips
175 and 173
can be positioned to optimize performance for specific and different liquid
flow rates
spraying from each ES probe tip. A system may also be configured with fast
flow
injection analysis using injector valves 186 and 196 and manual or auto
injectors 187 and
197 in alternating sequence. This alternating sample injection sequence
operating mode
increases the rate at which samples cam be mass analyzed by reducing the
relatively slow
injection rate cycle time of currently available auto injectors. An "open
access" system
can be configured with LC, CE and /or flow injection analysis to allow the
conducting of
multiple LC-MS, CE-MS or flow injection MS analysis with a single ES-MS
detector
system where no hardware reconfiguration is required.
More than three ES probe assemblies, each with different or similar
configurations, can be
mounted in ES chamber 160. Each ES probe assembly can be configured to
accommodate
different separation systems or sample injectors. One ES probe assembly may
interface to
an LC system, another to a CE or CEC system, another to an auto injector inlet
and yet
another to a calibration sample delivery system. Using multiple ES probe
assembly
configurations, an ES-MS or an ES-MS/MS system can be configured for a wider
range
47
CA 02590656 2007-06-04
of automation sample analysis techniques. Several widely diverse sample
analysis
techniques can performed in sequence or simultaneously with a single mass
analyzer in an
automated and unattended manner. Mass analyzers are generally more expensive
as
detectors than separation systems, consequently, the configuration of multiple
ES probes in
one ES source allows cost effective operation with multiple separation systems
connected
to a single API mass analyzer detector. Multiple ES probe assembly
configurations can
also save downtime due to component setup time by allowing simple switching
from one
analytical method to another.
Another embodiment of the invention is the configuration of an Atmospheric
Pressure
Chemical Ionization (APCI) source with multiple sample solution inlet probes
or
nebulizers interfaced to a mass analyzer. Each sample inlet probe can spray
solution
independently of other sample inlets either separately or simultaneously
during APCI
operation. APCI inlet probes or nebulizers can be configured to accommodate
solution
flow rates ranging from below 500 nL/min to above 2 mL/min. The invention
includes
configuring at least two APCI inlet probes with fixed or adjustable positions
which
independently spray solutions into a common vaporizer during APCI source
operation.
Solutions are delivered to the multiple APCI inlet probes configured with
pneumatic
nebulization through different liquid lines fed by individual liquid delivery
systems.
Different samples, mixture of samples and/or solutions can be sprayed
simultaneously
through multiple APCI inlet probes. The liquid delivery systems include but
are not
limited to liquid chromatography pumps, capillary electrophoresis separation
systems,
syringe pumps, gravity feed vessels, pressurized vessels, and/or aspiration
feed vessels.
Auto injectors and/or manual injection valves may be connected to one or more
APCI inlet
48
CA 02590656 2007-06-04
probe nebulizers for sample or calibration solution introduction. Similar to
the operation
of multiple ES probes in one ES source, multiple APCI nebulizers configured in
one APCI
source allow the introduction of multiple samples simultaneously or
sequentially with
different compositions and different liquid flow rates. A calibration solution
can be
introduced into an APCI source through one inlet probe with a sample solution
introduced
independently through a second inlet probe. Both calibration and sample
solutions flows
can be sprayed simultaneously without mixing chemical components in solution.
The
resulting sprayed droplet mixture is transferred into the APCI vaporizer. Ions
are
produced from the vaporized mixture in the corona discharge region of the APCI
source.
A portion of the ions produced from the vapor mixture are swept into vacuum
where they
are mass analyzed. The acquired mass spectrum of the ion mixture contains
peaks of ions
produced from compounds present in each sample and calibration solution. The
calibration peaks create an internal standard used for calculating the m/z
assignments of
sample related peaks. Simultaneously spraying from separate sample and
calibration
solutions allows the acquisition of mass spectra with internal standard peaks
without
mixing sample and calibration solutions prior to solution nebulization. The
multiple inlet
probe spraying prevents contamination of sample solution lines with
calibration compounds
and allows the selective and rapid turning on and off of calibration solution
flow. The
use of multiple solution inlet probes inAPCI sources can also be used to
introduce
mixtures of chemical components in the gas phase to investigate atmospheric
pressure gas
phase interactions and reactions of different samples and solvents without
prior mixing in
solution.
One embodiment of the invention is an APCI source, interfaced to a mass
analyzer,
49
CA 02590656 2007-06-04
configured with two sample inlet nebulizers assemblies shown in Figure 9. APCI
source
210 is configured with a heater or vaporizer 211, corona discharge needle 212,
a first
APCI inlet probe assembly 213, a second APCI inlet probe assembly 214,
cylindrical lens
215, nosepiece 216 attached to endplate 217, counter current gas heater 218
and capillary
220. Solution introduced through connecting tube 221 into APCI inlet probe
assembly
213 is sprayed with pneumatic nebulization from APCI inlet probe tip 222.
Nebulization
gas is supplied to APCI nebulizer probes 213 and 214 through gas delivery
tubes 227 and
228 respectively. APCI inlet probe assembly 213 is configured to spray
parallel (0213
=
0 ) with the APCI source centerline 223 into cavity 224. The sprayed liquid
droplets
traverse cavity 224, flow around droplet separator ball 225 and into vaporizer
211. The
sprayed liquid droplets evaporate in vaporizer 211 forming a vapor prior to
entering
corona discharge region 226. Corona discharge region 226 surrounds corona
discharge
needle tip 234. Additional makeup gas flow may be added independently into
region 224
or through APCI inlet probe assemblies 213 or 214 to aid in transporting the
droplets and
resulting vapor through the APCI source assembly 210. An electric field is
formed in
APCI source 230 by applying electrical potentials to cylindrical lens 215,
corona,
discharge needle 212, endplate 217 with attached nosepiece 216 and capillary
entrance
electrode 231. The applied electrical potentials, heated counter current gas
flow 232 and
the total gas flow through vaporizer 211 are set to establish a stable corona
discharge in
region 226 around and/or downstream of corona needle tip 234. The ions
produced in
corona discharge region 226 by atmospheric pressure chemical ionization are
driven by the
electric field against counter current bath gas 232 towards capillary orifice
233. A portion
of the ions produced are swept into vacuum through capillary orifice 235 where
they are
mass analyzed. In the embodiment shown, cavity 224 is configured with a
droplet
CA 02590656 2007-06-04
separator ball 225. Separator ball 225 removes larger droplets from the sprays
produced
by the nebulizer inlet probes preventing large droplets from entering
vaporizer 211.
Separator ball 225 is installed when higher liquid flow rates are introduced
typically
ranging from 200 to 2,000 microliters per minute. Separator ball 225 can be
removed
when lower solution flow rates are sprayed to improve sensitivity. A second
APCI inlet
probe assembly 214 is configured to spray at an angle of 45 (Q3214 = 450 )
relative to APCI
source centerline 223 into cavity 224 as shown in Figure 9. Solution flow
delivered to
both APCI inlet probes 213 and 214 through liquid delivery lines 221 and 236
respectively
can be controlled so that both APCI inlet probes can spray solution
simultaneously or
separately into cavity 224. Nebulizer spray performance for APCI probes 213
and 214 can
be optimized by adjusting solution delivery tube exit position with adjusting
screws 237
and 238 and locking nuts 239 and 240 respectively.
Different liquid flow rates and different solution types can be simultaneously
or separately
sprayed through APCI inlet probes 213 and 214. For example, the output of a
liquid
chromatography separation system can be sprayed through APCI inlet probe 213
at a flow
rate of I mL/min, while simultaneously a calibration sample solution is
sprayed from
APCI inlet probe 214 at a flow rate of 10 uUmin delivered through connecting
tube 236.
The sprayed droplet mixture forms a vapor mixture as it passes through
vaporizer 211. A
mixture of ions is formed from the vapor mixture as it passes through corona
discharge
region 226. A portion of the mixture of ions produced is swept into vacuum
along with
neutral gas molecules through capillary orifice 235 and the ions are mass to
charge
analyzed by a mass spectrometer. The acquired mass spectrum contains peaks of
ions
from the calibration sample which can be used as an internal standard to
improve mass
51
CA 02590656 2007-06-04
measurement accuracy and quantitation of the unknown sample peaks in the
acquired mass
spectrum. Alternatively, the second APCI inlet probe 214 can be used to
introduce a
sample solution that will create a desired solvent or ion mixture which will
interact
favorably in vaporizer 211 or corona discharge region 226 with the sample
vapor resulting.
from the solution sprayed from APCI inlet probe 213. It may not be desirable
to mix the
second solution with the sample solution prior to spraying. Spraying different
solutions
from multiple APCI probes can improve the APCI signal for an unknown sample or
interactions of gas phase mixtures of neutral molecules or ions can be studied
with
atmospheric pressure chemical ionization. To avoid mixing vaporized samples
molecules
or ions in the gas phase, APCI probes 213 and 214 can spray solutions in a
sequential
manner. For example, a calibration solution flow delivered to APCI inlet probe
214 can
be turned off while a mass spectrum is acquired from a sample solution
delivered to the
APCI source through APCI inlet probe 213. The calibraion solution flow
delivered
through connecting tube 236 to APCI probe 214 is then turned on to acquire an
external
standard calibration mass spectrum while the sample solution flow id turned
off.
Calibration mass spectrum can be acquired sequentially and/or simultaneously
with the
mass spectrum acquired for an unknown sample by turning on and off the
appropriate
solution flows during APCI source operation. Introducing calibration solution
through a
separate APCI inlet probe avoids contaminating the sample solution inlet line
and probe in
analytical applications requiring APCI. The mass spectra of the known and
unknown
samples can be added together in the data system to create a pseudo internal
standard.
Alternatively, sequentially acquiring mass spectra with and without an
internal standard
allows a direct comparison between the acquired sample mass spectra to check
for any
undesired effect that the calibration solution may cause to the acquired
sample ion
52
CA 02590656 2007-06-04
population.
An example of the APCI-MS operation of a dual probe APCI source as configured
in
Figure 9 is shown in Figure 10. Mass spectra 250, 252 and 255 shown in Figure
10 were
acquired with dual probe APCI source interfaced to a quadrupole mass analyzer.
Mass
spectrum 250 of a sample solution was acquired while infusing 2 pmole/ul of
leucine
enkephalin in a 1:1 solution of methanol:water with 0.1% acetic acid at a flow
rate of
100u1/min. The leucine enkephalin solution was delivered from a syringe pump
though
liquid delivery line 221 to APCI inlet probe nebulizer 222 during APCI
operation. No
liquid flow or nebulizer gas was delivered to APCI probe 214 during the
acquisition of
mass spectrum 250. Mass spectrum 250 contains protonated molecular ion peak
251 of
leucine enkephalin. Mass spectrum 252 of a calibration solution was acquired
from a
mixture of 50 pmoUul each of tri-tyrosine and hexa-tyrosine in an solution of
80:20
water:iso-propanol, 2% propionic acid at a flow rate of 5 ul/min. The
calibration solution
was delivered from a solution reservoir through delivery line 236 pulled by
the venturi of
pneumatic nebulizer 241 configured in APCI inlet probe 214. Mass spectrum 252
contains
calibration peaks 253 and 254 of protonated tri-tyrosine and hexa-tyrosine
respectively.
Sample liquid flow to APCI inlet probe 213 was turned off during the
acquisition of mass
spectrum 252. Mass spectrum 255 of Figure 10 was acquired while simultaneously
spraying sample and calibration solutions from APCI inlet probes 213 and 214
respectively. Solution compositions and flow rates were the same as was
described above
for individual spraying. Mass spectrum 255 contains internal standard peaks
256 and 258
of protonated tri-tyrosine and hexa-tyrosine respectively and sample compound
peak 257
of protonated. leucine enkephalin. The calibration peaks acquired as internal
standards can
53
CA 02590656 2007-06-04
be used to improve the calculated mass measurement of sample related peak 257.
Electrospray ionization, an APCI source creates sample and solvent molecule
vapor prior
to ionization. The APCI ionization process, unlike Electrospray, requires gas
phase
molecule-ion charge exchange reactions. Consequently, mixing samples, via
multiple inlet
probe introduction, in the gas phase in an APCI source may allow enhanced
opportunity to
study neutral molecule and ion molecule reactions which occur in the gas phase
while
avoiding solution chemistry effects. Gas phase sample interaction can be
avoided, if
desired, by introducing sample sequentially through multiple APCI inlet
probes. The
nebulizer gas can remain on or be turned off when the liquid sample flow
through an
APCI inlet probe is turned off. The venturi effect from the nebulizing gas at
the tip of an
APCI inlet probe may be used to pull the sample from a reservoir to the APCI
inlet probe
tip. This technique avoids the need for an additional sample delivery pump.
Multiple
APCI probes can be fixed in position as diagrammed in Figure 9 or can have
adjustable
sprayer positions relative to each other, cavity 224 or vaporizer 211. Each
APCI inlet
probe is removable and a single APCI source assembly can be configured with
one or
more APCI inlet probes mounted in a variety of positions. It is clear to one
skilled in the
art that more than two A.PCI inlet probes can be added to APCI source 210.
Each APCI
inlet probe can be configured at different angles relative to the APCI source
centerline and
each APCI inlet probe position can be fixed or adjustable during operation of
the APCI
source. APCI inlet probe tips can be configured at any position axially and
radially
upstream of vaporizer 211 or even configured to spray directly into corona
discharge
region 226. Multiple vaporizers and corona discharge needles can also be
configured into
APCI source 210. The relative radial positions of multiple APCI nebulizers
spraying into a
54
CA 02590656 2007-06-04
vaporizer can be set at any desired angle, radial position and tilt angle
relative to the
vaporizer centerline. The tips of each APCI inlet probe can be positioned to
optimize
nebulizer performance for a given solution flow rate and analytical
application.
An alternative embodiment of the invention is diagrammed in Figure 11 which
shows a
dual inlet probe APCI source with two inlet probes configured to spray in a
direction
parallel to the APCI source axis. APCI source chamber 271 of APCI source 260
is
configured similar to APCI source chamber 230 of APCI source 210 diagrammed in
Figure 9. APCI source 260 is configured with two pneumatic nebulization APCI
inlet
probes 264 and 265 which connect to liquid delivery lines 266 and 267
respectively.
Nebulizer gas lines 268 and 269 supply nebulization gas separately to APCI
inlet probes
264 and 265 respectively. In the embodiment shown, both APCI inlet probes 264
and 265
are configured such that axis of each pneumatic nebulizer sprayer axis is
positioned to be
approximately parallel with APCI vaporizer 261 axis 270. Different solutions
are sprayed
individually or simultaneously from both inlet probes 264 and 265 into region
262. A
portion of the sprayed droplets pass around separator ball 263 and flow into
vaporizer 261.
The sprayed liquid droplets evaporate in vaporizer 261 and ions are formed
from the vapor
as it passes through corona discharge region. A portion of the ions produced
pass into
vacuum through capillary orifice 273 and are mass to charge analyzed with a
mass
spectrometer and ion detector. Alternatively, APCI source 260 can be
configured with
more than two APCI inlet probes positioned in parallel and spraying in a
direction parallel
to vaporizer axis 270 into region 262. A set of parallel APCI inlet probes
positioned near
and spraying parallel with vaporizer axis 270 can be configured with single or
multiple off
axis angled APCI inlet probes. Multiple APCI inlet probes can be connected to
a variety
CA 02590656 2007-06-04
of liquid reservoirs, delivery systems or separation systems supplying
separate sample
solutions and/or calibration solutions to each individual APCI inlet probe.
Alternatively,
the axis 270 of vaporizer 261 may be configured at an angle from axis 274 of
capillary
275. Axis 270 of vaporizer 261 and, onsequently the axis of inlet probes 264
and 265 can
be configured at an angle from 0 to over 120 degrees relative to axis 274 of
capillary 275.
As will be shown in an alternative embodiment of the invention, off axis APCI
vaporizer
and inlet probe positioning allows the configuration of multiple APCI
vaporizer, inlet
probe and corona discharge APCI sources.
Similar to the Electrospray ionization source diagrammed in Figure 8 with
multiple ES
probes, multiple separation systems can be configured to deliver sample
solutions into an
APCI source configured with multiple inlet probes. As described for the ES
source,
sample throughput can be increased using a single APCI-MS detector for
multiple sample
separation or inlet systems. Multiple sample inlet probes configured in an
APCI source
can extend the range of analytical procedures which can be automatically or
manually run
sequentially or simultaneously with one APCI-MS instrument. The configuration
of
multiple APCI inlet probes in one APCI source can also minimize the time and
complexity
required to reconfigure and re-optimize an APCI source for different
analytical
applications.
An alternative embodiment of the invention is the combination of at least one
Electrospray
probe with at least one Atmospheric Pressure Chemical Ionization probe and
vaporizer
configured in an Atmospheric Pressure Ion Source interfaced to a mass
analyzer. It is
desirable for some analytical applications to incorporate both ES and APCI
capability in
56
CA 02590656 2007-06-04
one API source. Rapid switching from ES to APCI ionization methods without the
need
to reconfigure the API source minimizes the time and complexity to conduct API-
MS or
API-MS/MS" experiments with ES and APCI ion sources. The same sample can be
introduced sequentially or simultaneously through both APCI and ES probes to
obtain
comparative or combination mass spectra. Acquiring both ES and APCI mass
spectra of
the same solution can provide a useful comparison to assess any solution
chemistry
reactions or suppression effects with either ES or APCI ionization. Both ES
and APCI
probes can have fixed or moveable positions during operation of the API
source.
Alternatively, different samples can be introduced through the ES and APCI
probes
individually or simultaneously. For example, a calibration solution can be
introduced
through an ES probe while an unknown sample is introduced through an APCI
probe into
the same API source. The ES and APCI probe can be operated simultaneously or
sequentially in this manner when acquiring mass spectra to create an internal
or an
external standard. The combination of ES and APCI probes configured together
in an API
source minimizes probe transfer and setup time and expands the range of
analytical
techniques which an be run with a manual or automated means when acquiring
data with
an API MS instrument. Several combinations of sample introduction systems such
as
separations systems, pumps, manual injectors or auto injectors and / or sample
solution
reservoirs can be connected to the multiple combination ES and APCI probe API
source.
This integrated approach allows fully automated analysis with multiple
ionization
techniques, multiple separation systems and one MS detector to achieve the
most versatile
and cost effective analytical tool with increased sample throughput and little
or no
downtime due to instrumentation reconfiguration.
57
CA 02590656 2007-06-04
Figure 14 is a diagram of an embodiment of the invention which includes
individual or
simultaneous ES and APCI ionization capability configured together in an API
source
interfaced to a mass analyzer. APCI inlet probe and ionization assembly 280
and an
Electrospray probe assembly 281 are configured in API source assembly 282.
APCI probe
and ionization assembly 280 comprises dual inlet probes 283 and 284, spray
region 286,
optional separator ball 285, vaporizer 287 and corona discharge needle 288
with needle tip
289. APCI inlet probes 284 and 285 are configured to spray at an angle of
(02s3 & 284 =
00 ) relative to vaporizer 287 centerline 291. APCI inlet probes 283 and 284
are
configured with separate solution delivery lines 294 and 295 and separate
nebulizer gas
lines 294 and 295 respectively. Electrospray probe assembly 281 comprises
three layer
spray tip 296 with gas delivery line 297, sample solution delivery line 298
and layered
liquid flow delivery line 299. The ES probe tip 296 is configured to spray at
an angle of
(0296 = 70,D) relative to centerline 300 of API source 282. The position of ES
probe tip
296 is adjustable using adjuster knob 301. Alternatively, ES probe assembly
281 may be
configured with two or more ES probe tips positioned to spray at an angle
relative to API
source centerline 300.
API source 282 is additionally configured with cylindrical lens 120, endplate
303 with
attached nosepiece 304, capillary 305, counter current drying gas flow 306 and
gas heater
307. ES probe tip 296 is positioned a distance ZES axially from nosepiece 304
and
radially rEs from API source centerline 300. Electrical potentials applied to
cylindrical
lens 302, endplate 303 with nosepiece 304, capillary entrance electrode 308,
ES tip 296
and APCI corona needle 288 can be optimized to operate both the ES and APCI
probes
separately or simultaneously. Counter current drying gas flow 309, the
nebulization gas
58
CA 02590656 2007-06-04
flow from ES probe tip 296 and the nebulizer, makeup and vapor gas flow
through APCI
vaporizer 291 can be balanced to optimize performance of simultaneous ES and
APCI
operation. Alternatively, the ES and APCI probes can be operated sequentially
with fixed
positions by turning on and off the solution and/or nebulizing gas flow for
each probe
sequentially. Mass spectra with ES ionization can be acquired with solution
flow and
voltages applied to the ES probe tip 296 turned on while solution flow to APCI
inlet probe
283 and/or 284 and voltage applied to corona discharge needle 288 are turned
off. Liquid
flow and voltage applied to ES probe tip 296 can then be turned off with
liquid flow to
APCI inlet probes 283 and/or 284 and voltage applied to corona discharge
needle 288
turned on prior to acquiring mass spectra with APCI ionization.
Different solutions or the same solutions can be delivered through the ES and
APCI
probes during acquisition of mass spectra. The electrical potentials applied
to elements in
the API source may be adjusted for ES and APCI operation to optimize
performance for
each solution composition and liquid flow rate. Also, voltages applied to
elements or
positions of elements in the API source may be changed and then reset to
optimize ES or
APCI operation. For example, if APCI assembly 280 operating and no sample is
being
delivered through ES probe 281, the voltage applied to ES probe tip 296 can be
set so that
tip 296 will appear electrically neutral to avoid interfering with the
electric field in corona
discharge region 290. Similarly, when ES probe 281 is operating and solution
flow to
APCI assembly 280 is turned off, voltage can be applied to corona discharge
needle 289
such that it does not interfere with the Electrospray process or actually
improves the
Electrospray performance. For example, voltage applied to corona discharge
needle 289
can aid in moving or focusing Electrospray produced ions toward capillary
orifice 310.
59
CA 02590656 2007-06-04
Alternatively, the position of APCI corona discharge needle 288 can be moved
temporarily
during ES probe operation to minimize interference with the Electrospray
ionization
process. APCI corona discharge needle 288 can then be moved back into position
during
operation of APCI probe assembly 280. Simultaneous ES and APCI operation can
be
configured to produce ions of opposite polarity. Ions produced in the APCI
corona region
290 can be of one polarity, while spraying the ES needle at the corona needle
can
produce opposite polarity ES ions. Voltages applied to API source elements to
achieve
positive APCI generated ions and negative ES generated ions can be capillary
entrance
electrode 308 (-4,000V), endplate 303 and nosepiece 304 (-3,000V), cylindrical
lens 302 (-
2,000V), corona discharge needle 288 (-2,000V) and ES probe tip 296 (-5,000V).
A
portion of the resulting mixture of ions reacting at atmosphere of one
polarity is enters
vacuum through capillary orifice 310 and subsequently mass analyzed. Several
combinations of sample inlet delivery systems, as have been described earlier,
can be
interfaced to the combination ES and APCI API source. Multiple ES and multiple
APCI
inlet probes can be included in an API source assembly. The ES and APCI probe
assemblies can be configured to mount through the API source chamber walls,
within the
API chamber or through the API chamber back plate.
Figures 15A through 15D include mass spectra acquired from a combination API
source
configured similar to API source 282 diagrammed in Figure 14 interfaced to a
quadrupole
mass spectrometer. Mass spectrum 320 shown in Figure 15A was acquired with
APCI
ionization of a sample or 82 pmol/ul of reserpine in a 1:1 methanol:water with
0.015%
formic acid solution sprayed from APCI probe 283 at a liquid flow rate of 200
ul/min.
Mass spectrum 320 contains peak 321 of the protonated molecular ion of
reserpine.
CA 02590656 2007-06-04
Solution flow to ES probe tip 296 was turned off during the acquisition of
APCI-MS
generated mass spectrum 320. Mass spectrum 322 shown in Figure 15B was
acquired
with Electrospray ionization of 10 pmoUul of cytochrome C in a 1:1
methanol:water, 0.1%
acetic acid solution spraying from ES tip 296 with pneumatic nebulization
assist at a liquid
flow rate of l0ul/min. Mass spectrum 322 contains primarily the Electrosprayed
multiply
charged peaks 323 of cytochrome C. Solution flow to APCI inlet probe 283 was
turned
off during the acquisition of ES-MS spectrum 322. Mass spectrum 324 shown in
Figure
15C was acquired from the same cytochrome C solution Electrosprayed into API
source
282 with pneumatic nebulization assist. During the acquisition of mass
spectrum 324,
containing peaks 325 of Electrospray generated multiply charged cytochrome C
ions, the
nebulizing gas was supplied to APCI inlet probe 283 with the vaporizer 287
heater turned
on but with no high voltage applied to corona discharge needle 288 and no
reserpine
solution flowing to APCI inlet probe 283. Mass spectrum 326 shown in Figure
15D was
acquired with the same conditions as mass spectrum 324 with high voltage
applied to
corona discharge needle 288 and the same reserpine solution as above sprayed
from APCI
inlet probe 283. Both peak 327 of the protonated molecular ion of reserpine
and peaks
328 of multiply charged protonated cytochrome C ions appear in mass spectrum
326
acquired with simultaneous ES and APCI ion production occurring in API source
assembly
282. Mass spectra 320, 322, 324 and 326 were acquired sequentially with no
position
adjustment of API source 282 hardware. Raid switching between individual or
simultaneous ES and APCI operating modes with combination source 282 shown in
Figure
14.
An API source with multiple ES or APCI probes or combinations of ES and APCI
probes
61
CA 02590656 2007-06-04
can be configured to allow the study of ion-ion interactions at atmospheric
pressure.
Many of the combination and multiple inlet probe API source configurations
shown above
can be operated using methods and techniques that will allow the study of gas
phase ion-
ion interactions at atmospheric pressure. Alternative embodiments of multiple
inlet probe
API sources configured specifically to allow the simultaneous production of
opposite
polarity ions will be described below. One embodiment of a multiple ES probe
API
source configured for studying ion-ion interactions at atmospheric pressure is
diagrammed
in Figure 16. ES probe assembly 340 is configured with ES probe tip 344
located near
axis 341 of API source 342 (0340 = 0 ) spaced a distance of Z344 from API
source
nosepiece 347. Solution is Electrosprayed from ES probe tip 344 with pneumatic
nebulization assist. The polarity of the Electrosprayed ions produced is
determined by the
relative potentials set on the electrostatic elements comprising API source
342. For
purposes of discussion assume that the API source potentials and gas flows
applied are set
to produce positive ions from solutions Electrosprayed from ES probe tip 344,
A second ES probe assembly 345 is mounted with ES probe tip 346 positioned at
a
distance along API source axis 341, Z346, from API source nosepiece 347 and
radially,
r346, from API source axis 341, The angle of the spraying axis of ES probe tip
346 is
positioned approximately at 110 degrees (0346 = 110 ) relative to API source
centerline
341. The voltage applied to ES probe tip 346 is set such that negatively
charged liquid
droplets are produced from solution Electrosprayed from ES probe tip 346 with
pneumatic
nebulization assist. The positive and negative ions produced from the positive
and
negative charged liquid droplets Electrosprayed from ES probe tips 344 and 346
respectively mix and interact in region 348 of API source 342. This positive
and negative
62
CA 02590656 2007-06-04
ion-ion interaction at atmospheric pressure will cause the neutralization of
some but not all
of the mixed ion population. A portion of the resulting positive ion
population will be
driven to capillary entrance 349 by the electric fields present. A portion of
the positive
ions which enter capillary orifice 349 are swept through capillary bore 350
into vacuum
and subsequently mass to charge analyzed with a mass spectrometer and
detector.
Reversing voltage polarities in API source 342, will cause negative ions to be
produced
from solution Electrosprayed from ES probe tip 344 and positive ions to be
produced from
solution Electrosprayed from ES probe tip 346. With polarities reversed,
negative product
ions will be move toward capillary entrance orifice 349, be swept into vacuum
through
capillary bore 350 and subsequently mass to charge analyzed.
Several geometries of ES probes can be configured to achieve multiple sample
ion-ion
interaction from different solutions Electrosprayed from multiple ES probe
assemblies.
More than two ES probes can be configured in an API source positioned at
angles, 01 ... i
ranging from 0 to 180 degrees and rotation angles 81 ... i ranging from 0 to
360 degrees.
Selected neutral gas composition can be added to nebulizer or counter current
drying gas
to study ion-neutral reactions in relation to ion-ion interactions. Unlike the
opposite
polarity ion-ion interactive studies conducted in partial vacuum reported by
Smith et. al.,
the embodiment of the invention described allows the production of ES ions in
one API
source chamber with ion-ion interaction conducted in higher ion and gas
densities at
atmospheric pressure.
An embodiment of an API source configured with a dual APCI vaporizer, corona
discharge needle and probe assembly is diagrammed in Figure 17. One APCI probe
63
CA 02590656 2007-06-04
assembly 366 is positioned off-axis, 0366 90 , at a distance Z366 from API
source
nosepiece 375. APCI probe assembly 366 comprises pneumatic nebulizer sample
inlet
probe assembly 367, optional droplet separator ball 368, vaporizer 369, and
corona
discharge needle 370. Sample solution supplied from liquid delivery system 372
is
sprayed from inlet probe assembly 367. Sprayed droplets pass around separator
ball 368
and into vaporizer 369 where the droplets evaporate to form a vapor. The vapor
exiting
vaporizer 369 is ionized in the corona discharge region at the tip of corona
discharge
needle 370. A second APCI probe assembly 360 is also positioned off-axis, 0360
90 ,
spaced a distance Z360 from API source nosepiece 375. In the configuration
shown
dimension Z360 is shorter than Z366. APCI probe assembly 360 comprises
pneumatic
nebulizer sample inlet probe assembly 362, optional droplet separator ball
363, vaporizer
364, and corona discharge needle 365. Inlet probe 362 sprays sample solution
delivered
from liquid delivery system 373 into APCI probe assembly 360. For purposes of
discussion, assume that the applied API source element electrical potentials
and gas flows
are set to produce positive ions from solutions sprayed, vaporized and ionized
through
APCI probe 366 and negative ions from solutions sprayed vaporized and ionized
through
APCI probe 360. The positive ions produced in the corona discharge region
surrounding
the tip of corona discharge needle 370 are drawn towards the capillary 361,
end plate 375,
and corona discharge needle 365 due the applied electrical potentials. The
negative ions
produced in the corona discharge region surrounding the tip of corona
discharge needle
365 are drawn towards corona discharge needle 370 due to the applied
electrical potentials.
The positive and negative ions interact and react at atmospheric pressure in
region 371.
The positive and negative ion interaction at atmospheric pressure will result
in the
neutralization of some the positive and negative ions, however, some positive
ions after
64
CA 02590656 2007-06-04
reacting can be re-ionized and subsequently drawn towards nose piece 375 and
capillary
361 by the applied electrical potentials. Positive ions are swept into vacuum
through the
bore of capillary where they are mass analyzed by a mass spectrometer located
in vacuum
region 374. A higher number of positive solvent ions may be introduced from a
higher
solution flow rate through APCI probe assembly 366 compared with the solution
flow
rate delivered to APCI probe assembly 360. The higher abundance of positive
solvent
ions ion in mixing region 371 will increase the efficiency of re-ionization of
positive ions
after a neutralization reaction with a negative ion. Reversing voltage
polarities in API
source, will allow negative ions to be produced from solution delivered to
APCI probe
assembly 366 and positive ions to be produced from solution delivered to APCI
probe
assembly 360. A portion of the reacted negative ion population will be swept
into vacuum
and mass to charge analyzed.
Variations of APCI probe locations can be configured to achieve multiple
sample ion-ion
interaction from different solutions sprayed from multiple APCI probe
assemblies. More
than two APCI probes can be configured in an API source positioned at angles
(Pi i
ranging from 0 to 180 degrees and rotation angles 91 ... i ranging from 0 to
360 degrees.
Selected neutral gas composition can be added to nebulizer or counter current
drying gas
study ion-neutral reactions in relation to ion-ion interactions.
An embodiment of an API source configured with three APCI probe assemblies
positioned
to facilitate the study of ion-ion interactions at atmospheric pressure is
shown in Figure 18.
APCI probe assembly 380 is positioned at angles 0380 90 and , 9380=270 with
electrical
CA 02590656 2007-06-04
potentials applied relative to grid 381 to produce negative ions in the corona
discharge
region surrounding the tip of corona discharge needle 392. A second APCI probe
assembly
382 is positioned at angles 10382=90 and 0,$2=90 with electrical potentials
applied relative
to grid 384 to produce negative ions. A third APCI probe assembly 385 is
positioned at
angles 0 = 0 and 9= 0 with electrical potentials applied relative to grid 390
to produce
positive ions. The positive and negative ions produced from APCI probe
assemblies 380,
382 and 385 pass through grids 381, 384 and 390 respectively and interact at
atmospheric
pressure. Two grids 381 and 384 are positioned between APCI probe assembly 385
and
the entrance of capillary 386. Interaction between ions of opposite polarity
results in the
cause the neutralization of the positive and negative ions, however, the
positive sample and
solvent ions supplied from APCI probe assembly 385 can re-ionize reacted
product
molecules. The newly formed ion will be drawn towards nose piece 389 and
capillary 386
by the applied electric fields. Ions swept through the bore of capillary 386
into vacuum
are mass analyzed with a mass spectrometer and ion detector. The applied
voltage
polarities can be switched to enable the mass analysis of a negative reacted
ion population.
One or more APCI probes assemblies configured in the embodiment shown in
Figure 18
can be removed or replaced with Electrospray probe assemblies. API sources
configured
with multiple APCI probe assemblies can be used to study a range of ion-ion
interactions
and reactions.
Multiple ES and APCI inlet probe configurations as diagranimed in Figures 1,
2, 3, 5, 6,
8, 9, 11, 14, 16, 17 and 18 show individual solution delivery systems
connected to each
inlet probe tip. atternatively, multiple sample delivery systems can be
switched directed to
66
CA 02590656 2007-06-04
supply solution to ati individual inlet probe tip. The cotnbinatioii of
tiiultiple sample inlet
lines a.ticl multiple iiebulizers can be configurecl in a single API source
assenlbly. Several
comhiriatiotis of multiple probe tip positions can be coal~igured by one
skilled in the art
atid the inveiition is not li.nlited to those .multiPle ES atlcl APCI probe
embodiments
specifically described hereiri.
Having described this invention witli respect to specific embocliments, it is
to be
understood that the clescr.iption is tiot nieant as a lirnitatiori sitlce
furtlier nioditications
atid variations niay be apparent or may suggest theiliselves to tliose skilled
in the art. It is
intended that the preseiit application cover all such modifications and
variations as fall
withiri the scope of the appended clainis.
Refere-ices Cited:
The references referred to in this clocutnent iticlude:
U.S. Palejlt Docculients:
4,542,293 Sept. 17, 1985 Penrt, John I3.; Yatnaslzita, Nlasaniichi;
W.liitellouse, Craig.
5,495,108 Feb. 27, 1996 A.p1.11e1, Jatnes; Werlich, Mark;
Bectach, Jnnies. Publicatior.ts:
R. Kostiatliiien aad A.P. J3fuiizs, Pt-oceedings of the 41s' ASMS C-onference
oil Mass
Spectrometry, 744a, 1993.
67
CA 02590656 2007-06-04
R. R. Ogorzalek Loo, Harold R. Udseth, and Richard Smith, Proceedings of the
39th
ASMS
Conference on Mass Spectrometry and Allied Topics, 266-267, 1991.
R. R. Ogorzalek Loo, Harold R. Udseth, and Richard Smith, J. Phys. Chem., 6412-
6415,
1991.
Richard D. Smith, Joseph A. Loo, Rachel R. Ogorzalek Loo, Mark Busman, and
Harold R.
Udseth, Mass Spectrometry Reviews, 10, 359-451,1991.
Bordoli, Woolfit and Bateman, Proceedings of the 43th ASMS Conference on Mass
Spectrometry and Allied Topics, 98, 1995.
68
