U.S. patent application number 11/776392 was filed with the patent office on 2008-07-24 for methods and apparatus for the ion mobility based separation and collection of molecules.
This patent application is currently assigned to Excellims Corporation. Invention is credited to Ching Wu.
Application Number | 20080173809 11/776392 |
Document ID | / |
Family ID | 38924137 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080173809 |
Kind Code |
A1 |
Wu; Ching |
July 24, 2008 |
METHODS AND APPARATUS FOR THE ION MOBILITY BASED SEPARATION AND
COLLECTION OF MOLECULES
Abstract
This invention describes an apparatus for the separation and
collection of components in a sample of interest comprising: an
ionization source; an ion mobility separator and an ion collector
positioned to receive ions leaving the ion mobility separator. The
ion mobility separator having an inlet to supply at least one
separating substance which comprises particles which selectively
interact with at least one analyte component of interest to certain
degree different from the others. The analyte component of interest
may be enantiomers, diastereomers, stereoisomers, isomers, etc. The
ion collector can be used to conduct analytical, preparative, and
semi-preparative separation. In addition, a combined primary
electrospray and secondary electrospray ionization source is
disclosed to enhance ionization efficiency of interest.
Inventors: |
Wu; Ching; (Acton,
MA) |
Correspondence
Address: |
CHING WU
20 Main Street
ACTON
MA
01720
US
|
Assignee: |
Excellims Corporation
Maynard
MA
|
Family ID: |
38924137 |
Appl. No.: |
11/776392 |
Filed: |
July 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60807031 |
Jul 11, 2006 |
|
|
|
60891532 |
Feb 26, 2007 |
|
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Current U.S.
Class: |
250/283 ;
250/288 |
Current CPC
Class: |
H01J 49/165 20130101;
C07B 63/00 20130101; G01N 27/622 20130101 |
Class at
Publication: |
250/283 ;
250/288 |
International
Class: |
H01J 49/26 20060101
H01J049/26 |
Claims
1. An apparatus for the separation of analyte components in a
sample comprising: (a) an ionization source; (b) an ion mobility
separator in fluid communication with the ionization source;
comprising a separating substance inlet to supply at least one
separating substance which interacts with at least one analyte
component of interest to a degree different than the separating
substance interacts with at least one other analyte component of
interest; and (c) an ion collector in fluid communication with the
ion mobility separator and positioned to receive ions leaving the
ion mobility separator.
2. The apparatus of claim 1, wherein the analyte components are
isomers.
3. The apparatus of claim 1, wherein the analyte components are
stereoisomers.
4. The apparatus of claim 1, wherein the analyte components are
diastereomers.
5. The apparatus of claim 1, wherein the analyte components are
enantiomers.
6. The apparatus of claim 1, wherein the separating substance is
chiral and/or nonchiral.
7. The apparatus of claim 1, wherein the separating substance
further comprises a plurality of components that interact with the
analyte components to different degrees.
8. The apparatus of claim 7, wherein the plurality of components
are supplied sequentially or simultaneously.
9. The apparatus of claim 1, wherein the ion mobility separator
comprises a symmetric ion mobility separator.
10. The apparatus of claim 1, wherein the ion mobility separator
comprises an asymmetric ion mobility separator.
11. The apparatus of claim 10, wherein the asymmetric ion mobility
separator is a field asymmetric ion mobility spectrometer.
12. The apparatus of claim 10, wherein the asymmetric ion mobility
separator is a differential mobility spectrometer.
13. The apparatus of claim 1, wherein the ionization source
comprises a single or plurality of primary ionization sources
combined with a single or a plurality of secondary ionization
sources.
14. The apparatus of claim 13, wherein the primary ionization
source may include but is not limited to: electron beam, MALDI,
electrospray, secondary electrospray, surface, corona discharge,
radioactive, photo, laser, laser ablation/desorption, DESI,
DART.
15. The apparatus of claim 13, wherein the secondary ionization
source may include but is not limited to: electron beam, secondary
electrospray, surface, corona discharge, radioactive, photo, laser,
DESI, DART.
16. The apparatus of claim 1, further comprising at least one
sample inlet for supplying samples that are gas, liquid or
solid.
17. The apparatus of claim 16, wherein the samples are from other
separation devices, including but not limited to gas chromatograph,
supercritical fluid chromatograph, liquid chromatograph,
electrophoresis, on solid surface, or in a medium.
18. The apparatus of claim 1, wherein the separating substance
comprises one or more of: (R)-(-)-.alpha.-(Trifluoromethyl)benzyl
alcohol, (S)-(+)-.alpha.-(Trifluoromethyl)benzyl alcohol,
(R)-Tetrahydrofuran-2-carbonitrile,
(S)-Tetrahydrofuran-2-carbonitrile, (2R,6R)-2,6-Heptanediol,
(+)-Ethyl D-lactate, and (-)-Ethyl L-lactate.
19. The apparatus of claim 1, wherein the separating substance
comprises one or more of S-(+)-2-butanol and R-(-)-2-butanol.
20. The apparatus of claim 1, wherein the ion collector comprises a
static ion collector.
21. The apparatus of claim 20, wherein the static ion collector
comprises a Faraday plate.
22. The apparatus of claim 20, wherein the static ion collector
comprises a non-flowing liquid.
23. The apparatus of claim 1, wherein the ion collector comprises a
dynamic ion collector.
24. The apparatus of claim 23, wherein the dynamic ion collector
comprises one or more of a moving belt and a moving Faraday
plate.
25. The apparatus of claim 23, wherein the dynamic ion collector
comprises a flowing liquid.
26. The apparatus of claim 20 and 23, wherein ion collectors are
segmented.
27. The apparatus of claim 1, further comprising: a mass
spectrometer disposed at the end of the ion mobility separator; and
an interface structure disposed between the ion mobility separator
and the mass spectrometer.
28. The apparatus of claim 27, wherein the mass spectrometer may
include but is not limited to: a quadrupole, an ion trap, a time of
flight mass analyzer.
29. A method for the separation of analyte components in a sample
comprising the steps of: (a) ionizing the sample of interest
comprising two or more analyte components of interest to produce an
ionized sample; and (b) transporting under the influence of an
electrical field at least a portion of the ionized sample through a
neutral medium containing at least one separating substance, the
separating substance interacting with at least one analyte
component of interest to a degree different than the separating
substance interacts with at least one other analyte component of
interest; and (c) collecting with an ion collector at least a
portion of the ionized sample transported through the neutral
medium containing the separating substance.
30. The method of claim 29, wherein the step of ionizing comprises
ionizing the sample with a primary ionization source followed by
ionizing a un-ionized neutral sample with a secondary ionization
source.
31. The method of claim 29, wherein the electrical field is a
substantially static electrical field during the step of
transporting.
32. The method of claim 29, wherein the electrical field is a
substantially dynamic electrical field during the step of
transporting.
33. The method of claim 32, wherein the electrical field comprise
an AC component and a DC component.
34. The method of claim 33, wherein the DC component is applied for
a time period; after which at least two analyte components of
interest acquire different drift times.
35. The method of claim 29, wherein the separating substance
comprises one or more of: (R)-(-)-.alpha.-(Trifluoromethyl),
(S)-(+)-.alpha.-(Trifluoromethyl),
(R)-Tetrahydrofuran-2-carbonitrile,
(S)-Tetrahydrofuran-2-carbonitrile, (2R,6R)-2,6-Heptanediol,
(+)-Ethyl D-lactate, and (-)-Ethyl L-lactate.
36. The method of claim 29, wherein the separating substance
comprises one or more of S-(+)-2-butanol and R-(-)-2-butanol.
37. The method of claim 29, wherein the step of collecting
comprises impinging at least a portion of the ionized sample, that
was transported through the neutral medium containing the
separating substance, upon a surface.
38. The method of claim 37, wherein the surface is a solid
surface.
39. The method of claim 38, wherein the solid surface is a moving
surface.
40. The method of claim 37, wherein the surface is a liquid
surface.
41. The method of claim 40, wherein the liquid surface is flowing
liquid surface.
42. An apparatus for the separation of analyte components in a
sample comprising: (a) an ionization source; (b) an ion mobility
separator in fluid communication with the ionization source; and
(c) an ion collector in fluid communication with the ion mobility
separator and positioned to receive ions leaving the ion mobility
separator.
43. The apparatus of claim 42, wherein the ion mobility separator
comprises a symmetric ion mobility separator.
44. The apparatus of claim 42, wherein the ion mobility separator
comprises an asymmetric ion mobility separator.
45. The apparatus of claim 44, wherein the asymmetric ion mobility
separator is a field asymmetric ion mobility spectrometer.
46. The apparatus of claim 44, wherein the asymmetric ion mobility
separator is a differential mobility spectrometer.
47. The apparatus of claim 42, wherein the ion collector comprises
a static ion collector.
48. The apparatus of claim 47, wherein the static ion collector
comprises a Faraday plate.
49. The apparatus of claim 47, wherein the static ion collector
comprises a non-flowing liquid.
50. The apparatus of claim 42, wherein the ion collector comprises
a dynamic ion collector.
51. The apparatus of claim 50, wherein the dynamic ion collector
comprises one or more of a moving belt and a moving Faraday
plate.
52. The apparatus of claim 47 and 50, wherein the ion collectors
are segmented.
53. The apparatus of claim 50, wherein the dynamic ion collector
comprises a flowing liquid.
54. An apparatus for the ionization of samples comprising: (a) at
least one primary ionization source; (b) at least one secondary
ionization source; (c) a ionization chamber containing a electric
field that guides charged particles from the secondary ionization
source into a ionization chamber and/or extracts ionized analyte
components from the ionization chamber; (d) at least one sample
inlet; (e) a gas flow that carries neutral analyte components; (f)
at least one analyzer in fluid communication with the ionization
chamber.
55. The apparatus of claim 54, wherein the analyzer is an ion
mobility separator or a mass analyzer.
56. The apparatus of claim 55, wherein the mass analyzer may
include but is not limited to: a quadrupole, an ion trap, a time of
flight mass analyzer.
57. The apparatus of claim 54, wherein the sample inlet is for a
gas phase sample.
58. The apparatus of claim 57, wherein the gas phase sample
comprises elutents from a GC or SFC.
59. The apparatus of claim 54, wherein the sample inlet is for a
liquid phase sample.
60. The apparatus of claim 54, wherein the analyzer is interfaced
to the ionization chamber between substantially zero and
substantially one hundred eighty degrees from the direction ion
traveling axis.
61. The apparatus of claim 55, wherein the primary ionization
source and the secondary ionization source may include but is not
limited to: electron beam, MALDI, secondary electrospray, surface,
corona discharge, radioactive, photo, laser, laser
ablation/desorption, DESI, DART, SESI, APCI.
62. The apparatus of claim 61, wherein the ionization source
further comprises chemical modifiers.
63. The apparatus of claim 54, wherein the sample inlet is an open
inlet that allows direct ionization of samples from a surface with
the primary ionization source and introduction neutral samples into
the ionization chamber with the gas flow.
64. A method for the ionization of samples comprising the steps of:
(a) introducing a sample into a ionization chamber; (b) ionizing
the sample by a primary ionization source; (c) ionizing a neutral
sample in a gas flow by a secondary ionization source; and (e)
extracting an ionized sample into an analyzer with a electric
field.
65. The method of claim 64, wherein the step of ionizing comprises
introducing a chemical modifiers into the ionization source.
66. The method of claim 64, wherein the step of introducing the
sample comprises a continuous or pulsed flow.
67. The method of claim 64, wherein the step of ionizing the
neutral sample comprises bringing charged particles generated by
the secondary ionization source into the ionization chamber
continuously or as pulses
68. The method of claim 64, further comprises (a) ionizing at least
a portion of the sample in the ionization chamber (b) separating
the ionized sample from an un-ionized sample with the electric
field (c) introducing the charged particles into the ionization
chamber to ionize the un-ionized sample (d) extracting the ionized
samples into the analyzer
69. The method of claim 68, wherein the steps of (b) and (c) are
repeated until all the sample is ionized
70. The method of claim 64, further comprises directly ionizing
samples from a surface and introducing neutral samples on the
surface into the ionization chamber with the gas flow.
71. An apparatus of ion gate for an ion mobility separator
comprising a segmented Bradbury-Nielson that contains multiple
sections of Bradbury-Nielson gate.
72. The apparatus of claim 71, wherein the segmented
Bradbury-Nielson gate is a second gate in a time-of-flight type ion
mobility separator.
73. The apparatus of claim 71, wherein the segmented
Bradbury-Nielson gate comprises a variety of geometries which may
include but is not limited to: parallel, rectangular,
concentric.
74. The apparatus of claim 71, wherein the ion mobility separator
further comprises a segmented ion collector where a plurality of
sections of ion collector is inline with the sections of the
segmented Bradbury-Nielson gate.
75. A apparatus for the interface between an IMS and MS comprising:
a resistance tube; and a high voltage power supply operatively
connected to the resistance tube.
76. The apparatus of claim 75, wherein: the resistance tube has an
inner diameter in the range between about 1 micrometer and about 2
mm and the high voltage power supply is configured to apply a
voltage gradient in the range between about 1 and about 40,000
volts across the inner diameter of the resistance tube.
77. A apparatus for the interface between an IMS and MS comprising:
a first conductive member; a second conductive member, wherein the
first and second conductive members are substantially symmetrically
arranged about an ion transport axis and wherein the distance
between the ends of the first and second conductive members
proximal to an ion mobility separator is equal or less than the
distance between the ends of the first and second conductive
members distal to the ion mobility separator; a DC and RF power
supply operatively connected to one or more for the first and
second conductive members.
78. The apparatus of claim 77, wherein the first and second
conductive members are plates.
79. The apparatus of claim 77, wherein the DC and RF power supply
is configured to apply voltages between the first and second
conductive members in a means that is resemble to the operation of
an asymmetric ion mobility separator with two parallel plates,
where the first and second conductive members are closest.
80. A method for operating an ion mobility separator and a mass
spectrometer comprising: (a) measuring ion mobility of an analyte
component using an ion detector/collector at the end of the ion
mobility separator; (b) measuring ion mobility and mass to charge
ratio using ion detector of mass spectrometer; (c) correlating the
ion mobility data obtained from mass spectrometer with the ion
mobility dada from the ion mobility separator.
81. The method of claim 80, wherein mass identifying ions collected
on the ion collector at the end of the IMS using the correlated ion
mobility data.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of and priority
to corresponding U.S. Provisional Patent Application No. 60/807,031
and 60/891,532, filed Jul. 11, 2006 and Feb. 26, 2007 respectively,
the entire content of the applications are herein incorporated by
reference
BACKGROUND OF THE INVENTION
[0002] Enantiomerically pure compounds are of great interest in the
pharmaceutical industry and other fields. The rapid and efficient
separation and collection of chiral compounds is difficult since
they have the same physical properties. All of their physical
properties correspond, except the direction in which they rotate
plane-polarized light, i.e. they differ in a specific optical
activity. Furthermore, all of their chemical properties correspond,
except the reactivity toward other chiral compounds. Note that the
term chiral compounds are also often used as a general term that
refers to the molecules with a chiral center. Development of both
preparative and analytical scale separations has provided the tools
to determine the enantiomer composition for racemic mixtures,
further establishing evidence of the enantiomer rates of activity.
Although it has also extended into the agrochemical and food
industries, this technology has been primarily driven by the
pharmaceutical industry.
[0003] The processes traditionally employed for enantiomer
preparation, however, suffer from several drawbacks. For example,
one process is liquid or gas chromatography. In this process, the
analysis mixture is mixed with an externally prepared carrier
medium and separated in a separating column as a function of the
different affinity of the enantiomers for the stationary phase of
the chromatographic column; and thus, the individual components
pass in succession through the chromatography column as a function
of their different retention times. This process, however, can be
very time consuming when multiple samples (such as might be desired
in high-throughput screening) are to be analyzed as elution times
of 20-30 minutes for one sample, are relatively common. A further
disadvantage of the chromatographic process is that the
enantiomeric molecules can often have very similar retention times,
leading to poor separation per pass.
[0004] One of two approaches is typically utilized for chiral
separation: 1) indirect and 2) direct separation methods. Indirect
separation methods incorporate a reaction between each enantiomer
and a chiral molecule to covalently form a new complex, which is
then separated from the other enantiomeric complex. This approach
is frequently utilized, especially in large-scale operations.
Direct methods are based on the formation of non-covalent
diastereomeric pairs of molecules using a chiral selector (CS) and
rely on differences in the energetics of the complex formation for
enantiomer resolution. The chiral selector can either be
incorporated into the stationary phase or as an additive in the
mobile phase. Chromatography and capillary electrophoresis (CE)
have been primarily exploited for chiral separations, both
prep-scale and microscale. Typically in chromatography, the
stationary phase is chiral (CSP) but chiral additives may also be
added to the mobile phase (in liquid chromatography). The first
analytical separation of two enantiomers occurred with gas
chromatography, but due to the required analyte volatility for gas
chromatography, its applications are limited. It is for this reason
that liquid chromatography is more commonly employed. In CE, a
chiral selector (CS) is added to the electrolyte solution.
[0005] Both CE and HPLC have received considerable attention,
however, a major difficulty with both techniques is that prediction
of the separation conditions remains difficult. For example, in
HPLC, there are over 200 CSP's commercially available, yet no clear
method to determine which CSP will provide a good separation. This
can lead to both time-consuming and costly method development. The
fact that HPLC and sometimes CE require longer analysis times
(minutes to hours) combined with the lengthy method development
creates a real need for analytical tools which either are
predictable in the separation capabilities or have faster analysis
times, specifically in the early stages of drug development.
SUMMARY OF THE INVENTION
[0006] In various aspects, the present inventions provide apparatus
and method for separating and collecting chiral molecules using ion
mobility, preferably in the gas phase. In comparison to
chromatographic separations, gas phase separations can be conducted
rapidly, e.g. on the order of milliseconds to tens of seconds as
opposed to the tens of minutes typically found in chromatographic
approaches. In the case of other compounds which are very similar
to each other, for example very similar proteins. These compounds
may contain two or more chiral centers that are not related as an
object and its mirror image, separation and collection can be
enhanced by adding a separating substance where their physical
properties are nearly identical.
[0007] In various embodiments, the present inventions provide an
apparatus for the separation and collection of analyte components
in a sample of interest comprising: an ionization source; an ion
mobility separator and an ion collector positioned to receive ions
leaving the ion mobility separator. The ion mobility separator
having an inlet to supply at least one separating substance which
comprises particles which to certain degrees selectively interact
with at least one analyte component in the sample of interest.
[0008] In various embodiments, the ion source employs electrospray
ionization (ESI) to form ions. Other methods of ionization and
suitable ionization sources include, but are not limited to, matrix
assisted laser desorption ionization (MALDI), electrospray
ionization (ESI), secondary electrospray ionization (SESI),
desorption electrospray ionization (DESI), surface ionization,
corona discharge ionization, electron beam ionization, radioactive
ionization, photo ionization, laser ionization, laser ablation
ionization, direct analysis in real time (DART) ionization and
possible combination of multiple ionization principles. In various
embodiments, the combined ionization source disclosed in this
invention may eliminate ionization suppression in the primary
ionization source and enhance over all ionization efficiency.
[0009] In various embodiments, the ion mobility separator comprises
a device that separates ions on the basis of their mobility through
a medium, where the medium is a gas, a liquid, a supercritical
fluid, and/or other fluidic materials. It is to be understood, that
in the present inventions that this mobility need not be a
steady-state ion mobility nor a field independent mobility. The
term ion mobility separators (IMS), and ion mobility spectrometers
(IMS), includes two broad classes of separators, those that employ
a substantially symmetric field (often referred to simply as ion
mobility spectrometers although this term is also used to refer to
all types of IMS instruments) and those that employ an asymmetric
electrical field, often referred to as differential mobility
spectrometers (DMS) or field asymmetric ion mobility spectrometers
(FAIMS). In the present inventions, both symmetric IMS and field
asymmetric IMS can be used.
[0010] In various embodiments, the ion collector comprises a moving
belt collector. In various embodiments, a moving belt collector
includes a belt and accurate motor. Other suitable ion collectors
include, but are not limited to, single or multiple Faraday plate,
Faraday plate with selective chemical coating, solution phase ion
collection, or ion collection/detection method in high vacuum, such
as mass spectrometer or electronmultiplier ion detector.
[0011] The foregoing and other aspects, embodiments, objects,
features and advantages of the invention can be more fully
understood from the following description in conjunction with the
accompanying drawings. In the drawings like reference characters
generally refer to like features and structural elements throughout
the various figures. The drawings are not necessarily to scale,
emphasis instead being placed upon illustrating the principles of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a schematic of an example of a moving belt ion
collector for sample recovery;
[0013] FIG. 2 is a schematic of an example of an access collection
plate or plates for sample recovery;
[0014] FIGS. 3A and 3B are schematics of a asymmetric IMS for
analyte separation and ion collection;
[0015] FIG. 4 is a schematic of a combined primary electrospray and
secondary electrospray ionization source;
[0016] FIG. 5 is a schematic of a solid phase sampling, ionization,
and detection process;
[0017] FIG. 6 is a schematic example of a segmented
Bradbury-Neilson gate for ion collection;
[0018] FIG. 7 shows examples of chiral modifiers (separating
substances);
[0019] FIG. 8 shows examples of various embodiments of ambient
pressure and vacuum interfaces for sampling ions from ambient
pressure;
[0020] FIG. 9 shows simulation of equal potential lines for a 143
V/cm (100V over 0.7 cm), 10000 V/cm (2000V over 2 mm) inside the
resistive interface;
[0021] FIG. 10 shows a schematic example of an embodiment of
asymmetric IMS with the pressure gradient in the device serving as
the interface of the mass spectrometer for the sampling of
atmospheric ions. The interface can be used, e.g., to sample ions
from IMS or directly from atmospheric ionization sources;
[0022] FIG. 11 shows a schematic example of a data acquisition
scheme for an IMS-MS systems. Ion Gate 1 and Gate 2 are designed to
select an ion of interest and to deposit such on the sample
collector, direct to a mass spectrometer for further analysis or
both. Ion mobility spectrometer and mass spectrometer data can be
generated through two separate channels and correlated in the data
acquisition software;
[0023] FIG. 12 shows a schematic diagram of an ESI-IMS-MS. The ion
mobility spectrometer contains an electrospray ionization source; a
desolvation region and an ion drift region separation by a
Bradbery-Nelson ion gate; it was operated at atmospheric pressure
in the examples;
[0024] FIG. 13 shows a graph of superimposed IMS spectra of racemic
mixtures of valinol, threonine, penicillamine, tryptophan,
methyl-.alpha.-glucopyranoside and atenolol, where pure nitrogen
was used as the drift gas. As expected, separation of the
enantiomers in pure nitrogen drift gas was not achieved;
[0025] FIG. 14 shows two graphs of the gas phase separation of
atenolol enantiomers. The upper graph shows the superimposed
spectra of S- and R-atenolol obtained after introduction of
S-(+)-2-butanol as the chiral modifier in the drift gas. The bottom
graph demonstrates the IMS separation of an enantiomeric mixture of
S- and R-atenolol;
[0026] FIG. 15 shows a graph showing the effects of chirality and
flow rate of the chiral modifier on the drift times of the
methionine enantiomers in CIMS. A better separation of methionine
enantiomers was observed with S-(+)-2-butanol compared to
R-(-)-2-butanol. The order of elution was reversed when the
chirality of the modifier was reversed. Preferred chiral modifier
flow rate was at 45 .mu.L/min corresponding to 10 ppm in the drift
gas;
[0027] FIG. 16 shows two graphs of the CIMS-MS separation of L- and
D-tryptophan. The upper graph shows the superimposed spectra of L-
and D-tryptophan obtained independently. The bottom graph
demonstrates the separation of the enantiomeric mixture of L- and
D-tryptophan;
[0028] FIG. 17 shows two graphs of CIMS-MS separation of L- and D-
and Methyl-.alpha.-glucopyranoside Ion mobility spectra of sodium
adduct of D- and L-Methyl-.alpha.-glucopyranoside enantiomers. The
upper graph shows the superimposed spectrum of D- and
L-Methyl-.alpha.-glucopyranoside enantiomers obtained
independently. The bottom graph demonstrates the separation of the
enantiomeric mixture of D- and
L-Methyl-.alpha.-glucopyranoside;
[0029] FIG. 18 shows the graph of an IMS separation of the
enantiomeric mixture L- and D-penicillamine;
[0030] Table 1 shows examples of gas phase enantiomeric separation
using IMS-MS; and
[0031] Table 2 shows examples of selected chiral molecules.
DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS
[0032] Unless otherwise specified in this document the term
"chiral" is intended to mean a particle with at least one
stereogenic center or chiral center. It should be noted that
"chiral" as used herein below may be, but not limited to,
chemicals, biologicals, enantiomers, diastereomers, and
atropisomers.
[0033] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
[0034] Unless otherwise specified in this document the term
"separating substance" is intended to mean single or plurality of
particle which to certain degrees selectively interacts with single
or plurality of analyte component of interest to be separated.
[0035] Unless otherwise specified in this document the term
"particle" is intended to mean chemical and/or biological single or
plurality of atom, molecule, large or macro molecule, nanoparticle,
or other matters that are vapor, droplets, aerosol, liquid, solid
that follow a mobile medium, where the medium can be a gas, a
liquid, supercritical fluid and/or other fluidic materials.
[0036] Unless otherwise specified in this document the term
"analyte component" is intended to mean various particles, charged
particles, and charged particles derived from atoms, molecules,
particles, sub-atomic particles, and ions.
[0037] In the present invention, one or more volatile chiral
compounds (e.g. chiral modifier, also referred to as a separating
substance) are infused into the drift gas stream and introduced
into the ion mobility separator. Without being held to theory, it
is believed that during the analyte-separating substance
collisions, transient diastereomeric complexes may form. The
hypothesis is that enantiomers can have slightly different
equilibrium constants for the diastereomeric complex formation. As
the transient diastereomeric complexes formation and deformation
process rapidly repeat in the ion mobility separator,
stereostructure specific separation of enantiomers can be observed.
The contribution of the ion-chiral modifier to the average measured
mobility shift should be concentration dependent and analytically
quantifiable. The degree of interaction between the enantiomeric
ions and the chiral modifiers can also be altered by altering the
type and concentration of the chiral modifiers and gas temperature,
pressure and flow rate in the drift tube.
[0038] IMS may potentially replace chiral SFC and HPLC in the many
applications as a chiral molecule separation and collection
technique where analysis time is a critical consideration. A more
powerful tool can be developed, e.g., based on a chromatography-ion
mobility separator-mass spectrometry for characterization of
complex mixtures where chiral separation is required. In one
aspect, the present inventions provide an instrument with the size
comparable to commercial analytical HPLC or SFC. In various
aspects, the present inventions provide an IMS system for
separation and collection of chiral molecules. A broad range of
applications of such a system can be developed to support
biomedical research: such a system, e.g., can be used to directly
confirm the enantiomeric excess of chiral ingredients in
pharmaceutical products; providing, for example, one or more of the
following: [0039] 1) an increase in the throughput for chirality
measurements, specifically in the initial stages of drug
development when hundreds of drug compounds are being screened as
drug candidates; [0040] 2) monitoring of the performance of
preparative chiral separation processes, such as SFC and HPLC based
separation; [0041] 3) detection for chromatography with non-chiral
columns; providing, e.g. researchers with more flexibility when
choosing chromatographic conditions for the analysis of biomarkers,
metabolites or other biological samples; or a detector for chiral
chromatograph as a complimentary separation method to resolve
enantiomers that cannot be separated by given chiral stationary
phase, especially for molecules with multiple chiral centers.
[0042] 4) preparation of a substantially pure single enantiomer
compound
[0043] The ion mobility base chiral separation methods of the
present inventions in various embodiments, can be used for
analytical separation, to conduct preparative, semi-preparative
separation of chiral compounds or combinations thereof. For
example, after being introduced into the gas phase and separation
by ion mobility separators, the separated chiral molecules can be
collected onto a surface or by liquid solutions. The collection or
sample preparation method can be operated as either an online
method, a offline method or combinations thereof.
[0044] In various embodiments, the methods of the present invention
comprise a full profile collection method (FIGS. 1, 3). Profile
collections implies collecting samples on-the-fly during mobility
separation. For example in one embodiment shown in FIG. 1, after
analyte component ions created in ionization source 102, introduced
to ion mobility separator through an ion gate as used for
analytical purpose IMS. For online collection of from an ion
mobility separator 103, a moving belt 101 can be used as the ion
collector. An optional ion gate 105 could be placed in front of the
moving belt or any other kind of collector for selective collection
of ions with a mobility of interest. To operate the moving belt ion
collector, motor operable wheels 107 and 108 turning in the range
of several thousand RPM may be required. As the ions reach the belt
surface, the belt speed is preferably set to correspond with the
resolution of the IMS. After the samples are collected on different
locations of the belt, the samples can be recollected by dissolving
them back in suitable solvents; by separately removing samples from
specific locations on the belt, different analyte components can be
separately collected. In various embodiments, the belt has marks
that correspond to the arrival time in a specific ion mobility
separation device and a specific section of the belt can be cut,
disassembled, washed, etc., to isolate compounds of interests. For
example, a segmented belt could be dissolved back in liquid phase
in the same or different solutions. Such recollected sample could
be used for, e.g., further chemical analysis.
[0045] In various embodiments, a selective method (FIGS. 2, 3) can
involve collecting samples ionized by ionization sources 201 on a
collector 202 from an ion mobility separator 205, and then removing
the collected samples from the instrument and, e.g., the separated
samples recovered for further study. For example, a metal plate at
a set potential can inserted in the spectrometer, and mobility
selected ions collected on this plate. The ion mobility separator,
e.g., could be a two ion gate TOF-IMS or DMS or FAIMS. In a various
embodiments, the analyte components that are separated and
collected on the ion collectors, as shown in FIG. 1-3, can be
treated with a matrix and further analyzed by MALDI mass
spectrometer. Alternatively, the collected samples can be analyzed
by mass spectrometer using DART or DESI or other ionization
methods. Apparatuses for sample collection after a ion mobility
separator, including but not limited to the features described in
FIG. 1-3, can be used with a variety of separation devices,
separating substance is not necessary to be used with such
devices.
[0046] Most common ionization sources used for ion mobility and
mass analysis can be used to ionize molecules. Electron beam
ionization, matrix assisted laser desorption ionization (MALDI),
secondary electrospray ionization (SESI), desorption electrospray
ionization (DESI), surface ionization, corona discharge ionization,
radioactive ionization, photo ionization, laser ionization, laser
ablation ionization, DART ionization, and possible combination of
multiple ionization principles.
[0047] In this invention, a new ionization source is disclosed to
enhance to ionization efficiency. FIG. 4 shows a combined ESI and
SESI source. The ionization source could be interfaced to the ion
mobility separator or mass analyzer 418 and 422 from different
angles. In principle, when liquid samples 420 (e.g. eluents from a
HPLC) are ionized by the primary electrospray ionization source
401, certain amount of sample in the electrosprayed droplets are
not ionized due to limited amount of available charges and surface
area. The proposed combined source uses additional electrosprayed
(solvent) droplets introduced from separate electrosprayer(s) 405
to interact with un-ionized neutral sample molecules in the
ionization chamber 412 to improved ionization efficiency,
instrument sensitivity and/or sample recovery efficiency.
[0048] In addition, when a mixture of sample is introduced to an
ordinary electrospray ionization source 401 (primary ionization) as
show in FIG. 4, the samples have higher charge affinity may have a
better ionization efficiency; and such high charge affinity
compounds may suppress the ionization of other co-existing
compounds resulting in certain classes of chemicals that cannot be
ionized or suffer from significant sensitivity loss in IMS or MS.
The combined ionization source allows separation of ions from high
charge affinity compounds from un-ionized low charge affinity
compounds by applied electric fields on the guard ring 403
surrounding the ionization chamber 412. After extracting the
ionized high charge affinity compounds, the low charge affinity
compounds are subsequentially ionized by electrosprayed solvent
droplets that are introduced by the secondary ionization source 405
in this region.
[0049] The combined source may use one or multiple primary
electrospray ionization sources 420 and one or multiple secondary
electrospray ionization sources 405, a set of guard rings 403, a
gas phase sample inlet 408, ion gates 410 and 415, and interface to
mobility or mass analyzer. Ion gate 410 controls the amount of
electrosprayed solvent droplet introduced into the source chamber
412. Ion gate 415 controls the timing for ionized sample to be
introduced to the mobility or mass analyzer 418. Note the ions can
be extracted into an ion mobility separator or mass spectrometer
422 by applying a kick out voltage on segmented guard rings 403;
the kick out voltage can force ions in the ionization chamber 412
travel substantially perpendicular to drift direction defined by
the electric field before extraction occurred. For liquid phase
samples 420, the primary electrospray source introduces charged
droplet into the ionization chamber and the droplets are
subsequentially desolvated by high temperature gas in the
ionization chamber 412. During the desolvation process, sample ions
form from the charged droplets. As portion of the sample (low
charge affinity samples) are not ionized in this process, they stay
as neutrals and flow with the gas toward the secondary electrospray
ionization source. Once interacting with the solvent droplets in
this chamber, these neutral molecules are ionized via "secondary
electrospray ionization" process. The solvent droplets can be
introduced into the chamber 412 as a continuous source or pulsed
"plug" of charges drifting under the effluence of electric field
created by the guard ring electrodes 403. Gas flow rate can be in a
range of substantially slow during the ionization and substantially
fast during the clean up process. Certain gas flow pattern may be
created to suspend neutral molecules or particle of different
sizes. As long as the unionized substance stay in the gas flow,
they could ionized by the secondary ionization source. When charged
droplets, created from such as organic solvents, doped solvents or
other liquid mixtures, are introduced into the ionization chamber
continuously, maximum ionization efficiency can be achieved by the
secondary ionization process. Alternatively, when charged droplets
are introduced into the ionization chamber as pulses, each pulse of
charged droplets can be used to selectively ionization neutrals
with different charge affinities step by step; as the higher charge
affinity neutrals extract from the ionization chamber as ions, the
next pulse will ionize next high charge affinity neutral. The
process can be repeated until all samples in the mixture are
ionized. As the ionized samples are all extracted and analyzed
using an ion mobility separator or mass spectrometer, the ion
mobility spectra or mass spectra could be process and/or
reconstructed to provide qualitative and quantitative information
about the sample mixture. The analyzers can locate in-line with the
guiding electric field 418 or with a designed angle that is from
zero to 180 degrees, e.g. perpendicular to the guiding electric
field in the source 422. Both primary and secondary electrospray
ionized samples are kicked out into the mobility or mass analyzer
either sequentially or simultaneously.
[0050] Similarly, gas phase sample 408, e.g. eluents from a GC or
SFC, can be introduced to the ionization chamber 412 via the gas
sample inlet. These samples will interact with the droplet or ions
created by the secondary electrospray ionization source and become
ionized in this chamber. Note that the secondary electrospray
ionization source may introduce ions or charged droplet to the
combined source ionization chamber 412 depending on the gas
temperature and drying time allowed before ion gate 410. Controlled
pulse of solvent ions can be used to ionize chemicals with
different charge affinities at different spatial location in the
ionization chamber when these chemicals 408 are introduce into the
ionization chamber as a pulse of neutral samples.
[0051] The ionization process not only depends on charge affinity,
the selective ionization mechanism can be used to resolve
"suppression" problem in common ionization source based other
chemical properties. For example, when Cl.sup.- is doped in the
secondary electrospray solvent, the ionization efficiency of
chemicals that may form stable chloride adduct could be further
enhanced. Not only electrospray ionization source can be used for
multiple step ionization operation mechanism, other combined
primary and secondary ionization method could also be used to
reduce suppression and improve ionization efficiency of mixtures;
chemical modifiers can be used in SESI source to create different
chemical properties that may selectively ionize compounds of
different class with different chemical properties.
[0052] For the asymmetric ion mobility separator, FIG. 3A-B shows
similar concept where the secondary ionization is used to enhance
the ionization efficiency of the primary ionization source. Voltage
offset between primary 301 and secondary 303 sources can help
extracting ions formed in the primary source faster then the
neutral molecules, where the driving force to move neutral
molecules is the gas flow; with the assistance of the electric
field, ions can be moved out from the primary source faster then
the neutrals. Thus, the suppressed ionization process for molecules
with less charge affinities can be resolved similar to the
mechanism described in FIG. 4. The combined ionization source could
be, but not limited to, an electrospray and secondary electrospray
ionization source. It may be advantageous for asymmetric IMS by
using a plasma ionization source where both positive and negative
ions are generated. Depending on the principle of ionization,
sample flow 308 shown in the figure may represent a liquid flow,
for electrospray ionization, for example; or a gas flow for
radioactive ionization, for example.
[0053] Similar to the combined electrospray ionization source, FIG.
5 shows the ionization method for solid samples. In this case the
primary ionization source 501 can be desorption electrospray
ionization (DESI), Direct Analysis in Real Time (DART) ionization,
laser ablation/desorption ionization, MALDI and other method for
ionization of solid samples. The secondary ionization source 503
can be SESI or other gas phase chemical ionization methods. For
solid samples 502, air flow may be added to assist removing samples
away from a surface. When the sample is desorbed from the surface
as ions in the primary ionization region 514, the ions are
extracted into secondary ionization region under influence of gas
flow 505 or electric field, or both; the electric field is created
by guard rings 522. When the sample on the surface is desorbed as
neutral molecules, they are extracted into the secondary ionization
region by gas flow 505. Depending on sample's physical and chemical
properties, the secondary ionization source may employ a variety
different kind of charged droplets (that could be altered by
chemical modifiers) to interact with the sample that has been
brought into the gas phase. As shown in this figure, secondary
electrospray ionization process is the main mechanism for the
secondary ionization; alternatively, for relative small molecules,
atmosphere chemical ionization (APCI) may be used as the secondary
ionization mechanism. In various embodiments, other ionization
methods, such as photo ionization, electron beam ionization or
laser ionization can also be method of choice for secondary
ionization.
[0054] FIG. 5 illustrates an example of the solid phase sampling,
ionization, and detection process. Sampling target 510 is a surface
where the sample 502 of interest may be located on. The sampling
and detection apparatus may also have a sealing material 512 when
it is intended to be used as in contact with sampling target,
however, non-contact sampling is preferable in many application of
this apparatus. For instance, when DESI method is used as the
primary desorption/ionization method, both ionized and neutral
molecules are extracted from the surface and brought into the
secondary ionization region 518; in this area, the charged solvent
droplets introduced by the SESI source 503 interacts with the
neutral molecule by either dissolving them in to the solvent
droplets or transferring charges to these molecules. The ionized
samples are introduced to an IMS through an ion gate 520. The
unionized neutral molecules, carrier gas 505, and drift gas 515
from mobility separator are exhausted 516. As a result, the
combined ionization source can enhance the ionization efficiency
beyond the primary ionization methods alone. Alternatively, after
the secondary ionization process, ions can also be introduced to a
mass analyzer 508; the mass analyzer can either located at the end
of an ion mobility separator, as shown in FIG. 5, or directly
mounted to the combined ionization source.
[0055] In a symmetric IMS device (sometimes referred to as
TOF-IMS), a propelling DC field gradient and a counter gas flow are
set and an ionized sample is released into the field which flows to
a collector electrode. Ion species are identified based on the DC
field strength and time of flight of the ions to the collector. At
low values of E/N ion mobility is typically a constant value, where
E is the electrical field and N is the gas density (often referred
to as number density) in the drift tube.
[0056] Time of flight ion mobility spectrometry is a gas phase
analytical technique that separates ions based on both size and
shape. In IMS, ions are created and then subjected to an electric
field, causing the ions to accelerate through the ion mobility
drift tube while colliding with neutral drift gas molecules
(typically an inert gas such as nitrogen). As the ions travel
through the drift tube, they undergo random collisions and
accelerations until reaching the end of the drift region, where
they are either detected by a Faraday plate or transmitted through
an interface to a mass spectrometer where they are mass separated
and detected. An ion's mobility through the drift tube is defined
as the ratio of the average ion velocity (v.sub.d) to the applied
electric field (E) (when operating in the low-field region). IMS
then takes advantages of mobility differences to separate ions.
[0057] Experimentally, an ion's mobility (K) can be determined by
the following equation:
K = v d E = L 2 t d V ( 1 ) ##EQU00001##
where L is the length of the drift region, t.sub.d is the time the
ion travels through the drift region (drift time), and V is the
voltage applied to the drift region. The ion mobility can be
related to the ion-drift gas collision processes at the molecular
level by the following:
.OMEGA. = ( 3 16 N ) ( 2 .pi. .mu. kT ) 1 / 2 ( ze K ) ( 2 )
##EQU00002##
where .OMEGA. is the average ion-drift gas collision cross section,
z is the number of charges on the ion, e is the charge of one
proton, N is the number density of the drift gas, .mu.[=mM/(m+M)]
is the reduced mass of an ion (m) and the neutral drift gas (M), K
is the ion mobility and k is Boltzmann's constant. When the
experimental parameters are held constant, the mobility is
dependent on the ion charge, the ion-drift gas reduced mass and the
collision cross section as follows:
K .varies. z .OMEGA. .mu. ( 3 ) ##EQU00003##
For ions more massive than the drift gas molecule, the reduced mass
is nearly equal to M and the mobility is primarily proportional to
z and .OMEGA..
[0058] Assuming that the ion is more massive than the drift gas
molecule and that the ion charge can not be altered, a change in
the ion mobility would require a change in the collision cross
section. The collision cross section term is a function of the
interaction between the ion and the neutral drift gas molecule, the
collision dynamics and the size and shape of the ion and neutral
molecule. The drift gas can be thought of as a weak stationary
phase for the ion mobility experiment and by adjusting the
stationary phase, the separation characteristics can be adjusted.
The collision cross section can be altered by changing the drift
gas, both due to the size contribution of the drift gas and the
degree of interaction between the ion and neutral molecule. Varying
the temperature of the experiment can also affect the interaction
between the ion and neutral molecules.
[0059] In an asymmetric IMS device, ion species are identified by
mobility behavior in a high asymmetric RF field, where ions flow in
a carrier gas and are shifted in their path by an electric field.
Various asymmetric IMS devices operate with a selected RF field at
Vmax and species detections are correlated with a pre-set, or
scanned, DC compensation voltage (Vc). Species are identified based
upon correlation of Vmax and Vc with historical detection data. For
a given ion species in a sample, as the amplitude of the asymmetric
RF voltage (at Vmax) changes, the amplitude of the DC compensation
voltage (Vc) required for passage of that species through the
filter field also changes. The amount of compensation depends upon
species characteristics.
[0060] Various asymmetric IMS devices include a pair of opposed
filter electrodes defining a gap between them in a flow path (also
known as a drift tube). Ions flow into the analytical gap. An
asymmetric RF field (sometimes referred to as a filter field, a
dispersion field or a separation field) is generated between the
electrodes transverse to the carrier gas/ion flow in the gap.
Electrical field strength, E, varies as the applied RF voltage
(sometimes referred to as dispersion or separation voltage, or Vrf)
and size of the gap between the electrodes. Such systems can
operate at atmospheric pressure.
[0061] Ions are displaced transversely by the RF field, with a
given species being displaced a characteristic amount toward the
electrodes per cycle. DC compensation (Vc) is applied to the
electrodes along with Vrf to compensate for the displacement of a
particular species. The applied compensation is used to offset the
transverse displacement generated by the applied Vrf for that
particular ion species. The result is zero or near-zero
substantially transverse displacement of that species, which
enables that species to pass through the filter for detection. All
other ions undergo a net displacement toward the filter electrodes
and eventually undergo collisional neutralization on one of the
electrodes.
[0062] If the compensation voltage is scanned for a given RF field,
a complete spectrum of ion species in the sample can be produced.
The recorded image of this spectral scan is sometimes referred to
as a "mobility scan", as an "ionogram", or as "DMS spectra". The
time required to complete a scan is system dependent. Relatively
speaking, a prior art IMS scan might take on the order of a second
to complete while and a prior art DMS might take on the order of 10
seconds to complete.
[0063] An asymmetric IMS operates based on the fact that an ion
species will have an identifying property of high and low field
mobility in the RF field. Thus, an asymmetric IMS detects
differences in an ion's mobility between high and low field
conditions and classifies the ions according to these differences.
These differences reflect ion properties such as charge, size, and
mass as well as the collision frequency and energy obtained by ions
between collisions and therefore enables identification of ions by
species.
[0064] In various aspects, the present inventions employ asymmetric
IMS to separate chiral molecules, a similar approach as described
for the symmetric IMS is used. FIG. 3A schematically illustrates an
overview of such a device and method. A chiral modifier or chiral
modifiers are introduced to the device from either the sample flow
inlet 311 or an additional gas inlet 313 located between ionization
source and the ion mobility separator or from both inlets. The IMS
separator can, for example, be two parallel plates 318 (as shown in
FIG. 3A), concentric cylinders, or other shapes. The ion mobility
separator can be used for, for example, analytical purposes,
preparative purpose or both. For analytical purposes, in various
embodiments, separated chiral molecule(s) are collected on a
different set of electrodes 316 after passing through the mobility
analyzer. For preparative purpose, in various embodiments, the
mobility analyzer plates can also be used as ion collectors; and
compounds with different mobility properties can be collected a
different location on these plates. These compounds can be
recovered after the separation process. This practice is not
limited to chiral separation; it can be used to recover samples for
general chemical isolation purposes without chiral modifier or any
other separating substances. The collector plates can be made of,
but not limited to, metal plates or metalized non-conductive
plates. Each of these plates can have one or multiple electrodes
for ion collection and generating electric field for ion
separation. FIG. 3B shows the surface of upper 322 and lower 324
ion collection plates of a parallel plate asymmetric ion mobility
separator, where multiple electrodes 325 and 327 are used in ion
mobility separator and detector region, respectively. The
electrodes on ion mobility separator plates 318 (FIG. 3A) can be
segmented in uneven sizes according the mobilities of targeted
analyte components and the resolution required. These electrodes
can be individually set to different voltages or removed from the
sample collection. The multiple electrode approach can provide the
capability of setting different dc or rf potentials to enhance
mobility based separation.
[0065] Similar to the moving belt configuration for the symmetric
IMS, the preparative collection plate (e.g. FIG. 2 item 202 and
FIG. 3A item 318) can be removed from the device and cut into
slices according to the spatial separation of the samples of
interest. Slices of collected sample can be dissolved in solution
for further investigation or study. Alternatively, the collection
plates can also be directly analyze by MALDI-, DESI-, DART-, other
ionization method with either mass spectrometers or ion mobility
spectrometers; the collection plates can also be further analyzed
by other spectroscopic methods, including but not limited to NMR,
IR, UV-Vis methods. For selective collection purpose, the ions of
interest can also be collected on different sets of plates 316 that
are used as the ion detector for analytical asymmetric IMS. The
selective collection plate can also be segmented and have
electrodes to enhance the selectivity by spatially removing
overlapping samples on the collective plate.
[0066] An underlying principle for separating and collecting chiral
and nonchiral molecules in the asymmetric IMS is the gas phase
selective interaction between chemical modifiers and the analyte
components of interest. In an asymmetric IMS device, the time for
such interaction is during the CV applied period. During this
period of time, the ions are at low E/N conditions. The asymmetric
IMS is preferably operated to have an extended CV period compared,
for example, to traditional DMS and FIAMS device operation. In
various embodiments, the CV period is 1150 ns where 37,500
collisions may occur under ambient pressure conditions.
[0067] In various embodiments, operating conditions such as, but
limited to, pressure, temperature, electric field strength, and
carrier or drift gas flow rate. For example, operation of the ion
mobility separator at a relatively low pressure, e.g., about 1 to
about 700 Torr, can provide for an easier interface to a MS,
however it is preferred that the gas phase concentrative of chiral
modifier is adjusted to achieve similar separation performance to
higher pressure operation.
[0068] Above described ion mobility base chiral separation methods
of the present inventions in various embodiments, can be also used
for the separation of nonchiral compounds; in this case, the chiral
modifier can be replace with other chemical modifiers that
selectively interact with analyte components of interest. The
sample collection methods already described in previous sections
can be used to collect these compounds. For example, separation of
stereoisomers, such as proteins and lipid, that can be enhanced by
adding a separating substance where their physical properties are
nearly identical. If, for example, two proteins having very similar
drift times are to be separated from each other in an ion mobility
separator, a suitable separating substance can be selected which is
known to have a significantly greater interactive cross section
with one of the protein molecules than with the other protein. The
sample collection methods already mentioned can be used to collect
these compounds.
[0069] In various embodiments for symmetric IMS, an ion focusing
method can be employed to guide ions to a target collection area on
the collector. Suitable focusing methods may include, but are not
limited to, static electric field focusing and ion funnel focusing.
An ion collector can be segment to facilitate, e.g., collection of
ions with specific ion mobility (drift time) or a certain range of
mobilities on to different segment of the ion collectors.
[0070] In various embodiments of IMS instruments, wherein the
Bradbury-Nielson gate can be segmented. A variety of geometries,
including but not limited to parallel, rectangular, concentric ring
shape, can be used for the segmentation, referring to FIG. 6,
various embodiments can use parallel segmentation. Each segment of
the ion gate, for example, 601, 603, and 605, can be controlled to
open at a different time. Such a segmented ion gate can be used as
either first or second ion gate in a time-of-flight type ion
mobility separator. While it is used as the second ion gate in a
IMS, multiple portions of ions with different drift time are allow
to pass through segmented ion gate, thus collected on different
section of ion collectors, and recovered separately if desired.
[0071] In various embodiments, an apparatus of ion gate for an ion
mobility separator comprising a segmented Bradbury-Nielson that
contains multiple sections of Bradbury-Nielson gate. The segmented
Bradbury-Nielson gate can be used as a second gate in a
time-of-flight type ion mobility separator. The segmented
Bradbury-Nielson gate comprises a variety of geometries which may
include but is not limited to: parallel, rectangular, concentric.
The ion mobility separator further comprises a segmented ion
collector where a plurality of sections of ion collector is inline
with the sections of the segmented Bradbury-Nielson gate.
[0072] The collection surface for an ion collector of the present
inventions can be, for example, a solid surface. This surface can
be coated with different materials to facilitate collection,
removal, detection, etc. The surface can be set at an appropriate
potential to electrically neutralize (convert ions to neutrals)
ions collected thereon. The surface can be, e.g., a metal belt, a
metalized non-conductive material, such as ceramic or polymers, or
combinations thereof. FIG. 1 shows a schematic of moving belt 101
configuration and FIG. 2 shows a schematic of a configuration
having an accessible ion collector plate 202. Coating materials,
applied, for example, to the belt or plate, can be used to enhance
the usability of the collection method. For example, a collector
coated with a chemical agent that can form a chemical bond, e.g.,
covalent bonds, with ions of interest can be used to the enhance
the selectivity of the separation and purification process; such
collector can, e.g., be chemically washed to removed unwanted
interferences, thus only sample that had right mobility properties
and chemical reactivity toward the coating material will be left on
the plate. Any chemical agent, or a portion thereof, that remains
attached to the analyte component of interest can later be removed
for further investigation.
[0073] An ion mobility separator and methods of use thereof, of the
present inventions, can use a liquid phase, e.g., a solution, as an
ion collection device. Such a solution, or solutions, can be
static, for example, retained in a container or can be dynamic, for
example, flowing on a surface. An electrical potential can be
applied to the solution or solutions, e.g., to facilitate an ion
neutralization, collection, or reaction with a chemical agent in
the solution(s).
[0074] The collection solution can be conductive or non-conductive.
With a conductive solution, e.g., a voltage can be applied to the
solution and ions can enter the solution directly, so, in various
embodiments, the solution can behave like a solid. For a
non-conductive solution, other effects can be used to assist the
ion collection. Such effects include, but are not limited to, gas
flow toward the collection solution, a high electric field ion
acceleration in front of collection solution, and usage of a
polarizable liquid. After ions of interest are collected in the
solution, the solution can be removed from the IMS device and used,
e.g., for further investigation subject to purification, etc. The
ion collector can use more than one collection container with
different solutions. Sample solution or solutions can contain a
reagent (e.g., chemical agent) that may be reactive to the ions to
be collected; on-the-fly reactions between collected ions and the
added reagent, or the solutions themselves, can be accomplished in
the IMS device. The collected samples, e.g., can be used for
further analysis, such as IR spectroscopy, NMR spectroscopy, and
MS, for synthetic reactions, etc. The collected samples can be used
to prepare a pharmaceutical formulation.
[0075] In various embodiments of a static collection solution,
mobility selected samples are collected into the solution for a
period of time and the solution is removed from the device for
further investigation and/or use in various embodiments of a
dynamic collection solution, the solution moves on the surface. The
movement of the liquid can be either across the surface, flow
inside, flow outside of the surface, or combinations thereof. The
fluid can be mechanically moved, e.g., by creating a pressure
difference, by electroosmotic force, etc. In various embodiments, a
micro-machined collection plate, having multiple channels for
liquid flows can be used. With the dynamic solution collection
methods, the collected flow can be led to a flow cell or other
fluid guides of any kind to communicate with other instruments.
[0076] With a preparative IMS, a sample is first prepared in a
suitable format, gas, liquid, solid phase, for example, or
combinations thereof. A sample is introduced to the ionization
source where it is ionized; subsequently the ionized sample is
separated by a mobility separator. The sample can be a mixture of
many different molecules and chiral molecule(s) thereof. The
mobility separated sample can be collected on a full-profile
collector, such as a moving belt, selectively collected on a
partial collector, such Faraday plates or collection solutions or
combinations thereof. For example, ions corresponding to molecules
not of interest could be collected in a static manner, e.g., all on
substantially the same solution or location on a belt. The ion
collector could then be operated in a dynamic mode, for example
moving a belt, flowing solution, etc., to separately collect
samples, e.g., different chiral molecule(s) of interest. After the
samples are collected on the collector for certain amount of time,
they are removed from the IMS and reused in their pure form.
[0077] Selecting an effective chiral modifier (also referred to as
a separating substance herein) for the ion mobility separator can
be accomplished as follows. Without being held to theory it is
believed that the interaction force among chiral molecules may
involve hydrogen bonding, dipole-dipole, .pi.-.pi., acid/base,
stereo-repulsion, etc. As illustrated in the Examples of the
present application, R- and S-2-Butanol can be used to separate
enantiomeric mixtures. Without being held to theory it is believed
that the primary force for a butanol based chiral modifier is
hydrogen bonding. In various embodiments a series of chiral
modifiers, examples are as shown in FIG. 7, are selected for
separation of targeted bioactive chiral molecules. The modifiers
are chosen to demonstrate different possibilities of gas phase
interactions. Accordingly, it is to be understood that suitable
chiral modifiers for the present inventions are not limited to
those specifically shown.
[0078] Among gas phase interaction forces, gas phase hydrogen
bonding is typically the strongest; and the addition of other
interaction forces may further enhance the chiral based separation.
Using (R)-(-)-2-Butanol 701 and (S)-(+)-2-Butanol 703 as a baseline
study, see FIG. 7, we choose
(R)-(-)-.alpha.-(Trifluoromethyl)benzyl alcohol 709 and
(S)-(+)-.alpha.-(Trifluoromethyl)benzyl alcohol 711 for its
possible .pi.-.pi. interaction; (R)-Tetrahydrofuran-2-carbonitrile
715 and (S)-Tetrahydrofuran-2-carbonitrile 713 for its possible
stereo-hindrance effects; (2R,6R)-2,6-Heptanediol 717 for its
symmetric multiple chiral center interaction; (+)-Ethyl D-lactate
705 and (-)-Ethyl L-lactate 707 for its enhancement of hydrogen
bonding, as these molecules can be a hydrogen bond donor as well as
acceptor. Using the principles of the present application a larger
selection of the chiral modifiers can be screened to build a
knowledge base for chiral modifier selection and/or choice are
based on different interaction forces.
[0079] In the separation and collection process, multiple chiral
modifier gases can be used simultaneously, sequentially, in turns
or combinations thereof. The instrument design for the IMS can
include, e.g., a fast switch mechanism to infuse different a chiral
modifier one at a time, selective chiral modifiers at the same
time, or combinations thereof in to the IMS. The fast switch
mechanism is preferred for rapid chiral separation and collection
using IMS. Different chiral modifier gases are preferably switched
from one to another within seconds or minutes and typically the
effectiveness of the separating substance can be observed within
same amount of time. The chiral modifier concentration during the
ion mobility separation process can either be set to a
substantially constant or change with time. A chiral modifier
concentration gradient in the IMS can be used, e.g., to alter the
separation characteristics. By this means, e.g., a sequence of
mobility spectra could be obtained under different chiral modifier
concentration conditions. A software module can be used to monitor
the ion mobility peak shifting and other characteristics of the
chiral molecule ions. The separation and collection of chiral
molecule structure can be tracked, e.g., by the software to find
preferred separating conditions. Similarly, a pressure or
temperature gradient, together with the chiral modifiers), can be
used in the ion mobility separator to further refine or alter
separation and collection conditions.
[0080] In addition, it is important to note that the mobility based
separation happens in an ion mobility separator, any reactions,
such as charge transfer or cluster formation, between chiral
molecule ions and chiral modifiers can cause ion mobility peak
broadening and may produce unpredictable results. Charge transfer
in the ion mobility separator is a major concern, thus, chiral
molecules with relatively weak charge affinity are preferably
chosen. Similarly, less reactive chiral molecules are preferred
instead of those that form a stable complex that may permanently
convert the targeted chiral molecule(s) to another chemical form.
In various embodiments, the chiral modifiers can be chosen to
selectively react to one of the chiral molecule(s) in the mixture.
In various embodiments, such a reaction can occur in the ionization
source, and the separation in IMS can become separating one chiral
molecule from the other chiral molecule in the cluster. This
approach, in various embodiments, can provide a means for
determining the chiral structure (S-, R-, L- or D-) of the molecule
since the chiral modifier structure is known.
[0081] The ion mobility separator described in the present
inventions can be interfaced with a chromatographic separation
method, e.g., a supercritical fluid chromatography (SFC), high
performance liquid phase chromatography (HPLC), electrophoresis
systems, etc. With the combined ionization sources described
herein, elutents from an HPLC, electrophoresis systems, etc., can
be directly electrosprayed into the IMS device; and the elutents
from SFC or GC can be introduced to the heated gas sample inlet of
the ionization source (see e.g. FIG. 4 item 408).
[0082] The IMS systems and methods of the present inventions can be
powerful tools for chiral separation and collection; combining with
chromatographic system can open a broad range of instrumentation.
For example, using chiral separation IMS with non-chiral
chromatographic or electrophoresic systems can facilitate the
separation of complex mixtures with a more flexible choice of
stationary phases, mobility phases, and other chromatographic or
electrophoresic conditions. The chiral separation and collection
IMS device can be linked with a chiral separation chromatographic
or elctrophoresic systems to further purify the chiral molecule(s)
of interest. For complex mixtures, for example, interfacing
chromatographic or elctrophoresic systems to ion mobility
separator, and to mass spectrometric systems can provide a powerful
tool for analytical and preparative separation.
[0083] The IMS based chiral separation methods of the present
inventions can also be used to monitor other preparative or
semi-preparative chiral separation methods, such as, e.g., SFC and
HPLC methods. For example, by splitting the flow in these systems
to the IMS, IMS can provide an online monitoring method for the
preparative separation method.
[0084] IMS-MS provides a powerful tool for sample analysis. The ion
mobility separator can be interfaced to, but limited to a
quadrupole, an ion trap, or time of flight mass spectrometer.
Existing IMS-MS interfaces typically suffer from low transportation
efficiency. The present application provides unique IMS-MS
interface designs that facilitate overcoming this limitation of
traditional interfaces.
[0085] Historically, the ion transportation rate of an IMS-MS is
one of the bottlenecks for instrument sensitivity. Because of the
large pressure barrier between IMS and MS operating conditions,
effective transport of ions from the IMS to the MS through an open
interface with minimal time delay has been difficult. The present
application, provides several interface designs. The interface
described in this invention can be used independently from rest of
the instruments and methods described in this invention.
[0086] In one aspect, the present inventions provide an interface
using high field ion extraction with short resistive glass tube or
pinhole interface 800. Resistive pinhole or resistive capillarity
tube interface 800 of the present inventions can be used for
transporting ions from atmospheric pressure to high vacuum. For
example, conductive glass from Burle Industry can be used for a
glass capillary tube. Examples are shown in FIG. 8 where first
voltage 810 and second voltage 811 are applied across the tubing or
pinhole. The size and shape of the resistive interface is made to
maximize the ion transportation. An alternative shape of the
resistive interface 803 is shown in FIG. 8. Simulation of the
electric field is shown in FIG. 9. The resistive glass tube can be
used to generate a high electric field inside the pinhole and the
electric field strength inside and outside the pinhole can create a
local focusing effect that can bring more ions into the vacuum.
Multiple resistive tubes or pinholes, e.g., in parallel, can be
used on the same device to enhance the sensitive. Resistive glass
is one material that can withstand the temperatures typically
required for an IMS-MS interface. Beyond the electric field created
in the pinhole region, the ion focus electric field can be extended
to further distance for the local pinhole region, in order, e.g.,
to focus more ions into the pinhole region, and thus transport them
into the vacuum chamber for mass analysis
[0087] In various embodiments, the interface between an IMS and MS
uses a resistance tube; and a high voltage power supply operatively
connected to the resistance tube. The resistance tube has an inner
diameter in the range between about 1 micrometer and about 2 mm and
the high voltage power supply is configured to apply a voltage
gradient in the range between about 1 and about 40,000 volts across
the inner diameter of the resistance tube.
[0088] In one aspect, the present inventions provide an interface
using a transverse electric field at the interface 1000 of the
ambient pressure and vacuum region (FIG. 10). The two conductive
plates 1002 and 1004 are arranged with a small angle that is equal
or greater than zero degree; the E/N ratio is preferably kept
substantially the same in this interface by balancing the pressure
gradient and distance between these two plates. In various
embodiments, an electric field correction electrode 1006 can be
placed at a location outside the interface 1000 with an appropriate
potential with respect to the plates. Similar arrangements can be
achieved in the cylindrical fashion. Alternatively, the segmented
electrodes on non-conductive plates can also be used to create
similar transverse electric field that could function as asymmetric
ion mobility separator at the IMS-MS interface. The segmented plate
(electrode) approach provides flexible control parameters. The
asymmetric ion mobility separator interface could also be optimized
for ion focusing during the ion transportation in the IMS-MS
interface.
[0089] The interface between an IMS and MS uses a first conductive
member and a second conductive member, wherein the first and second
conductive members are substantially symmetrically arranged about
an ion transport axis and wherein the distance between the ends of
the first and second conductive members proximal to an ion mobility
separator is equal or less than the distance between the ends of
the first and second conductive members distal to the ion mobility
separator and a DC and RF power supply operatively connected to one
or more for the first and second conductive members. The first and
second conductive members are plates. The DC and RF power supply is
configured to apply voltages between the first and second
conductive members in a means that is resemble to the operation of
an asymmetric ion mobility separator with two parallel plates,
where the first and second conductive members are closest.
[0090] For IMS-MS analysis, ion mobility is preferably measured
outside the vacuum chamber (under a uniform pressure conditions)
for better mobility resolution and increased accuracy. FIG. 11
schematically illustrates using measured mobility outside the
vacuum chamber to, for example, correct mobility measured inside
the vacuum chamber with a MS. With common MS design, addition drift
time is added to the mobility measurement when using MS as a
detector; ions have travel through a pressure gradient in the
IMS-MS interface and low vacuum ion optics where addition collision
occurs. With the IMS-MS system shown in FIG. 11, a control and data
acquisition module locates on a computer 1115; Signals 1117
communicated to the ion mobility separator 1101 control the first
gate 1103 and second gate 1105 of the ion mobility separator, at
least a portion of the ions are allow to enter the ion mobility
separator and then allowed to pass through the second gate 1105.
Drift time (or mobility) of ions are first measured at the ion
detector/collector 1107; the measured ion signal is processed with
preamp 1113 and the data acquisition modules on the computer 1115;
After a portion of the ions travel through the IMS-MS interface,
and then mass separated in MS 1109 and detected on the MS ion
detector 1110. The measured ion signal is then process with preamp
1111 and the data acquisition modules. Ion mobility spectra
generated at ion detectors 1107 and 1110 are processed by the data
acquisition module and mobility correction can be made for each
individual ions based on their mobility measured outside the vacuum
chamber. In various embodiments, a software module can be used to
realize such correction/calibration. This procedure is preferred
when using an ion collector outside the vacuum chamber for sample
collection and MS for ion monitoring and identification.
[0091] In various embodiments, the ion detector 1107 used to
measure ion mobility outside the vacuum chamber, could also be used
as an ion collector that collects at least a portion of the samples
for further analysis or other use. In various operation mode of the
IMS-MS device, selected ions may be allowed to pass the second ion
gate 1105. As a large portion of the selected ions are collected on
the ion collector 1107, a small portion of the selected ions may be
detected by the MS to identify their mobilities and mass to charge
ratio. Similarly, when asymmetric ion mobility spectrometer used as
ion mobility separator, selected ion are allowed to pass through
the IMS and detected either on the ion detection/collection plates
316 or a MS located in the vicinity of the detection plates, the
rest of the ions are collected at different location of the full
profile ion collection plate 325. In various embodiments, the
instrument operating parameters, e.g. compensation voltage and RF
frequency, may be used to correlate the location of ions collected
on the full profile ion collection plate and ions detected by the
MS or ion detection plates.
[0092] If the drift time measured outside the vacuum chamber is
t.sub.out and the m/z data is acquired at t.sub.ms, then the
measured m/z data can be correlated to the mobility data by the
factor of a delay time in the interface for each individual
ions.
[0093] If the drift time measured outside the vacuum chamber is
t.sub.out and the m/z data is acquired at t.sub.ms, then the
measured m/z data can be correlated to the mobility data by the
factor of a delay time in the interface for each individual
ions.
[0094] The method for operating an ion mobility separator and a
mass spectrometer may include: (a) measuring ion mobility of an
analyte component using an ion detector/collector at the end of the
ion mobility separator; (b) measuring ion mobility and mass to
charge ratio using ion detector of mass spectrometer; (c)
correlating the ion mobility data obtained from mass spectrometer
with the ion mobility dada from the ion mobility separator. The
ions collected on the ion collector at the end of the IMS are mass
identified using the correlated ion mobility data.
[0095] The IMS-MS instrument of the present inventions can be
operated, in various embodiments, as a combined preparative or
analytical chiral separation and sample recovery system. For
example, with segmented or un-segment Faraday collection plates
mounted in the front of MS, a majority of the sample separated by
the IMS can be collected on the Faraday plate(s) under high
pressure conditions and a small portion of the mobility separated
sample can be transported through an interface to the MS. The
collection plate can have an opening that matches the geometry of
the IMS-MS interface design. The MS can be used as an online
monitoring device for what is collected on the collection plate.
Selective collection on this plate can be achieved by using
asymmetric IMS as an ion filter, by adding a second ion gate for a
symmetric IMS, or both. For example, ions with one mobility
property (to the best resolution of a given device) are collected
on a plate, and used for preparative, analytical purposes, or both.
Furthermore, if a transverse electric field at the interface for MS
is used; multiple stage ion mobility based separation can be
achieved according to ions symmetric or asymmetric ion mobility
properties. In various embodiments, this tandem ion mobility
separation can produce high mobility separation efficiency.
[0096] Smaller pressure difference between IMS and quadrupole MS is
preferred for better ion transportation efficiency. In addition,
quadrupole MS is also preferred for quantitative measurements.
Finally, as a practical matter, the quadrupole MS often has the
lowest development and manufacturing cost compared to other MS in
the current MS market. From an instrument application point of
view, IMS-QMS can be a preferred choice as a fast screening tool
for enantiomeric excess measurement. The optical purity is
numerically equivalent to the enantiomeric excess, which is defined
as: Enantiomeric excess %=[mole fraction (major enantiomer)-mole
fraction (minor enantiomer)].times.100. In various embodiments, it
can increase the throughput in these measurements, specifically in
the initial stages of the drug discovery process where hundreds or
thousands of drug compounds are being screened as potential drug
candidates. In various embodiments, it can also be used as a rapid
QA/QC method of pharmaceutical products. In addition, various
embodiments of the chiral separation IMS-QMS of the present
inventions are compatible detection methods for current preparative
chiral separation methods, such as SFC and HPLC.
[0097] In various embodiments, a chiral separation IMS-MS of the
present inventions can be a desktop unit that has a comparable size
with current analytical HPLC systems. An integrated data
acquisition system can be used to control both IMS and MS. Multiple
infusion points on the drift gas inlet manifold can be implemented
to allow multiple chiral modifiers to be introduced into the
IMS-QMS system. In various embodiments, rapid switching among
chiral modifiers with different chemical structures can reduce
method development time from days to weeks for chiral
chromatography to several minutes on the CIMS-QMS.
[0098] The present inventions also contemplate a stand alone Chiral
Ion Mobility Separator (CIMS) system without MS. In various
embodiments, CIMS can be used as a rapid and low cost chirality and
enantiomeric excess detector of known samples. This configuration,
e.g., can be preferred when used as a portable QA/QC equipment for
pharmaceutical products. A chiral separation IMS-time of flight
mass spectrometer system is one of the possible embodiments for
chiral separation IMS systems; interfacing with a chromatographic
system it could be a method of choice for the analysis biomarkers,
metabolites or other complex biological samples while chirality of
these molecules is of interests.
[0099] As already mentioned, the present invention is applicable in
principle also to the separation and collection of nonchiral
analyte components such as; isomers, stereoisomers, but not limited
to these. If, for example, the analyte components contain a double
bond (olefin) in which the first analyte components' double bond is
in the cis configuration and the second analyte components' double
bond is in the trans configuration, then the separating
substance(s) can interact selectively to some degree with either
the cis or trans configuration enhancing analyte component
separation. In addition and already mentioned, the present
invention is applicable to using nonchiral separating substance(s)
for the separation of chiral and/or nonchiral analyte components.
If, for example, the separating substance can be a particle such as
helium, argon, nitrogen, carbon dioxide, but not limited to these.
It has been shown that these gases used as the drift gas have
differing polarizability values and do not affect all ions equally
[Asbury, G. Reid; Hill Jr., Herbert H.; Anal. Chem. 2000, 72,
580-584 and Beegle, Luther W.; Kanik, Isik; Matz, Laura; Hill Jr.,
Herbert H.; International Journal of Mass Spectrometry 216 (2002)
257-268]. This effect can be exploited in order to alter the
separation factors between different analyte components by mixing
the particle (separating substance(s)) into the drift gas.
[0100] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
[0101] The preliminary data of the following examples was obtained
on an electrospray ionization-ion mobility spectrometer-quadrupole
mass spectrometer (ESI-IMS-QMS) system. The system has been
modified for continuous infusion of the chiral modifier into a
preheated drift gas. The system is schematically shown in FIG. 12,
the chiral modifier was pumped into the drift gas supply line
through a "T" fitting 1201 located behind the preheating element
1203. The transfer line after the infusion point is maintained at
substantially the same temperature to prevent condensation of the
chiral modifier. A substantially constant chiral modifier
concentration in the drift gas was maintained in the about 1 to
about 20 ppm range for the experiments unless indicated otherwise.
The temperature of the drift region, desolvation region and the
drift gas was set at 200.degree. C. for all experiments.
[0102] In the experiments, the analyte enantiomers were directly
electrosprayed into IMS. The enantiomeric ions are formed and
desolvated in the desolvation region, and separated in the drift
region of the IMS. After mobility based separation, the ions were
mass identified by the quadrupole mass spectrometer. In most of the
experiments, the MS was operated in the single ion monitoring mode
to selectively detect targeted enantiomers. Typically, the
separation in the drift tube can be accomplished within 30
milliseconds and the ion identification in MS can be achieved
within a few milliseconds. To achieve a desired signal to noise
ratio, the spectrometer was set to signal average multiple ion
mobility spectra for a few seconds in total.
Example 1
[0103] FIG. 13 shows superimposed ion mobility spectra of racemic
mixtures in a pure nitrogen drift gas (no chiral modifier added).
Each enantiomer in the electrospray solution was at a concentration
of 100 ppm. Samples were introduced into the IMS via the
electrosprayer with a flow rate of 1 .mu.L/min. The enantiomeric
mixtures showed in FIG. 13 are D- and L-valinol, D and L-threonine,
D- and L-penicillamine, D and L-tryptophan, D- and
L-methyl-.alpha.-glucopyranoside and R- and S-atenolol. These
spectra represent data that can be obtained using a conventional
ion mobility spectrometer. Even though these test enantiomeric
mixtures could be separated from each other in the IMS, no
enantiomeric separation was observed for the racemates in nitrogen
drift gas without chiral modifier.
Example 2
[0104] Atenolol is from a class of drugs called beta-blockers
mainly prescribed alone or in combination with other medications to
treat high blood pressure and lower heart rate, to prevent angina
and to reduce the risk of recurrent heart attacks. Chiral Ion
Mobility Separator (CIMS) separation of S- and R-atenolol
enantiomeric mixture is illustrated in FIG. 14. When no chiral
modifier was introduced to the drift gas, drift times for the S-
and R-enantiomers were almost identical at 24.56 and 24.51 ms,
respectively. In the CIMS, The drift times of S- and R-atenolol
were 24.61 ms and 25.04 ms respectively when analyzed individually;
the drift times of S- and R-atenolol were 24.66 ms and 25.06 ms
when analyzed as a mixture. It was observed that drift time shift
of R-atenolol was more significant compared to S-atenolol.
Example 3
[0105] To illustrate how a chiral modifier affects separation in a
CIMS, the drift times of individual enantiomers, L- and
D-methionine, were recorded as a function of infusion flow rate of
the chiral modifiers introduced into the drift gas. In the
experiments, .mu.L/min of a liquid chiral modifier, S-(+)-2-butanol
or R-(-)-2-butanol, was pumped into and volatilized in the
preheated nitrogen gas stream. The results of this investigation
are shown in FIG. 15.
[0106] The drift times of methionine enantiomers increased with the
introduction of chiral modifier, S-(+) or R-(-)-2-butanol. When
S-(+)-2-butanol was used as the chiral modifier the drift time of
both enantiomers, D- and L-methionine increased as a function of
the concentration of the chiral modifier in the nitrogen drift gas.
However, no difference in the drift time of the enantiomers could
be seen until the chiral modifier flow rate reached about 30
.mu.L/min. With only nitrogen as the drift gas, the drift time of
both methionine enantiomers was 21.52.+-.0.04 ms. With a chiral
modifier flow rate at 5 .mu.l/min, the drift time of methionine
enantiomers shifted to 22.12 ms. At a chiral modifier flow rate of
45 .mu.l/min, the drift times were 23.83.+-.0.03 ms for
D-methionine and 23.64.+-.0.04 ms for L-methionine. A 0.8% change
in the separation factor between the enantiomers was observed,
where the separation factor is defined as the ratio of the
t.sub.2/t.sub.1; t.sub.1 and t.sub.2 are drift time of the two
enantiomers.
[0107] With a chiral modifier flow rate below about 30 .mu.l/h,
both L- and D-methionine drift time increased with the gas phase
concentration of chiral modifiers. The drift time increase was
caused, it is believed without being held to theory, by drift gas
composition change, which reflects changes of ion-molecular
interaction in the CIMS. However, it is believed that this
interaction is not related to molecular chirality. When chiral
modifier concentrations reached a certain level, the selective
interaction between enantiomers and chiral modifiers could be
observed. The drift time shifts started to show differences
according to their chiralities. Significant change in separation
factor was observed beyond the flow rate of about 45 .mu.l/h of
S-(+)-2-butanol. The flow rate of 45 .mu.l/min of S-(+)-2-butanol
corresponds to a mixing ratio of approximately 10 ppm of
S-(+)-2-butanol in nitrogen at standard temperature and
pressure.
[0108] A smaller shift in drift time was observed with
R-(-)-2-butanol as the chiral modifier. The maximum shift in
separation factor was about 0.4% between the enantiomers. However,
with R-(-)-2-butanol, L-methionine drifted longer than
D-methionine. The enantiomers were identified by measuring the
drift time of each enantiomer separately under substantially
identical experimental conditions. Based on this data,
S-(+)-2-butanol was chosen as the chiral modifier at a flow rate of
45 .mu.L/min for the remainder of these experiments.
[0109] Similarly, tryptophan enantiomers were used to demonstrate
CIMS separation under substantially the same experiment conditions.
FIG. 16 illustrates the gas phase separation of tryptophan
enantiomers when S-(+)-2-butanol was used as the chiral modifier in
the drift gas. The upper graph of FIG. 16 shows the drift times of
L- and D-Tryptophan are 23.28 ms and 23.66 ms, respectively, when
analyzed individually. The bottom graph shows the separation of the
enantiomers from a mixture of L- and D-Tryptophan. The measured
drift times of L- and D-Tryptophan were 23.22 ms and 23.58 ms,
respectively, when measured as mixture. With no chiral modifier in
the gas, drift times of L- and D-Tryptophan were nearly identical,
22.02 ms and 21.99 ms respectively. In this case, both enantiomers
interacted significantly with the chiral modifier but the
interaction between D-Tryptophan and S-(+)-2-butano was stronger
than L-Tryptophan, thus the separation of L- and D-Tryptophan
became possible.
Example 4
[0110] FIG. 17 shows CIMS separation of the sodium adducts of D-
and L-Methyl-.alpha.-glucopyranoside. The difference in drift time
shifts of D- and L-Methyl-.alpha.-glucopyranoside were significant.
Strong chiral selective interaction of the S-(+)-2-butanol and
Methyl-.alpha.-glucopyranoside enantiomers was observed. It is
believed, without being held to theory, that the stronger
interaction was the result of multiple-point selective interaction
between the chiral modifier and Methyl-.alpha.-glucopyranoside
enantiomers with multiple chiral centers.
Example 5
[0111] FIG. 18 shows CIMS separation of the D- and L-penicillamine.
Without aromatic rings in the penicillamine structure, the observed
drift time difference between D- and L-penicillamine was relatively
small.
[0112] Table 1 Summarizes the results of most of the enantiomers
studied in these examples. It shows a comparison of the drift times
and mobilities of enantiomers with and without chiral modifier
infused in the drift gas. The first column lists measured m/z of
identified enantiomeric ions; the second column identifies the test
compounds; the third column shows their drift times in pure
nitrogen; the fourth column shows their drift times with the
presence of chiral modifier, S-(+)-2-butanol; the fifth and sixth
columns show calculated reduced mobility values according to
measured drift time. The data demonstrates that when chiral
modifier is added to the drift gas, the drift time of both
enantiomers are elongated, however, one enantiomer always has a
greater shift compared to the other due to structure selective
interaction between chiral modifier and the enantiomers.
[0113] Using S-(+)-2-butanol as the chiral modifier at a infusion
flow rate of 45 .mu.L/min, Table 1 reports the drift times and
mobilities of a variety of enantiomers. When the drift gas was
mixed with chiral modifier, the drift times of enantiomers were no
longer the same. A 2-28% shift in the drift times of the analyzed
samples was observed when the chiral modifier was added to the
nitrogen drift gas. Compared to the drift time in pure nitrogen,
the maximum observed drift time difference was about 5.2 ms for
methionine when S-(+)-2-butanol was introduced. This suggests that
methionine had the strongest interaction with the chiral modifier
as compared to other analyzed samples. The minimum shift was
observed for atenolol, which was about 0.6 ms. It was observed that
even though methionine experienced interaction in chirally modified
drift gas, the difference in the drift times of the enantiomeric
ions was the least among the samples studied. The maximum
difference in the drift times between the enantiomeric pair was
observed between D- and L-methyl-.alpha.-glucopyranoside and D- and
L-serine of the samples studied. On average, a 2% deviation in
drift times between the enantiomers was observed in IMS.
[0114] It is believed, without being held to theory, that the above
observations indicate that the interaction between chiral modifier
and targeted enantiomers can be divided into two categories, chiral
selective or non-chiral selective interactions. The drift time
shift caused by non-chiral selective interaction may include
enhanced elastic collision or other long-range gas phase
interactions between the modifier and analyte ions. Even though the
interaction force can be strong, the drift time of enantiomers
shifted at the same degree because the enantiomers had identical or
substantially identical properties involved in the interactions.
CIMS relies on the chiral selective interaction that involves, it
is believed, the functional group(s) around a chiral center of the
enantiomers. Multiple chiral centers in D- and
L-methyl-.alpha.-glucopyranoside seem to enhance the chiral
selective interaction significantly.
[0115] The gas phase separation of each enantiomeric pair was
repeated for a statistically significant number of times to
determine reproducibility. Overall the reproducibility of the drift
times was excellent and similar to that achieved when no chiral
modifier was introduced. The standard deviations of the
measurements ranged from 0.03 to 0.05 ms. Thus, the drift time and
ion mobilities were measured reproducibly within a RSD of 0.2%.
[0116] The present examples indicate that the parameters that tend
to have the most impact on governing the performance of the ion
mobility separator for chiral compounds are separator temperature
and chiral modifier concentration. In the present examples,
methionine was used to explore required chiral modifier
concentration to separate enantiomeric ions.
[0117] It was observed in common ion mobility experiments that
ion-molecular interaction was more significant when the
spectrometer is operated under lower temperature (<100.degree.
C.) as opposed to higher temperatures. Most of the data was
obtained at under 200.degree. C. conditions.
[0118] Biologically active molecules can also be separated by CIMS.
A few selected examples of biologically active molecules suitable
for testing of and evaluating CIMS-MS performance include, but are
not limited to, those in Table 2. This group of chiral analytes can
be prepared in a solution that can be electrosprayed into CIMS. The
solutions may contain single enantiomers or mixtures with known
enantiomeric ratio. These samples can be used, e.g., for system
optimization, chiral modifier selection, performance comparison
with other separation methods, etc.
[0119] In general, IMS is referred as a semi-quantitative method.
Quantitative measurement capability of IMS is limited by the
ionization process as charge transfer reactions are commonly used
to ionize target molecules. The charge competition process in
electrospray ionization source, for example, can prevent target
molecule from being completely ionized in a mixture. As a result
the measured peak height cannot be quantitatively related back to
liquid phase concentration when charge competition processes exist.
However, for the purpose of chiral separation and detection, the
charge competition will not affect the measured enantiomeric excess
because the enantiomers have identical or substantially identical
charge affinity and should have an equal or a substantially equal
probability of being ionized. As one set of practical tests, common
chiral drugs, such as atorvastatin, clopidogrel, olanzapine, etc.,
can be prepared in suitable electrospray solvents, and then
introduced to an IMS system for the assessment of enantiomeric
excess.
[0120] The data acquired was analyzed to seek information and
develop a fuller understanding of the relationship between drift
time shift and chirality. Some indication has been found in the
data that the drift time shift of L- and D-Methionine was reversed
when the chirality of the modifier is reversed.
[0121] The elution order of the chiral molecules from the CIMS is
determined by running a single enantiomer standard. However, the
chiral modifiers in CIMS can be easily switched from one chirality
to another within seconds. If the chirality is predicted with the
information obtained using multiple chiral modifier with multiple
chirality, the same result will be obtained much faster than
testing single enantiomer standards separately.
[0122] All literature and similar material cited in this
application, including, but not limited to, patents, patent
applications, articles, books, treatises, and web pages, regardless
of the format of such literature and similar materials, are
expressly incorporated by reference in their entirety. In the event
that one or more of the incorporated literature and similar
materials differs from or contradicts this application, including
but not limited to defined terms, term usage, described techniques,
or the like, this application controls.
[0123] While the present inventions have been described in
conjunction with various embodiments and examples, it is not
intended that the present inventions be limited to such embodiments
or examples. On the contrary, the present inventions encompass
various alternatives, modifications, and equivalents, as will be
appreciated by those of skill in the art.
[0124] While the inventions have been particularly shown and
described with reference to specific illustrative embodiments, it
should be understood that various changes in form and detail may be
made without departing from the spirit and scope of the inventions.
By way of example, any of the disclosed ionization sources, ion
mobility separators and ion collectors can be combined in any
combination to provide an apparatus for the separation of chiral
molecules in accordance with various embodiments of the invention.
Therefore, all embodiments that come within the scope and spirit of
the inventions, and equivalents thereto, are claimed. The
descriptions and diagrams of the methods, systems, and assays of
the present inventions should not be read as limited to the
described order of elements unless stated to that effect.
TABLE-US-00001 TABLE 1 KO OF CHIRALITY OF KO ENANTIOMERS IONS TEST
CHIRALITY AND TD, OF ENANTIOMERS AND T.sub.D ENANTIOMERS IN
S-(+)-2- (M/Z) COMPOUNDS ENANTIOMERS IN N2 WITH S-(+)-2-BUTANOL IN
N2 BUTANOL 267 R AND S- (S), (R), (S), (R), 1.18 1.18 1.18 1.16 (M
+ H)+ ATENOLOL 24.56 .+-. 0.03 24.51 .+-. 0.03 24.66 .+-. 0.04
25.06 .+-. 0.05 205 D AND L- (D), (L), (D), (L), 1.32 1.32 1.25
1.23 (M + H)+ TRYPTOPHAN 22.02 .+-. 0.03 21.99 .+-. 0.04 23.22 .+-.
0.05 23.63 .+-. 0.05 150 D AND L- (D), (L), (D), (L), 1.56 1.56
1.23 1.22 (M + H)+ METHIONINE 18.61 .+-. 0.04 18.66 .+-. 0.04 23.59
.+-. 0.04 23.83 .+-. 0.06 120 D AND L- (D), (L), (D), (L), 1.69
1.69 1.51 1.48 (M + H)+ THREONINE 17.22 .+-. 0.03 17.20 .+-. 0.04
19.22 .+-. 0.05 19.61 .+-. 0.05 217 D- AND L- (D), (L), (D), (L),
1.30 1.30 1.15 1.12 (M + NA)+ METHYL-A- 22.42 .+-. 0.05 22.40 .+-.
0.05 25.33 .+-. 0.08 25.87 .+-. 0.07 GLUCOPYRANOSIDE 203 D- AND L-
(D), (L), (D), (L), 1.30 1.30 1.23 1.21 (M + NA)+ METHYL-A- 22.35
.+-. 0.04 22.32 .+-. 0.03 23.61 .+-. 0.05 23.98 .+-. 0.04 GLUCOSE
150 D- AND L- (D), (L), (D), (L), 1.53 1.53 1.42 1.40 (M + H)+
PENICILLAMINE 18.94 .+-. 0.03 18.92 .+-. 0.05 20.48 .+-. 0.03 20.78
.+-. 0.04 104 D AND L- (L), (D), (L), (D), 1.74 1.74 1.62 1.60 (M +
H)+ VALINOL 16.72 .+-. 0.04 16.75 .+-. 0.03 17.84 .+-. 0.04 18.26
.+-. 0.04 166 D AND L- (L), (D), (D), (L), 1.45 1.45 1.31 1.28 (M +
H)+ PHENYLALANINE 20.07 .+-. 0.04 20.05 .+-. 0.04 22.22 .+-. 0.03
22.61 .+-. 0.05 106 D AND L- (D), (L), (D), (L), 1.73 1.73 1.55
1.52 (M + H)+ SERINE 16.82 .+-. 0.03 16.83 .+-. 0.04 18.72 .+-.
0.04 19.11 .+-. 0.05
TABLE-US-00002 TABLE 2 AMINO NUCLEOTIDE ACID/PEPTIDE ALKALOID
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
CARBOHYDRATE DRUG CARCINOGEN ##STR00006## ##STR00007## ##STR00008##
##STR00009## ##STR00010## ##STR00011##
* * * * *