CN115295394A - Electrospray ionization interface for high pressure mass spectrometry and related methods - Google Patents

Abstract

Embodiments of the present application relate to electrospray ionization interfaces and related methods for high pressure mass spectrometry. An electrospray ionization (ESI) mass spectrometer analysis system of an embodiment of the present application includes: an ESI device having at least one emitter configured to electrospray ions; and a mass spectrometer in fluid communication with the at least one emitter of the ESI device. The mass spectrometer includes a mass analyzer held in a vacuum chamber. The vacuum chamber is configured to have a relatively large (background/gas) pressure of about 50 mtorr or more during operation.

Description

Electrospray ionization interface for high pressure mass spectrometry and related methods

The application is a divisional application of an invention patent application No.201580079949.8 entitled electrospray ionization interface and related method for high pressure mass spectrometry at the stage of entering China national phase of PCT national application No. PCT/US2015/030380 with application date of 2015, 5, month 12.

Technical Field

The invention relates to mass spectrometry, and is particularly suitable for high-pressure mass spectrometers.

Background

Mass Spectrometry (MS) is a powerful analytical technique due to its sensitivity, versatility and ability to provide chemical and structural information of molecules; because of this, it is often the detection method of choice for a wide variety of applications. Electrospray ionization (ESI) significantly expands the scope of mass spectrometry, extending it to include biomolecules and other liquid analytes. ESI provides a convenient method for coupling liquid chromatography separations such as Liquid Chromatography (LC) or Capillary Electrophoresis (CE) with MS detection. Therefore, LC-MS has become a widely used analytical tool in fields such as proteomics, environmental monitoring, drug development, and clinical diagnosis. However, conventional LC-MS systems are typically limited to specialized laboratories because they are large, expensive, complex, and require a large amount of power. Conventional mass spectrometers are not suitable for these situations because of their large size, weight and power consumption (SWaP). See, e.g., rapid commu. Mass spectra.2004, 18, 1749-52 by whittten et al. The miniaturization of LC-MS systems is limited by the need for robust systems of pumps, valves and piping, while mass spectrometers are limited by low pressure operation, which traditionally requires bulky, fragile and expensive turbomolecular pumps.

One of the difficulties associated with coupling an ESI source and an MS system is that ions must be transported into a vacuum for mass analysis. See, e.g., page J.S. et al, "Ionization and Transmission Efficiency in an electrophoresis analysis-mass Spectrometry interface," J.Am.Soc.Mass.Spec.,2007,1 β (9), 1582-1590. The ion flow transmitted from the ESI source through the capillary inlet system can be reduced by up to three orders of magnitude. These losses occur primarily in the transfer region from the higher pressure to the lower pressure (i.e., on either side of the capillary inlet), and two or more of these regions are commonly used in conventional ESI-MS. See, e.g., rapid Communications in masses Spectrometry,1997, 11, 1813-1817, by s.a.Shaffer, k.tang, g.a.Anderson, d.c.prior, h.r.udset, r.d.Smith. This presents a significant challenge to coupling ESI with HPMS.

Disclosure of Invention

Embodiments of the present invention provide electrospray ionization devices coupled to High Pressure Mass Spectrometry (HPMS). The mass spectrometer may have an atmospheric conduction inlet in electrical communication with a dc power supply to introduce ions from the ESI device into the mass spectrometer. The HPMS may have a single chamber structure or a dual chamber structure. A mass analyzer such as a miniature cylindrical ion trap (mini-CIT) may reside in a vacuum chamber of a single vacuum chamber or a dual vacuum chamber design.

An electrospray ionization (ESI) mass spectrometer analysis system comprising: an ESI device having at least one emitter configured to electrospray ions; and

a mass spectrometer in fluid communication with at least one emitter of the ESI device. The ESI device can include a mass analyzer held in a vacuum chamber, wherein the vacuum chamber is configured to have a high pressure of about 50 mtorr or more (e.g., up to about 1 torr, about 10 torr, or about 100 torr) during operation; and a detector in communication with the mass analyzer in a vacuum chamber having a mass analyzer. During operation, the ESI device is configured to: (a) Electrospray ions into a spatial region external to the vacuum chamber at atmospheric pressure adjacent to an inlet device attached to the vacuum chamber, wherein the inlet device draws in electrospray ions external to the vacuum chamber with the mass analyzer and discharges the ions into the vacuum chamber with the mass analyzer. Or (b) electrospray ions directly into a vacuum chamber having the mass analyzer.

Embodiments of the present invention relate to electrospray ionization (ESI) mass spectrometer analysis systems. The system comprises: an ESI device having at least one emitter configured to electrospray ions; and a mass spectrometer in fluid communication with the at least one emitter of the ESI device. The mass spectrometer includes a mass analyzer held in a vacuum chamber. The vacuum chamber is configured to have a relatively large (background/gas) pressure of about 50 mtorr or more during operation. The mass spectrometer also includes a detector in communication with the mass analyzer. During operation, the ESI device is configured to: (a) Electrospraying ions into a region of space external to the vacuum chamber adjacent to an inlet device attached to the vacuum chamber at atmospheric pressure; or (b) electrospray ions directly into a vacuum chamber having the mass analyzer. For (a), the inlet device draws in electrospray ions outside of a vacuum chamber having a mass analyzer and discharges the ions into the vacuum chamber having the mass analyzer.

The detector may be held in a vacuum chamber with a mass analyzer.

The detector can be spaced apart from the mass analyzer in the vacuum chamber by a distance of about 1 mm to about 10 mm.

The ESI device can be configured to electrospray ions into a spatial region external to the vacuum chamber. The ESI device is positioned outside of a vacuum chamber having a mass analyzer. The inlet means may be spaced apart from the ESI means. An end portion of the entrance arrangement may reside inside a vacuum chamber having a mass analyzer spaced apart from an ion entrance of the mass analyzer by a distance of between 1-50 mm.

The inlet device may be tubular having at least one inlet aperture in fluid communication with at least one longitudinally extending passage extending therethrough. The system can include a dc voltage input to an inlet device external to the vacuum chamber having the mass analyzer.

The ESI device can be configured to electrospray ions into a spatial region external to the vacuum chamber adjacent to the inlet device at atmospheric pressure. The inlet device may be configured to position the inner end within the bore of the electrode in the vacuum chamber.

The ESI device can be configured to electrospray ions into a spatial region external to the vacuum chamber. The inlet device may comprise at least one inlet aperture and may have an external end spaced from the ESI device. The inlet device may be planar conductive and have a thickness of between about 0.100 millimeters and about 5 millimeters.

The system may include a compartment that holds the ESI device in an orientation and position in cooperative alignment with the inlet device. The compartment may comprise a buffer gas, such that during operation, the buffer gas may be conveyed via the inlet device into the vacuum chamber with the mass analyser.

The ESI device can be configured to electrospray ions directly into a vacuum chamber having a mass analyzer. The ESI device can be attached to a wall of a vacuum chamber such that at least one emitter is inside the vacuum chamber and one or more reservoirs of the ESI device are outside the vacuum chamber.

The at least one emitter may be spaced from the entrance aperture of the mass analyser by a distance of between 1-50 mm.

The ESI device can include a fluid microchip having at least one emitter. The at least one emitter may be positioned in a vacuum chamber having a mass analyzer and spaced apart from an entrance aperture of the mass analyzer by a distance of between about 1-50 millimeters.

During operation, the walls of the vacuum chamber may be held at electrical ground potential.

Only a portion of the fluid microchip may reside in a vacuum chamber having a mass analyzer.

The ESI device can be configured to electrospray ions into a spatial region external to the vacuum chamber adjacent to the inlet device at atmospheric pressure, and the at least one emitter can be spaced apart from an end of the inlet device external to the vacuum chamber by a distance of between about 1-10 millimeters.

The ESI device can be configured to electrospray ions into a spatial region external to the vacuum chamber. The system may include a Direct Current (DC) power supply connected to the inlet device at a location outside the vacuum chamber.

The system may include: a power supply configured to apply an electrical input to the ESI device during operation; and a vacuum pump in communication with the vacuum chamber having the mass analyzer.

The mass analyser may comprise an ion trap having an ejection end cap electrode, a ring electrode and an ejection end cap electrode. During operation, the vacuum chamber with the mass analyzer may be maintained at a gas pressure between 100 mtorr and 10 torr.

The inlet means may have an outer conical tip with at least one inlet aperture.

The at least one emitter may be spaced from the entrance aperture of the mass analyser by a distance of between 1-10 mm.

The system may include a tube or ion funnel electrode assembly in a vacuum chamber with the mass analyzer.

The mass analyser may comprise an ion trap mass analyser which may be: (a) A Cylindrical Ion Trap (CIT) having at least one of dimensions r0 or z0 less than about 1 millimeter; (b) A Stretched Length Ion Trap (SLIT) having a perforated central electrode extending along a longitudinal direction and surrounding the aperture in a transverse plane perpendicular to the longitudinal direction to define a transverse cavity for trapping charged particles. The aperture in the central electrode may be elongated in a transverse plane, the ratio of a major dimension to a minor dimension of the aperture being greater than 1.5.

Optionally, the minor dimension may be less than 10 mm (and may be about 1 mm or less), and/or the vertical dimension z of the transverse chamber ₀ And may be less than about 1 millimeter.

The mass analyser may be of dimension r ₀ A Cylindrical Ion Trap (CIT) between about 500 μm and about 100 μm.

The system can include a focusing electrode positioned in a vacuum chamber having a mass analyzer.

Other embodiments relate to methods of analyzing a sample. The method comprises the following steps: introducing sample ions into a vacuum chamber equipped with a mass analyzer by: (a) Electrospray ions from an electrospray ionization (ESI) device directly into a vacuum chamber having the mass analyzer with a gas pressure in the mass analyzer between 50 mtorr and 100 torr; or (b) electrospraying ions into a spatial region at atmospheric pressure external to the vacuum chamber, the spatial region being adjacent to an inlet device spaced apart from the ESI device, and then transporting the ions through the inlet device into the vacuum chamber holding the mass analyzer, wherein a gas pressure in the mass analyzer is between 50 mtorr and 100 torr. The method further comprises trapping ions in the mass analyser; selectively ejecting the ions from the mass analyser; detecting an electrical signal corresponding to the ejected ions using at least one detector; and generating data based on the detected electrical signals to determine information related to the sample.

The electrospray is from a tip of a microfluidic device having at least one electrospray emitter for electrospray of the ions.

The inlet device is attached to a wall of the vacuum chamber and may have an inner end that is located within the vacuum chamber and is about 1 mm to about 50 mm from an entrance aperture of the mass analyzer.

The mass analyser may comprise a miniature Cylindrical Ion Trap (CIT) and the mass analyser and detector may be held together in a vacuum chamber (no separate vacuum chamber is required).

The method may include transporting air as a buffer gas into the vacuum chamber using electric spraying.

The method may comprise maintaining the walls of the vacuum chamber at an electrical ground potential at least during electrospray.

The microfluidic device may be a microfluidic chip that performs step (a) and extends partially into the vacuum chamber to position at least one emitter of the microfluidic chip 1-50 mm from an entrance aperture of the mass analyzer.

The electrospray may be performed using the inlet device having one end extending a distance into the vacuum chamber, into the focusing electrode or into the aperture of the focusing electrode assembly. Ions may be directed from the end of the entrance arrangement towards a mass analyser by a focusing electrode or focusing electrode assembly prior to the trapping step.

It should be noted that aspects of the invention described with respect to one embodiment may be incorporated in a different embodiment, although not specifically described herein. That is, features of all embodiments and/or any embodiment can be combined in any manner and/or combination. The applicant reserves the right to amend any originally filed claim and/or to submit any new claim, including the right to be able to amend any originally filed claim to rely on and/or contain any other claim or claim, even though not originally claimed in that way. These and other objects and/or aspects of the present invention are explained in detail in the specification set forth below. Other features, advantages and details of the present invention will be appreciated by those of ordinary skill in the art from a reading of the following drawings and detailed description of the preferred embodiments, such description being merely illustrative of the present invention.

Drawings

FIG. 1 is a schematic diagram of an exemplary analysis system with a mass spectrometer with an electrified ejection ionization (ESI) interface, in accordance with an embodiment of the invention.

FIG. 2 is a schematic diagram of another embodiment of an exemplary analysis system having an ESI interface, in accordance with embodiments of the present invention.

3A-3C are schematic diagrams of other embodiments of exemplary analysis systems having dual vacuum chambers and ESI interfaces for differential pumping, according to embodiments of the present invention.

4A-4D are schematic diagrams of other embodiments of exemplary analysis systems having a single vacuum chamber with an ESI interface for a mass analyzer and a detector, in accordance with embodiments of the present invention.

Fig. 5A and 5B are enlarged schematic views of an exemplary electrospray device according to an embodiment of the present invention.

FIG. 6A illustrates an end view of an example inlet device, according to an embodiment of the invention.

Fig. 6B is a side view of the device shown in fig. 6A.

Fig. 6C is an end view of an alternative configuration of the inlet device shown in fig. 6A, according to an embodiment of the invention.

Fig. 7A is an end view of another embodiment of a tip portion of an exemplary inlet device, according to an embodiment of the present invention.

Fig. 7B is an end view of the opposite end of the device shown in fig. 7A.

Fig. 7C is a side view of the device shown in fig. 7B.

Figure 7D is a side view of an inlet tube, for example, having a conical end as shown in figures 7A or 7E, according to an embodiment of the invention.

Fig. 7E is an end view of an alternative configuration of the inlet device shown in fig. 7A, according to an embodiment of the invention.

Fig. 8A is a side perspective view of another exemplary inlet device according to an embodiment of the present invention.

Fig. 8B is an end view of the device shown in fig. 8A.

Fig. 8C is a side perspective view of a porous inlet device similar to the device shown in fig. 8A, in accordance with embodiments of the present invention.

Fig. 8D is a schematic diagram of an HPMS apparatus having a vacuum chamber and an inlet apparatus such as that shown in fig. 8A or 8C, in accordance with an embodiment of the invention.

FIG. 9A is a schematic diagram of another exemplary analysis system having an electrically injected ionization (ESI) interface for a mass spectrometer according to an embodiment of the invention.

FIG. 9B illustrates an end view of an ESI interface, in accordance with an embodiment of the present invention.

FIG. 10A is a block diagram of an analysis system including an ESI apparatus and a mass spectrometry system, in accordance with embodiments of the present invention.

FIG. 10B is another block diagram of an analysis system including an ESI device and a mass spectrometry system, in accordance with an embodiment of the present invention.

11A-11C are exemplary timing diagrams of analysis systems according to some embodiments of the inventions.

FIG. 12A is a flow diagram of operations that may be used to operate a mass spectrometry system, according to an embodiment of the invention.

FIG. 12B is another flow diagram of operations that may be used to operate a mass spectrometry system according to embodiments of the invention.

FIG. 13 is a schematic diagram of a data processing system according to an embodiment of the present invention.

FIG. 14 is a plot of normalized intensity versus mass-to-charge ratio (M/z) for HPMS (1.2 torr) perfusion-ESI spectra of four amino acids (100 μ M) with atmospheric interfaces, according to an embodiment of the present invention.

Fig. 15 is a plot of HPMS (1.3 torr) perfusion-ESI spectra versus (M/z) (Th) of 5 μ M thymopentin (V) using mini-CIT (r 0=250 μ M) and ambient air as a buffer gas for an example of the invention.

Fig. 16 is an electropherogram of normalized BPI (arbitrary units) versus time (minutes) for a 5 μ M peptide mixture comparing the signal from the Synapt G2 detection with the signal from the ESI-HPMS, according to an embodiment of the invention.

FIG. 17 is a plot of CE-ESI mass spectrum (normalized intensity, arbitrary units) versus m/z for bradykinin comparing signals from the Synapt G2 detection with those from ESI-HPMS, in accordance with an embodiment of the present invention.

FIG. 18 is a graph of normalized BPI (in arbitrary units) versus time comparing MS sampling rates of Synapt G2 detection and ESI-HPMS, according to an embodiment of the invention.

FIG. 19A is a plot of normalized intensity (in arbitrary units) versus M/z for 100 μ M histidine comparing the signal from ESI-HPMS to the signal from the mass set LC-ESI-qTOF (CID), according to an embodiment of the present invention.

FIG. 19B is a plot of signal (V) versus M/Z for infusion-ESI of an amino acid mixture (S, W, and M) for ESI-HPMS (1.3 torr) with ambient air as a buffer gas, according to an embodiment of the present invention.

FIG. 19C is a plot of signal (V) versus m/Z for infusion-ESI of peptides for ESI-HPMS (1.3 torr) with ambient air as a buffer gas, according to an embodiment of the invention.

Fig. 20 is a diagram illustrating the basic operating principle of the Cylindrical Ion Trap (CIT) and high-voltage ion trap theory.

FIG. 21 is a graph of normalized intensity (in arbitrary units) versus m/z at 1.0 Torr with ambient air as the buffer gas, different RF drive frequencies, and different critical r0 values, in accordance with an embodiment of the present invention.

Detailed Description

The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Like numbers refer to like elements throughout. Certain layers, components or features may be exaggerated in the figures for clarity and broken lines illustrate optional features or operations unless indicated otherwise. Additionally, the order of operations (or steps) is not limited to the order presented in the figures and/or claims unless otherwise specified. The thickness of lines, layers, features, components and/or regions may be exaggerated in the figures for clarity and broken lines illustrate optional features or operations unless specified otherwise. The abbreviations "fig." and "FIG" are used interchangeably with "figure" in the drawings and the description.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes" and/or "including," when used in this specification, specify the presence of stated features, regions, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y. As used herein, phrases such as "from about X to Y" mean "from about X to about Y".

It will be understood that when a feature, such as a layer, region or substrate, is referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when an element is referred to as being "directly on" another element, there are no intervening elements present. It will be understood that when an element or component is referred to as being "connected," "attached" or "coupled" to another feature or component, it can be directly connected, attached or coupled to the other component or intervening elements may be present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" to another element, there are no intervening elements present. Although described or illustrated with respect to one embodiment, features described or illustrated may be applied to other embodiments.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be understood that inputs such as those defined in commonly used dictionaries should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and/or clarity.

Spatially relative terms, such as "below," "beneath," "lower," "above," "upper," and the like, may be used herein to describe one element or feature's relationship to another element or feature as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the other elements or features. Thus, the exemplary term "below" can encompass both an orientation of above and below. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upward," "downward," "vertical," "horizontal," and the like are used herein for explanatory purposes only, unless specifically indicated otherwise.

It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another region, layer or section. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the present aspects.

The term "about" means that the number can vary from the value by +/-10%.

The term "analyte" refers to a molecule or chemical in a sample undergoing analysis. Analytes may include chemicals associated with any industrial product, process, or environmental hazard, such as, for example, toxins, organic compounds of toxic industrial chemicals or toxic industrial materials, and the like. In addition, the analyte may include a biomolecule found in a biological system or such as biopharmaceutical manufacturing.

The term "buffer gas" refers to any gas or mixture of gases having neutral atoms, such as air, nitrogen, helium, hydrogen, argon, and methane.

The term "mass resonance scan time" refers to the integrated signal acquisition time associated with mass selective ion ejection of an ion trap.

The term "mass" is generally to be interpreted as a mass-to-charge ratio, the meaning of which can be determined from the context. When the term is used in reference to mass spectrometry or mass spectrometry measurements, it denotes a measurement of the mass to charge ratio of an ion.

The term "microscale" with respect to the ion trap mass analyzer refers to small-sized ion traps having critical dimensions in the millimeter to submillimeter range, typically associated apertures having critical dimensions in one or more electrodes of the ion trap between about 0.001 millimeter and about 5 millimeters and any subrange thereof. The ion trap electrode central aperture may have a different geometry, for example a cylindrical or slit shaped void, and an array of voids is also possible.

The terms "miniature cylindrical ion trap", "miniature CIT" and "mini CIT" refer to cylindrical ion traps "CITs" having critical dimensions in the millimeter to sub-millimeter range, typically having associated apertures in one or more electrodes of the ion trap having critical dimensions between about 0.001 millimeter and about 5 millimeters and any subrange thereof. The ion trap electrode central aperture may have a different geometry, for example a cylindrical or slit shaped void, and an array of voids is also possible.

The term "microfluidic chip" is used interchangeably with "microchip" and refers to a fluidic sample processing device having a sub-millimeter sized fluid channel with at least one integrated emitter for processing a sample.

Mass spectrometry has historically been performed under high vacuum conditions. The reasons for this are: performance is improved if the ions do not collide with background gas molecules during the trajectory from the ion source through the mass analyser to the detector. Ion-molecule collision events scatter ions away from their intended trajectories, generally reducing mass resolution and signal intensity. Vacuum to achieve sufficient resolution in conventional systems can be normalized by the number of Knudsen (Knudsen). Mass spectrometry is typically performed under molecular flow conditions defined as Kn > 1, and in conventional practice, kn is between about 100 and 10,000 for mass analyzers of mass spectrometers.

Table 1 below is included at 10 ^-6 Calculated mean free path (mfp) for helium and nitrogen at a pressure range of 760 torr. The collision cross-sections of helium and nitrogen were determined by the respective van der waals volumes, and the average collision radii used in the calculation of mfp were 0.14nm and 0.18nm, respectively. See, e.g., knapman et al, int1.J. Mass spectra, 2010, 298, 17-23, the contents of which are incorporated herein by reference in their entirety. Calculate mfp value according to equation 1, where k is Boltzmann constant, T is the temperature in Kelvin, and d is the collision angleAnd P is air pressure. In Table 1, the temperature is assumed to be 300K.

10 ^-6 Torr or lower is a typical operating pressure for a linear quadrupole or time-of-flight mass analyzer, with critical length dimensions on the order of 100 millimeters. Such values result in Kn numbers of hundreds. Typical operating pressures for an ion trap mass spectrometer with a 10 mm ring electrode radius are about 10 ^-4 Torr, resulting in a Kn number of about 100. The operating regime of primary interest for the embodiments of the present application is a pressure greater than 50 mTorr and a critical length dimension z less than 1 millimeter ₀ Or r of less than 1 mm for some trap structures ₀ The value is obtained. In all cases listed in table 1, kn is less than 10 and all but one example is less than unit 1.

Table 1: knudsen number in micro-sized wells operated at high pressure

Embodiments of the invention perform mass spectrometry under non-conventional conditions, wherein the value of Kn is close to unit 1 and less (e.g., less than 10 and less than 1). At such pressures and fundamental length scales, the mean free path is similar to or less than the critical experimental length scale. Embodiments of the present invention may be particularly applicable to Paul trap mass analysers commonly referred to as ion trap mass analysers having a fundamental length dimension of less than 1 mm, e.g. radius r of the ring electrode ₀ Is 1 mm or less. Embodiments of the invention relate to high pressure mass spectrometers that can operate at pressures of 50 millitorr and above (e.g., to 1 torr, 10 torr, 100 torr, or 1000 torr) and/or that have Kn values of less than about 10 or even less than about 1.

The term "high resolution" refers to a mass spectrum that can be reliably resolved to less than 1Th, e.g., having a line width of less than 1Th (FWHM). "Th" is the Thomson unit of mass to charge ratio.

High resolution operations may allow the use of monoisotopic masses to identify the substance being analyzed. The term "high detector sensitivity" refers to detectors whose low end is capable of detecting signals of 1-100 charges in U.S.

The term "high pressure" refers to maintaining an operational (gas) background pressure in the vacuum chamber of the mass analyzer equal to or above about 50 mtorr, such as between about 50 mtorr to about 100 torr (thus, high pressure in the mass analyzer). In some embodiments, the vacuum chamber with the mass analyzer has a pressure between about 50 mtorr and about 10 torr, or between about 50 mtorr and about 1 torr, about 2 torr, or about 5 torr or less. In some embodiments, the high pressure may be about 50 mtorr, about 60 mtorr, about 70 mtorr, about 80 mtorr, about 90 mtorr, about 100 mtorr, about 150 mtorr, about 200 mtorr, about 250 mtorr, about 300 mtorr, about 350 mtorr, about 400 mtorr, about 450 mtorr, about 500 mtorr, about 600 mtorr, about 700 mtorr, about 800 mtorr, about 900 mtorr, about 1000 mtorr, about 1500 mtorr, or about 2000 mtorr.

Fig. 1 is a block diagram of an exemplary analytical system 100 having an electrospray ionization (ESI) device 20 (shown as a fluidic microchip device by way of example only) cooperatively aligned with a mass spectrometer 10. As is well known, the mass spectrometer 10 has three basic components: an ion source, a mass analyzer, and a detector. These components may take different forms depending on the type of mass analyzer. As shown in fig. 1, the ionizer includes an ESI device 20. The ESI device 20 can have different forms/structures including microfluidic chips, glass or quartz capillaries, drawn glass or quartz capillaries, metal capillaries, and combinations thereof.

The mass analyzer 30 resides in the vacuum chamber 12, the vacuum chamber 12 being maintained at an elevated pressure during operation. The mass spectrometer 10 may be a high pressure mass spectrometer that operates without the need for a turbopump, allowing for a more compact design relative to conventional high pressure systems. A detector 40 (which may include an electron multiplier and/or other type of detector) is located downstream of the mass analyzer 30. In some embodiments, the mass spectrometer 10 has a housing 10h that can have a second vacuum chamber 14 adjacent to the first vacuum chamber 12 and separated by a partition 102, which can be maintained at a different pressure than the first chamber 12, e.g., differential vacuum pumping.

In some embodiments, the first and second vacuum chambers 12, 14 can be maintained between 50 mtorr and 100 torr, with the second vacuum chamber 14 (where used) being maintained at a lower pressure than the first chamber 12. For example, the pressure in the vacuum chamber 12 may be about 100 torr, about 10 torr, about 1 torr, about 100 mtorr, or about 50 mtorr, while the second chamber 14 may be maintained at a lower pressure, such as about 10 mtorr or less. Where differential pumping is used, the second chamber 14 may be maintained at a pressure that is about 1 (one) or more orders of magnitude less than the first chamber 12. In some embodiments, the pressure differential may be a factor of 100 or more, depending on the leak rate and pumping capacity between the chambers 12, 14. For example, in certain embodiments, the high pressure chamber 12 may be at about 1 torr, while the low pressure (high vacuum) chamber may be at about 10 mtorr. However, other pressure differentials may be used, for example, the high pressure chamber 12 may operate at 100 torr and the low pressure chamber 14 at about 10 mtorr.

Although each chamber 12, 14 is shown connected to a vacuum pump 70 through a valve 71, in other embodiments a single vacuum pump may be used to provide different pressures for the two chambers 12, 14.

As shown in FIG. 1, the mass analyzer 30 can be mounted on a partition 102 that separates the vacuum chambers 12 and 14. The baffle plate 102 includes at least one aperture or open space 102a that fluidly connects the two chambers 12, 14, which allows buffer gas and ions to be transported from the vacuum chamber 12 to the chamber 14. The pressure drop created by the gas flow through the holes 102a creates a different pressure in the two chambers 12, 14. The mass analyzer 30 may be sealingly attached to the baffle 102 and may form a closed flow path between the two chambers 12, 14. In some embodiments, in the case of certain types of ion trap mass spectrometers, gas transport through the mass analyzer 30 can be used to enhance the ion signal. See, for example, co-pending U.S. provisional application serial No.62/010,050, the contents of which are hereby incorporated by reference as if fully set forth herein.

In some embodiments, as shown in fig. 1, 2 and 3A, for example, the ESI device 20 can electrospray an ion stream 20s from at least one emitter 20e of the ESI device 20 into the inlet device 15, and then through the inlet device 15 directly into the mass analyzer chamber 12 at high pressure. The inlet device 15 can be spaced a close distance or abutting contact with the emitter 20e while the emitter 20e releases (e.g., electrosprays) the sample into a spatial region at ambient pressure (e.g., atmospheric pressure) outside the vacuum chamber 12 and then into the inlet tube 15. Electrospray 20s may enter ambient (i.e., atmospheric) pressure, then enter the inlet aperture 15a at ambient pressure, then enter the vacuum chamber 12 with the mass analyzer 30. The mass analyzer chamber 12 may be in fluid communication with a vacuum pump 70 via a valve 71. While the mass analyzer vacuum chamber 12 is at high pressure, the outer end 15e of the inlet device 15 is at atmospheric pressure, facing the ESI emitter 20e. The inner end 15i of the inlet device 15 is held inside the mass analyser chamber 12. The inlet device 15 may be connected via a connector 18 (e.g., a vacuum fitting such as Ultra-Torr from Swagelok, solon, OH) ^TM Fittings) are sealingly attached to the wall 12w of the mass analyzer vacuum chamber 12.

The emitter 20e, which is an ion source, may be positioned to provide a relatively compact footprint. As shown in fig. 1, the outer and inner distance Di-m measured from the emitter tip 20e to the entrance of the mass analyzer 30 is typically between about 1cm and about 15cm, and more typically between about 5cm and about 12 cm, such as about 5cm, about 5.5cm, about 6cm, about 6.5 cm, about 7 cm, about 7.5 cm, about 8 cm, about 8.5 cm, about 9 cm, about 9.5 cm, about 10 cm, about 10.5 cm, about 11cm, about 11.5 cm and about 12 cm.

In some embodiments, the internal distance from the end of the device 15 defining the internal inlet 15i may be closely spaced to the inlet of the mass analyzer 30 to define an ion entrance distance from the internal ion source to the mass analyzer of between about 1 mm and about 50 mm, between about 1 mm and 40 mm, between about 1 mm and 30 mm, between 1 mm and 20 mm, or between about 1 mm and 10 mm. This distance may increase and/or maximize ion transport without the need for complex ion optics.

In certain embodiments, the inlet device 15 may be electrically conductive and in electrical communication with at least one power source 125. The inlet device 15 may be stainless steel or other suitable material. As shown, a voltage input 126 from a power supply 125 may be applied to an outer section of the inlet device 15 between the tip of the outer end 15e and the wall 12w of the chamber or the wall of the MS housing 10h holding the chamber 12. In some embodiments, the voltage input 126 may be between about 10V to about 500V, more typically between about 100V to about 250V. The voltage applied to the inlet device 15 may vary depending on one or more of the following factors: the length of the input device, the position of the inlet device relative to the mass analyzer (e.g., ion trap), the analyte of interest, the electrospray volume, the electrospray pressure, and the like. For example, the voltage may have a positive or negative polarity depending on, for example, the analyte of interest (e.g., cation versus anion).

The ESI device 20 can be held by an xyz stage or other support 112 (fig. 1) that can allow the apparatus 20 to be placed adjacent to the outer end of the inlet 15e, typically within about 1-50 millimeters, more typically within about 5-10 millimeters, with the corresponding at least one device emitter 20e in the correct orientation and position. Alternatively or additionally, the support 112 may be configured to rotate for rotational positioning to change the angular orientation of the emitter relative to the inlet 15 e.

In some embodiments, at least one emitter 20e may be positioned axially with the inlet 15e, preferably at least when low ESI flow rates are used, e.g., typically <1 μ L/min. In other embodiments, the at least one emitter 20e may be above, below, and/or to the side of the inlet 15 e.

In the embodiment shown in fig. 1, the inner end 15i of the inlet device may be in communication with the electrode 28. The inner end 15i of the inlet device and the electrodes 28 may be spaced from the entrance of the gated electrodes 38 and/or the mass analyzer 30 by about 1 mm to about 20 mm, more typically about 1 mm to about 10 mm, such as about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm and about 10 mm. In some embodiments, the electrode 28 is an accelerating electrode for ions.

In some embodiments, as shown in fig. 2, the mass spectrometer 10 can have a holding compartment 60 that holds the ESI device 20. In certain embodiments, the holding compartment 60 may be open to the surrounding atmosphere such that air acts as a buffer gas. In some embodiments, compartment 60 may be enclosed and filled with a buffer gas, such as helium, hydrogen, or dry nitrogen, for example from pressurized buffer gas supply vessel 160. The holding compartment 60 may include a support 62 that may hold the ESI device 20 in a desired (typically adjustable) orientation and position relative to the inlet device 15. The support 62 may be configured as an x-y-z stage 112 or may cooperate with the stage 112. The holding compartment 60 may be configured to enclose the emitter 20e and/or the entire ESI device 20 during operation.

In some embodiments, as also shown in FIG. 2, an electrical barrier 64 may be positioned around the ESI device 20 to shield the ESI emitter 20e from the voltage applied to the one or more reservoirs 20r on the ESI device 20. A segment of the ESI device 20 having the ESI emitter 20e (e.g., between about 1-10 millimeters in length) may extend through a slit 64s in the barrier 64. Barrier 64 may comprise a single-sided copper-clad circuit board (e.g., commercially available from m.g. chemicals of berlington, ontario, canada) or any other suitable barrier means known to those skilled in the art. In some embodiments, the barrier 64 may be maintained at a defined voltage for CE use and a reference ground potential (GND) for perfusion use.

Fig. 3A-3C and 4A-4C illustrate other examples of the analysis system 100.

As shown in fig. 3A and 4A, for example, the inlet device 15 may extend into a focusing electrode 48, which focusing electrode 48 is shown as a tube electrode 48t and is used in place of the accelerating electrodes 28 and gate electrodes 38 shown in fig. 1 and 2. The focusing electrode 48 may act as a "lens" to focus ions into the mass analyzer 30. The focusing electrode 48 may be operated with a DC voltage to focus the ions. The focusing electrode 48 may have an inner diameter of between about 3 mm and 6 mm and may have a length of between 3 mm-10 mm, typically about 5 mm. The focusing electrode 48 may be spaced a close distance from the front end of the mass analyzer 30 (e.g., the front end cap electrode of the ion trap), typically about 0.1 mm to about 2 mm, such as in some embodiments about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 1.1 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, and about 2 mm.

In some embodiments, the inner end 15i of the inlet device may be positioned a short distance within the focusing electrode 48, between about 0.1 mm and about 1 mm, typically about 0.2 mm, about 0.3 mm, about 0.4 mm, or about 0.5 mm.

The inner end 15i of the inlet arrangement 15 may be about 1-50 mm from the front of the mass analyser 30, for example from the front end cap of the ion trap. In some embodiments, the inner end 15i of the inlet device may be located about 1-10 mm or about 1-5 mm in front of the mass analyzer 30.

Although shown in fig. 1 and 2 with accelerating and gating electrode structures, and in fig. 3A and 4A with focusing electrodes 48 as tube electrodes 48t, other focusing/lens electrode arrangements may be used. The release end 15i of the inlet tube may extend a distance into the focusing lens and/or the electrode. For example, the focusing electrode 48 may include a single lens and/or an ion funnel 48f. Fig. 3C and 4C show that the mass spectrometer 10 can have a focusing electrode 48, which focusing electrode 48 includes an ion funnel electrode 48f upstream of the mass analyzer 30 in the vacuum chamber 12 holding the mass analyzer 30.

Accelerating electrodes, such as electrode 28 (fig. 1), are typically electrically connected to capillary inlet tube 15 and/or capillary ESI device 20t, and the resulting electric field accelerates ions toward mass analyzer 30 (e.g., ion trap). The "focusing electrodes" discussed above focus ions (which may have been accelerated by the "accelerating electrodes") into the mass analyser 30, for example an ion trap. Accordingly, the mass spectrometer 10 may include a variety of different ion optics (focusing or "lens" electrode structures).

Ion funnel 48f (fig. 3C, 4C) can increase ion transport by at least an order of magnitude compared to a simple capillary inlet. See, e.g., rapid Communications in masses Spectrometry,1997, 11, 1813-1817, of a.buffer, k.tang, g.a.anderson, d.c.prior, h.r.udset, r.d.smith. The ion funnel typically has a stack of ring electrodes of reduced inner diameter, using a combination of RF and DC potentials to focus the ions. See, e.g., kim, T., tolmachev, A.V., harkewicz, R., prior, D.C., anderson, G., udseth, H.R., smith, R.D., analytical Chemistry,2000, 72, 2247-2255, julian, R.R., mabbett, S.R., jarrold, journal of the American Society for Mass Spectrometry,2005, 16 (10), 1708-1712. However, some ion funnels may be planar. See, e.g., U.S. patent application publication No. 2013/0120894, the contents of which are incorporated by reference as if fully set forth herein. Ion funnels are conventionally operated at pressures ranging from 0.1 torr to 20 torr. An RF potential is applied to each of the other electrodes ("even electrodes") and an RF potential of the same magnitude and 180 ° in phase opposition is applied to the other electrodes ("odd electrodes"). A linear DC gradient is applied to both the even and odd electrodes, with the highest magnitude voltage applied to the entrance electrode and the lowest magnitude voltage applied to the exit electrode. A separate "dc-only" electrode may be placed between the funnel outlet and the mass analyser. See, for example, U.S. Pat. No.6,107,628 and U.S. Pat. No. 7,351,964, the contents of which are hereby incorporated by reference as if fully set forth herein.

The gate electrode is optional. In some embodiments, the tube electrode 48t may have a separate DC voltage applied to the tube electrode. The ion funnel 48f may have a combination of applied RF and DC potentials. When the mass spectrometer 10 includes the tube electrode 48t, the tube itself may also serve as a gate. When the mass spectrometer 10 includes the ion funnel 48f, ions can be gated in several ways (i.e., turning off the DC potential, switching one DC potential, etc.).

Fig. 4A-4D also illustrate that in some embodiments, the mass spectrometer 10 can have a single chamber 12 that maintains the mass analyzer 30 and the detector 40 at a common high pressure. Thus, mass analysis and detection are performed in a single common high pressure context, such as at or above 50 mtorr, more typically at or above 100 mtorr (e.g., between about 100 mtorr and 1 torr in a particular embodiment), optionally with ambient air as a buffer gas. In some embodiments, the holding compartment 60 (fig. 2) may be used to allow electrospray 20s and/or mass spectrometry using an alternative buffer gas as described above.

Fig. 3A and 4A show that in some embodiments, an inlet device 15 in communication with an ESI device 20 can be directly electrosprayed into a high pressure chamber 12 holding a mass analyzer 30.

Fig. 3B, 3C, 4B, 4C and 4D illustrate examples of ESI devices 20 sealed directly to a mass spectrometer 10 (e.g., the wall 12w of the vacuum chamber 12 holding the mass analyzer 30) with respective discharge ends of the emitter 20e inside the high pressure vacuum chamber 12 holding the mass analyzer 30 to discharge ions (e.g., electrospray) directly into the high pressure without the need for an inlet device 15 such as shown in fig. 3A, 4A.

For example, fig. 1, 2, 3A, 4D, 5A, and 5B illustrate that the ESI device 20 can be a fluid microchip 20c. However, as noted above, other ESI devices 20 may be used. Fig. 3B, 3C, 4B and 4C show a capillary tip 20t as an ESI emitter 20e. The emitter 20e is within the high pressure vacuum chamber 12 with the mass analyzer 30 rather than at atmospheric pressure. In some embodiments, the at least one emitter 20e may be located between about 1 millimeter and about 50 millimeters, more typically between about 1 millimeter and 20 millimeters. The distance may be about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, about 20 mm, about 25 mm, about 30 mm, about 35 mm, about 40 mm, about 45 mm, or about 50 mm from the ion entrance aperture/electrode of the mass analyzer 30.

In some embodiments, the ESI device 20 extends into the vacuum chamber 12 with the mass analyzer 30, for example as shown in fig. 3B, 3C, 4B, 4C as a capillary 20t, alternatively as an ESI microchip 20C as shown in fig. 4D. Thus, the microfluidic chip 20c can be placed directly in the vacuum chamber 12 without the need for an intermediate inlet device 15. The body of the microchip 20c can be sealed to the wall 12w of the vacuum chamber 12 holding the mass analyzer 30 such that the at least one emitter 20e is in vacuum and the reservoir 20r is outside of the vacuum chamber 12.

The wall of vacuum chamber 12w may include an aperture for receiving a segment of microchip 20 via vacuum seal 18. In some embodiments, the vacuum seal 18 may comprise an O-ring, gasket, or other seal that may extend around the outer surface of the microchip 20c. The shape of the seal member 18 may conform to the shape of the microchip 20c or the shape of the segment of the microchip. In some particular embodiments, the seal 18 may be rectangular. The orientation of the chip 20 relative to the vacuum chamber 12 may be horizontal, vertical, or even some angle between vertical and horizontal. A rectangular shape of the seal 18 may be appropriate, with the entire front end of the rectangular microchip 20c held in the vacuum chamber 12. The seal 18 may be located on the microchip 20 and/or on the chamber 12w and/or the wall 12w of the housing 10h, or in a vacuum fitting sized and configured to matingly and sealingly receive the ends of the microchip 20c.

As shown in fig. 4D, the vacuum chamber wall 12w may define an electrical barrier for the outer portion of the microchip 20c and may be at ground potential 127. Electrical and/or pressurized gas connections to one or more pressurized gas sources 120p and/or one or more power sources 120 may be made through the chip reservoir 20r and/or at the ESI at the chip reservoir 20r, which connections are used to allow the sample to be transported into the vacuum chamber 12 through the processing channel and/or electrospray.

For a metal ESI capillary 20t, an injection voltage may be applied to the capillary body. In the case of glass, quartz, and/or insulated capillaries, gold or other suitable conductive (typically metallic) coating can be applied to the spray tip, with the conductive coating exiting the seal 18 into the environment outside the vacuum chamber 12. In some embodiments, the analysis system 100 can include a liquid joint residing outside the vacuum chamber 12 where ESI voltages can be applied.

In some embodiments, ES [ device 20, shown as microfluidic chip 20c in, for example, fig. 1, 2, 3A, 4A and 4D, may instead be a capillary tube 20t with an emitter 20e, which emitter 20e resides outside of vacuum chamber 12 and operates in conjunction with inlet device 15.

Conventional mass spectrometry systems are typically around 10 ^-6 Torr, which is several orders of magnitude less than the operating pressure of the embodiments of the present invention. Considering electrospray to vacuum chambers at near atmospheric pressure (e.g., about 600 torr), these vacuum chambers are separate from the mass analyzer and utilize an inlet capillary into a commercial mass spectrometer, which results in ion loss. See, e.g., felton et al, automated High-through High input ESI-MS with Direct Coupling to a Micropter Plate, anal chem.2001, 73, pages 1449-1454; and High-through Microfabricated CE/ESI-MS by Zhang et al: the contents of the Automated Sampling from a Microwell Plate, anal Cham.2001, 73, 2675-2681, are incorporated herein by reference as if fully set forth. Instead, and advantageously, the new injection of ions directly into the high pressure vacuum chamber 12 holding the mass analyzer 30 can avoid such ion losses, e.g., ion losses are significantly reduced or avoided relative to a differential pressure interface to the (single) atmospheric-to-high pressure interface of the vacuum chamber with the mass analyzer.

As shown in fig. 3B, 3C, 4B, 4C and 4D, the ionic sample-releasing emitter 20e of the fluid handling device 20 may be closely spaced to the mass analyzer 30. The axial distance from the emitter 20e to the entrance of the mass analyser 30 (e.g. the first end cap electrode 31 of the ion trap in the case of the mass analyser 30), shown as Di-m in figures 3B, 3C, 4B, 4C and 4D, may be between about 1 mm and about 50 mm, between about 1 mm and about 40 mm, between about 1 mm and about 30 mm, between 1 mm and 20 mm or between 1 mm and 10 mm. In some embodiments, this spacing may maximize ion transport without the need for complex ion optics. In some embodiments, the at least one emitter 20e may be located at a distance of between about 1 mm and about 20 mm or between about 1 and about 10 mm from the inlet aperture (e.g., first end cap electrode 31) of the mass analyzer. In particular embodiments, the Di-m distance may be about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, and about 20 mm from the ion entrance aperture/electrode of the mass analyzer 30.

In the embodiments shown in fig. 1, 2, 3A, 3C, 4A-4D, the mass analyser 30 comprises at least one ion trap 30 having an array of closely spaced electrodes (conductors). The electrodes comprise a central (ring) electrode 33 located between the two end cap electrodes 31, 32. The electrodes may have axially aligned holes with a distance "b" between the centers of adjacent holes. The holes may be arranged in a regular pattern or may be random. The ring electrode 33 may have one or more apertures 33a, the apertures 33a being generally larger than the first or second end cap electrode apertures. The term "ring electrode" refers to the central electrode in the array of ion traps between the end cap electrodes 31 or end electrodes 32 and need not have a ring-shaped form factor, for example, at the periphery or in the boundary channel of the respective ion trap. As is well known, the respective ion trap 30 may have short tubular passages of different diameters aligned with end caps and annular rings. One or both of the end cap electrodes 31, 32 may comprise or may be in the form of a mesh electrode and/or conductive screen.

As shown in fig. 5A and 5B, for example, the ESI device 20 may be a microfluidic chip 20c comprising a reservoir 20r for a sample (S), sample Waste (SW), buffer (B) and/(electro-osmotic) pump (P) and a fluidic micro-channel and/or nano-channel 21. See, e.g., co-pending PCT/US2012/027662 and PCT/US2011/052127, which describe examples of micromachined fluidic devices. See also, mellors, J.S., gorbornov, V., ramsey, R.S., ramsey, J.M. "full integrated glass microfluidic device for performing high-efficiency capacitor and electrophoresis analysis", anal Chem 2008, 80 (18), 6881-6887. For additional information that may be useful for some designs, see also Xue Q, foret F, dunayevskiy YM, zavracky PM, mcGruer NE & Karger BL (1997), "Multi channel Microchip Electron Mass Spectrometry". Anal Chem 69, 426-430, ramsey RS &Ramsey JM (1997), "Generation Electron from Microchip Devices Using electrostatic Pumping". Anal Chem 69, 1174-1178, chambers AG, mellors JS, henley WH and Ramsey JM (2011), "Monolithic Integration of Two-Dimensional Liquid Chromatography-Capillary Electrophoresis and Electrolysis Ionization on a Microfluidic Device", analytical Chemistry 83, 842-849. Anal chem.2008, 80 (18), 6881-6887 of Mellors et al; anal. Chem.,2014, 86 (7) 3493-5000 to Batz et al; and U.S. Pat. No.9,006,648. The contents of these documents are incorporated by reference herein as if fully set forth herein.

Fig. 6A and 6B show an example of the inlet device 15. As shown, the inlet device 15 may have an elongated tubular body 15b extending between an inner end 15i and an outer end 15 e. The device 15 may have at least one (shown as a single) inlet hole 15a, the inlet hole 15a merging into a longitudinally extending fluid ("fluid" means liquid and/or gas ") channel 15c. The device 15 may be sized and configured with at least one capillary channel, for example configured as a capillary tube. The width and/or height dimension (shown as a circle having a diameter) of the at least one channel 15c may be between about 0.05 mm to about 0.50 mm (more typically between about 0.100 mm to about 0.250 mm), and in some embodiments may be about 0.125 mm. Other cross-sectional channel shapes may be used instead of circles.

Fig. 6C shows that the at least one inlet aperture 15a may be a plurality of inlet apertures 15a, each inlet aperture 15a merging into a respective inlet channel 15C. Alternatively, two or more inlets 15a may merge into a shared elongate channel 15c. Although five holes 15a are shown, more or fewer holes 15a may be used, for example, 2, 3,4, 6, 7, 8, 9, or 10.

In some embodiments, the outer diameter of the inlet device 15 may be between 1-5 millimeters, such as about 1 millimeter, about 1.2 millimeters, about 1.5 millimeters, about 1.6 millimeters, about 1.7 millimeters, about 1.8 millimeters, about 1.9 millimeters, and about 2 millimeters.

The length of the inlet device 15 may be between 1cm and 20cm, typically between 5cm and 15cm, for example about 5cm, about 6cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 11cm, about 12 cm, about 13 cm, about 14cm and about 15cm.

Fig. 7A-7D show that the outer end 15e may have a conical or conical skimmer device 15c with at least one inlet aperture 15a centered on the conical tip. In some embodiments, the conical shape may be frusto-conical with a flat forwardmost end that retains a bore 15a, which bore 15a retracts into the body of the inlet device 15 to form a conical tip. The outer tip portion 15e may be integral with the main body 15b of the inlet device or may be a separate component attached to the main body 15b of the inlet device 15. The at least one aperture 15a may have a width and/or height dimension (shown as a circle having a diameter) of between about 0.025 millimeters and about 0.50 millimeters, more typically between about 0.030 millimeters and about 0.125 millimeters, and in some embodiments may be about 0.100 millimeters, about 0.110 millimeters, or about 0.125 millimeters. Other cross-sectional channel shapes may be used instead of circles.

The conical head 15e may be a solid body having at least one bore and at least one axially extending fluid passage. In other embodiments, as shown in fig. 7B, the conical head 15e may be a shaped body of thin malleable or molded material having a hollow interior 15h, which is much larger than the bore 15a, and may be attached to the tubular longitudinally extending body 15B.

Fig. 7E shows that the inlet device 15 may have a plurality of inlet apertures 15a. Although three apertures 15a are shown, more or fewer apertures 15a may be used, such as 2, 4, 5, 6, 7, 8, 9, or 10. The plurality of inlet holes 15a may be respectively merged into a corresponding one of the plurality of inlet passages 15c. Alternatively, two or more inlet apertures 15a may merge into a shared elongate channel 15c.

Fig. 8A-8D show another embodiment of the inlet device 15. In this embodiment, the axial extent of the passage 15c is similar to the diameter of the bore 15a. The inlet device 15 may have a planar body 15p (e.g., a thinner plate). The planar body 15p may have a thickness of between about 0.100 millimeters and about 5 millimeters, and more typically between about 0.100 millimeters and about 0.50 millimeters. In some embodiments, the thickness may be between about 0.125 millimeters and about 0.30 millimeters, such as about 0.125 millimeters, about 0.150 millimeters, 0.200 millimeters, about 0.250 millimeters, and about 0.30 millimeters. For example, the holes 15a may have a diameter of between about 0.01 millimeters and 0.150 millimeters. In some embodiments, the axial extent or length of the passage 15c through the body of the plate 15p is about the same or no greater than about 50% relative to the diameter (or maximum cross-sectional dimension of the non-circular shape) of the inlet bore 12a (where one bore is used) or one of the inlet bores 12a (where more than one bore is used).

Figure 8D shows inlet device 15 sealably connected to mass spectrometer 10. In other embodiments, the inlet device may be integral with the wall of the housing 10h of the mass spectrometer 10 and/or the wall 12h of the vacuum chamber 12 holding the mass analyser 30 (monolithic). In some embodiments, a plate and O-ring seal 18p may be used to attach inlet device 15 to mass spectrometer 10. The inlet device 15 may be nested in a vacuum fitting that is screwed into the wall 12h having the small hole 15a for ions. The inlet device 15 can also be embodied as a vacuum fitting which is screwed directly into the wall 12w with the small hole 15a for the ions. The measured Di-m distance from the external transmitter 20e to the ion entrance of the mass analyzer 30 in the vacuum chamber 12 can be between 1-10 centimeters, for example, about 1 centimeter, about 2 centimeters, about 3 centimeters, about 4 centimeters, about 5 centimeters, about 6 centimeters, about 7 centimeters, about 8 centimeters, about 9 centimeters, and about 10 centimeters. In some embodiments, the distance Di-m is between 10 millimeters and about 150 millimeters.

Fig. 9A and 9B show that the analysis system 100 may have a multiple tube structure, each tube 15t providing at least one inlet aperture 15a, inhaling electrospray 20s at ambient (e.g., atmospheric) pressure during operation. The tubes 15t may be held as one assembly, each of which extends into the mass analyzer chamber 12 of the mass spectrometer housing 10h through at least one vacuum tight connector and/or fitting 18. Although five closely spaced tubes 15t are shown in fig. 9B, for example, less or more than five, such as 2, 3,4 or 6, may be used. The tubes 15t may be of the same or different lengths and located at common or staggered internal or external locations.

Where the inlet device 15 includes a plurality of inlet apertures 15a, such as shown in fig. 6C, 7D, 8C, 9A, 9B, each may have the same size or different size inlet apertures 15a and/or channel width/height (e.g., diameter where circular holes are used). Accordingly, the width and/or height dimension (shown as a circle having a diameter) of the respective apertures 15a may be between about 0.05 millimeters and about 0.50 millimeters, more typically between about 0.100 millimeters and about 0.250 millimeters, and in some embodiments may be about 0.100 millimeters, about 0.110 millimeters, or about 0.125 millimeters. Again, other cross-sectional channel shapes may be used instead of circular. Some of the holes 15a may be larger than others. The holes 15a may be regularly or irregularly spaced.

In some embodiments, the calculated electrospray inlet gas flow rate through the inlet device 15 may be between about 1sccm and 115sccm, but may be greater or smaller in some embodiments.

In some particular embodiments, the liquid flow rate from the ESI device 20 is typically between 50 and 300 nL/min. In some embodiments, ESI flow rates of, for example, typically <1 μ L/min may be used. The liquid flow rate of larger ESI emitters, such as glass, quartz or metal capillaries with internal diameters greater than 100 μm, can be greater than 1 μ L/min.

Embodiments of the present invention relate to compact or miniaturised configurations of ion trap mass analysers for use in apparatus for determining ion mass to charge ratios and may additionally provide information on the relative abundance of a plurality of ions within a range of mass to charge values. The specific examples described herein are particularly relevant to ion trap mass analyzers such as Paul trap, cylindrical Ion Trap (CIT), stretched Length Ion Trap (SLIT), and linear ion trap.

In the embodiments shown in fig. 1-4D, the mass analyzer 30 includes at least one ion trap, for example, in a corresponding array, such as between about 1-800, typically between about 5-256, more typically between about 5-50, including, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50, among others. In some embodiments, the ion trap 30 may have a Stretched Length Ion Trap (SLIT) configuration with a single trap or a plurality of such traps. For the latter, in the case of use, the number of traps may be between 2 and 50. See, for example, U.S. Pat. No. 8,878,127 to Ramsey et al, entitled "Miniature Charged Particle Trap With Elongated tracking Region For Mass Spectrometry", the contents of which are incorporated herein by reference as if fully set forth herein. However, other ion trap aperture shapes and aperture array configurations may be used.

The pump 70 may be any suitable pump, typically a small, lightweight pump. Examples of pumps include TPS Bench (SH 110 and Turbo-V81M pumps) compact pumping systems and/or TPS compact (IDP-3 and TurboV 81M pumps) pumping systems, such as from Agilent, santa Clara, calif. Operating pressures equal to or higher than 50 mtorr can be readily achieved by mechanical displacement pumps such as rotary vane pumps, reciprocating piston pumps or scroll pumps.

Fig. 4A-4D, 9A and 10B show that detector 40 may include a faraday cup detector 40C in communication with an amplifier such as a differential amplifier (908 Devices, boston, maryland). The ion signal can be collected on faraday cup detector 40C and amplified by amplifier 92 (fig. 10B). One example of amplifier 92 is from AmA250CF of ptek corporation

A charge sensitive preamplifier. Other detector configurations and other amplifiers may be used.

In some embodiments, the ions may be accumulated over a defined time of the respective scan, such as between about 1-30 milliseconds, typically between about 1-10 milliseconds, prior to analysis. For each analysis, successive scans may be averaged, typically between 20-1000 scans.

In some embodiments, the mass analyzer compartment/chamber 12 with only the mass analyzer 30 (in a dual vacuum chamber configuration) or the mass analyzer compartment/chamber 12 with both the mass analyzer 30 and the detector 40 in a single vacuum chamber configuration may be relatively small in volume, for example, between about 0.25 square inches to about 16 square inches, typically between about 1.3 square inches to about 10 square inches, such as about 1 square inch, about 2 square inches, about 3 square inches, about 4 square inches, about 5 square inches, about 6 square inches, about 7 square inches, about 8 square inches, about 9 square inches, about 10 square inches.

As shown in fig. 4A, for example, the chamber 12 may be located in a compact housing 20H having a length dimension L and a height (or width) dimension H. For example, the length dimension L may be between about 1-5 inches, typically between about 1-3 inches, such as about 1 inch, about 1.5 inches, about 1.75 inches, and about 1.85 inches. The height/width dimension H may be between about 0.5 inches and about 5 inches, typically about 1 inch. The depth or "z" dimension can be between 1-5 inches, typically about 1-3 inches.

In some embodiments, the front end of the ion trap 30 is spaced a close distance "Dd" from the detector 40, which is particularly advantageous for small mass spectrometry systems operating at high pressures due to the reduced mean free path experienced by the ejected ions at such pressures. In some embodiments, the spacing Dd (fig. 1, 2, 3A-3C, 4A-4C) is between about 0.01 inches (0.254 mm) to about 0.5 inches (13 mm), more typically between about 1 mm to about 10 mm.

Referring again to fig. 1, 2, 3A-3C and 4A-4C, where the mass analyser 30 comprises an ion trap, the ring electrode aperture will typically be larger than the first or second end cap electrode apertures and/or may be a grid pattern of end caps. When one or more of the end cap electrodes 31, 32 are implemented as mesh end caps, the electrodes may include holes covered by a fine mesh metal mesh, typically between 100-1000 wires per inch.

It is well known that corresponding ion traps have tubular passages of different diameters aligned with the end cap and the annular ring. The end cap electrodes 31, 32 are typically spaced apart from the ring electrode 33 by a distance d at symmetrical intervals. The specific spacing depends on the ring electrode thickness, but the distance separation of the end cap electrodes 31, 32 can be selected to optimize mass spectral performance. The end cap aperture or hole allows the implantation of ionizing energy or ions, while the other end cap aperture allows ejection of ions for detection purposes.

The electrode apertures 31, 32, 33 each have a radius r0 or average effective radius (e.g., the latter using shape and width/height dimensions to calculate average aperture diameter in the case of non-circular aperture shapes), and the traps 30 have a corresponding diameter or average cross-over distance 2r0 and effective length 2z0. The ion trap 30 can be configured to have a confinement ratio z0/r0 greater than 0.83. Note that z0 can be defined as the half height of the cavity. In some embodiments, the array of ion trap apertures has an effective length 2z0 measured as the distance between the inner surfaces of the end caps 31, 32. The array may be configured to have a defined ratio z0/r0 close to 1, but typically greater than 1, about 10% to about 30%. The r0 and z0 dimensions may be between about 0.5 μm and about 1cm, but for micro mass spectrometry applications contemplated by certain embodiments of the present invention, these dimensions are preferably 1 millimeter or less up to about 0.5 μm. The mass analyser 30 may be an ion trap having three stacked (metal) electrodes 31, 32, 33 separated by insulators. For further discussion of exemplary CIT configurations, see u.s.6,933,498 and u.s.6,469,298, the contents of which are incorporated by reference as if fully set forth herein. One example of a single electrode ionizer is described in Kornienko's anal. Chem.2000, 72, 559-562 and Kornienkorapid Commun. Mass Spectrum.1999, 13, 50-53, which are incorporated herein by reference in their entirety.

The distance "d" is typically chosen such that z0 is slightly greater than r0, usually 10-30% greater.

In some embodiments, the mass spectrometer system 100 can be configured with one or more mass analyzers 30. Where the ion trap is a mass analyser 30, the ion trap may comprise more than one trap. In some embodiments, mass ejection from each chamber may be detected by a single detector 40 to produce a composite (combined enhanced) mass spectral signal. In some embodiments, the signal used for detection may be based on outputs from a subset of the different traps. In some embodiments, the mass from each or one or more groups of cavities may be detected by a separate detector. Such a structure may be useful where each cavity or (subset of) groups of cavities has different trapping properties. For example, this type of arrangement may extend the range of ion masses that can be analyzed by the spectrometer system.

In some embodiments, a compact (small-scale) mass spectrometer 10 having multiple dual chamber devices or multiple single chamber devices can be configured to sample multiple samples simultaneously using a common or different detector or detectors 40.

In some embodiments, the mass analyzer 30 (such as, but not limited to, an ion trap mass analyzer) and the detector 40 may all be arranged as a releasably connected set or integrally connected unit of stacked planar conductor and insulator components, such as conductive and insulating films, substrates, sheets, plates and/or layers or combinations thereof, typically alternating in defining features for the desired function. See, for example, co-pending and commonly assigned U.S. patent application serial No. 13/804,911, the contents of which are incorporated by reference as if fully set forth herein.

The probe 40 may include a suitable transducer. The transducer typically includes an electron multiplier (fig. 1, 3A-3C, and 9A), but may be a planar detector, and in particular embodiments, as shown in fig. 4A-4C and 10B, the detector 40 includes a faraday cup detector 40C. However, other ion detectors may be used.

In some embodiments, detector 40 may include a planar detector for charge detection, which may be particularly attractive for small mass spectrometry systems because of their inherently small size and weight and the ability to operate at pressures from low vacuum to atmospheric pressure. The charge collected by the conductive film or other conductor associated with the detector 40 may be measured with an electrometer or charge-sensitive transimpedance amplifier. The term "electron collector" refers to an electronic circuit and/or device capable of detecting charge collected by a thin film sheet and/or a conductor.

For example, detector 40 may be configured to detect ions ejected in parallel from a planar CIT array having a planar electrode with a solid continuous conductive surface over the aperture of the end cap electrode. The gain of the detector amplifier 92 (fig. 10B), such as a charge-sensitive transimpedance amplifier, can be improved with reduced faraday cup capacitance.

The mass spectrometer system 10 can be lightweight, typically at about 1-25 pounds (including one or more vacuum pumps), and optionally battery powered. The housing 10h holding the mass spectrometer system and ESI inlet device 15 may be configured as a hand-held or desktop housing. In some embodiments, the portable housing may have a compatibility with Microsoft Windows

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A game machine or game controller may be similar in form factor or similar to that associated with an electronic notebook, PDA, IPAD or smartphone, and may optionally have a pistol grip. However, other configurations of housings and other arrangements of control circuitry may be used. The housing 10h generally holds a display screen 10d and may have a user interface 10i such as a graphical user interface ("GUI") (fig. 10A).

The system 100 may also include a transceiver, a GPS module, and an antenna, and may be configured to communicate with a smartphone or other personal computing device (laptop, electronic notebook, PDA, IPAD, etc.) to transfer data or control for operation, e.g., using secure APP or other wireless programmable communication protocol.

In some embodiments, mass spectrometer 100 is configured such that ESI device 20, as an ion source, delivers ions to inlet device 15 at atmospheric pressure, and mass analyzer 30 and detector 40 operate at near isobaric conditions and pressures greater than 100 mtorr.

As shown in fig. 10A and 10B, the analysis system 100 can include a spectrometer 10 having a function generator 82, the function generator 82 to provide a low voltage axial RF input 82i to the mass analyzer (e.g., ion trap) 30 during a mass scan for resonant ejection. The low voltage axial RF may be between about 100mVpp and about 12,000mVpp, typically between 100 and 10,000mVpp. During mass scanning, axial RF may be applied to either end cap 31 or 32, typically end cap 31, or between the two end caps 31 and 32 to promote resonant ejection. RF power supply 88 provides an input signal to ring electrode 33. The RF source 88 may include an RF signal generator 88g, an RF amplifier 88p, and an RF power amplifier 88a. The controller 100c may have control circuitry with an optional RF monitor. Some or all of these components may be held on a circuit board in a housing 10h that encloses a mass analyzer 30 in the chamber 12. In some embodiments, a waveform with a ramp amplitude may be provided as an input to the RF signal generator to modulate the RF amplitude. The low voltage RF may be amplified by an RF preamplifier and then amplified by a power amplifier to produce the desired RF signal. The RF signal can be between about 1MHz to 10GHz or 1MHz to 1000MHz depending on the size of the ring electrode features. RF frequency and annular electrode radius r, as is well known to those skilled in the art ₀ Are interdependent. r is ₀ At 500 μm, a typical radio frequency is 5-20MHz. The voltage can be 50V _0p To about 1500V _0p Between, typically up to about 500V _0p (As is well known to those skilled in the art, the "Op" subscript refers to 0 to half-peak).

As also shown, the system 100 includes: a voltage DC power supply 120 for the ESI device 20; and a Direct Current (DC) power supply 125 for the inlet device 15 alone (fig. 10B) or for both the inlet device 15 and the electrode compartment 12 (fig. 10A). The DC power sources 120 may optionally be controlled by a common controller 100c, or by separate controllers, or even manually. The ESI power supply 120 can be a high voltage power supply. The term "high voltage" refers to voltages in the kV range, typically between about 1-10kV, more typically between about 2-5 kV. For example, the ESI device 20 may be configured to employ a potential of a few kV, typically between about 1kV to about 5kV.

Ion detector 40 may be configured to register the number of ions emitted at different time intervals corresponding to a particular ion mass to perform mass spectrometric chemical analysis. Ion traps use the dynamic electric field generated by the RF drive signal to dynamically capture ions from a measurement sample. By varying the characteristics (e.g., amplitude, frequency, etc.) of the trapping Radio Frequency (RF) electric field, ions are selectively ejected in correspondence with their mass-to-charge ratio (mass (m)/charge (z)). The relative ion abundance at a particular m/z ratio can be digitized for analysis and can be displayed as a spectrum on-board and/or remote processor.

In its simplest form, a drive RF signal 88d of constant RF frequency may be applied to the central electrode 33 relative to the two end cap electrodes 31, 32. The amplitude of the central electrode signal may be ramped linearly to selectively destabilize different m/z ions held within the ion trap. Such amplitude ejection configurations may not yield optimal performance or resolution. However, this amplitude ejection method can be improved by applying the second signal differentially on the end caps 31, 32. In use, the axial RF signal causes dipole axial excitation, which may result in resonant ejection of ions from the ion trap when the secular oscillation frequency of ions in the trap matches the end cap excitation frequency.

The ion trap 30 or mass filter may have an equivalent circuit that appears to be almost purely capacitive. The voltage driving the ion trap 30 may be of high magnitude (e.g. 100V-1500V) and transformer coupling may be employed to generate the high voltage. The inductance of the transformer secondary and the capacitance of the ion trap may form a parallel tank circuit. It may be desirable to drive the circuit at a resonant frequency to avoid unnecessary losses and/or an increase in circuit size.

The supply of buffer gas may be sourced from a pressurized tank of buffer gas (e.g., 160 in fig. 2). However, any suitable buffer gas or buffer gas mixture including air, helium, hydrogen, or other gases may be used. Where air is used, it may be drawn from the atmosphere without the need for a pressurized tank or other source.

Fig. 11A and 11C illustrate exemplary timing diagrams that may be used to implement/control various components of analyzer system 10 with mass spectrometer 100. During ion implantation, a focusing electrode, such as lens 38 or 48 (if used), is turned on to focus ions to mass analyzer 30. The driving RF amplitude can then be held constant for a determined period of time (e.g., about 5 ms) to enable collisional cooling of trapped ions towards the trap center. The drive RF amplitude may be ramped linearly to perform mass instability scans and eject ions toward the detector 40 in order of increasing m/z. Data is acquired during a mass instability scan to generate a mass spectrum, and convective transport can enhance the signal for detection. Finally, the drive RF amplitude 88d may be reduced to a low voltage to clear any remaining ions from the trap 30 and prepare for the next scan. As is well known to those skilled in the art, many ion manipulation strategies may be applied to ion trap devices such as CIT. Different strategies of ejecting, separating or collisionally dissociating ions may be applied to the ion trapping structure.

Alternatively, as shown in fig. 11B and/or 11C, the axial RF signal may be applied synchronously with the start of the linear rise of the RF amplitude signal so as to gate substantially simultaneously to perform a resonant ejection during mass scanning to improve resolution and mass range.

The flowcharts and block diagrams of certain of the figures herein illustrate the architecture, functionality, and operation of possible implementations of mass spectrometers, or components and/or programs thereof, in accordance with the present invention. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of code, which comprises one or more executable instructions for implementing the specified logical function(s). It should also be noted that, in some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved.

As shown in fig. 10A and 10B, the mass spectrometer 10 may include a transmitter or transceiver 100t that allows it to communicate wirelessly with local and/or remote processors and/or servers using, for example, a LAN (local area network), a WAN (wide area network), an intranet, and/or the internet. Mass spectrometer 10 can be configured to generate an audible and/or visual alarm if an environmental, industrial, or other hazard is detected. The controller 100c may also or alternatively generate a local or remote alarm when buffer gas is detected as low, or based on a hypothetical usage/volume of consumable inputs. Alerts may also be automatically sent to one or more local or remote sites over the internet, a wide area network, a local area network, or an intranet to notify of potential hazards. The alert may be sent to a cellular phone, a landline phone, an electronic notebook or tablet, a portable computer, or other pervasive computing device.

The mass spectrometer 10 may include or be in communication with an analysis module and/or circuitry capable of identifying a substance by an acquired mass spectrum. The analysis module or circuit may be on the spectrometer arrangement 10 or at least partially remote from the spectrometer arrangement 10. If the latter is the case, the analysis module or circuitry may reside, in whole or in part, on the server. The servers may be provided using cloud computing, which includes providing computing resources on demand over a computer network. Resources may be embodied as various infrastructure services (e.g., computers, storage, etc.) as well as applications, databases, file services, email, and so forth. In the traditional computing model, the data and software are typically contained entirely on the user's computer; in cloud computing, a user's computer may contain little software or data (perhaps an operating system and/or a web browser), and may simply act as a display terminal for processes that occur on an external computer network. A cloud computing service (or aggregation of multiple cloud resources) may be generally referred to as a "cloud". Cloud storage may include a model of networked computer data storage in which data is stored on multiple virtual servers, rather than being hosted on one or more dedicated servers. The data transmission may be encrypted and any suitable firewall appropriate to the data collected may be used over the internet.

Fig. 12A is a flow diagram of exemplary actions that may be performed to analyze a sample, according to some embodiments. Ions from an electrospray ionization device are electrospray into a region of space at ambient (i.e., atmospheric) pressure (block 200). Electrospray ions are drawn into an inlet device at ambient (i.e., atmospheric) pressure (block 210). The ions are transferred into a vacuum chamber at a pressure of about 50 mtorr or higher (block 220) and flow into a mass analyzer in the vacuum chamber (block 230). At least one detector downstream of (and typically aligned with) the mass analyzer is used to detect signals from the ions (block 240).

During an electrospray process, a voltage may be applied to the ESI device while a lower voltage is applied to the inlet device (block 202).

The electrospray enters the air a distance in front of the inlet device (block 204).

The electrospray is performed from a tip of a microfluidic device having at least one electrospray emitter for electrospray of the ions (block 206).

The inlet device may have a plurality of inlet holes adjacent to but spaced from the ESI device (block 212).

The inlet device is sealably connected to a wall of the vacuum chamber and has an inner end portion at a distance of between about 1 mm and about 50 mm from an ion inlet of the mass analyzer (block 214).

When the vacuum chamber is between 50 mtorr and 100 torr, the ions are transferred directly into the vacuum chamber (block 222).

The mass analyzer may include a micro CIT ion trap (block 232).

The mass analyzer and the detector may both be maintained in the same vacuum chamber, which may be at a pressure between 100 mtorr and 10 torr (block 242).

Fig. 12B is another flow diagram of exemplary actions that may be performed to analyze a sample according to some embodiments. Ions from the fluid capillary electrophoresis device are released (e.g., electrospray) directly into a high pressure vacuum chamber housing a mass analyzer (block 250). The ions then flow into a mass analyzer in the vacuum chamber (block 260). Signals from the ions are detected using at least one detector downstream of (and typically aligned with) the mass analyzer (block 270).

In some embodiments, the high pressure may be between about 50 mtorr and 100 torr (block 255), and in typical embodiments between about 100 mtorr and about 10 torr.

The releasing may be performed by electrospray such that the end of the device from which ions are released in the vacuum chamber is at a position between about 1 mm to about 50 mm (and in some embodiments may be between about 1-10 mm or between about 1-20 mm) in front of the ion inlet of the mass analyzer (block 257).

The mass analyzer may be a miniature CIT, CIT array, SLIT, or SLIT array, and the first end cap electrode may be placed within about 1-50 millimeters in front of the ion outlet of the means for releasing ions (block 265).

The mass analyzer and detector may be housed in a single vacuum chamber at the same high pressure, typically between about 50 mtorr and 100 torr (block 275).

FIG. 13 is a block diagram of exemplary embodiments of a data processing system 305 that illustrates systems, methods, and computer program products in accordance with embodiments of the present invention. The processor 310 communicates with the memory 314 over a system bus 348. The processor 310 may be any commercially available or custom microprocessor. Processor 310 may be processor 100p. Memory 314 represents the general hierarchy of storage devices containing the software and data used to implement the functionality of data processing system 305. Memory 314 may include, but is not limited to, the following types of devices: cache, ROM, PROM, EPROM, EEPROM, flash, SRAM, and DRAM.

As shown in FIG. 13, the memory 314 may include several categories of software and data used in the data processing system 305: an operating system 352; the application programs 354; an auxiliary input/output (I/O) subsystem 358; ES 1-mass spectrometer control module 350; and data 356. Module 350 can be on the mass spectrometer, or s can be remote, or partially on the mass spectrometer and partially remote (e.g., in one or more servers, local or onboard or remote processors). The module 350 may communicate with the DC voltage supply 125 for ESI to the MS inlet device 15 and/or the power supply 120 for the ESI device 20.

As will be appreciated by those skilled in the art, the operating system 352 may be any operating system suitable for use with a data processing system, such as OS/2, AIX, or OS/390 from International Business machines corporation, armonk, N.Y., windows CE, windows NT, windows95, windows98, windows2000, or Windows XP, palm OS from Palm corporation, macOS, UNIX, freeBSD, or Linux from apple computer corporation, a proprietary operating system, or a proprietary operating system, such as for embedded data processing systems.

The I/O device drivers 358 typically include software routines accessed through the operating system 352 by the application programs 354 to communicate with devices such as I/O data ports, data storage 356 and certain memory 314 components and/or the image acquisition system 320. The application programs 354 are illustrative of the programs that implement the various features of the data processing system 305 and may include at least one application that supports operations according to embodiments of the present invention. Finally, the data 356 represents the static and dynamic data used by the application programs 354, the operating system 352, the I/O device drivers 358, and other software programs that may reside in the memory 314.

While the present invention is described, for example, with reference to the module 350 of the application program in FIG. 13, those skilled in the art will appreciate that other configurations may be used while still benefiting from the teachings of the present invention. For example, the module 350 may also be incorporated into the operating system 352, the I/O device drivers 358 or other such logical division of the data processing system 305. Thus, the present invention should not be construed as limited to the configuration of FIG. 13, which is intended to encompass any configuration capable of carrying out the operations described herein.

Embodiments of the present invention will be further described with reference to the non-limiting examples provided below.

Examples of the invention

The feasibility of a fully miniaturized CE-ESI-MS prototype system was investigated using a miniature CIT-based mass spectrometer, with emphasis on small biomolecules including amino acids, peptides and proteins. One application of the compact CE-ES1-MS system for biomolecular analysis is the monitoring of amino acids, which is used for process control in bioreactors for the production of biopharmaceuticals. Monitoring the amino acid concentration can be used to optimize growth conditions and monitor cell activity in cell cultures or bioreactors. Another application of this technology is the analysis of small molecule peptides, which can be used for quality assurance/quality control of biopharmaceuticals, identification and characterization of proteins, or for a more thorough understanding of cellular function. Thus, amino acids and peptides are selected as target analytes.

Experiment of

Reagents and materials.

HPLC grade acetonitrile obtained from Fisher Scientific (Fairlawn, NJ) and formic acid (99.9%). Purified deionized water was obtained using a Nanopure Diamond water purifier (Barnstead International, dubuque, IA). 3-aminopropyl) diisopropylethoxysilane (APDIPES) was obtained from Gelest (Morrisville, pa.). Amino acids were obtained from Fisher Scientific for analysis. The peptides bradykinin, methionine-enkephalin, thymopentin and angiotensin II obtained from American Peptide Company (Sunnyvale, CA). The background electrolyte for all experiments was 50% acetonitrile, 49.9% water and 0.1% formic acid (v/v/v, pH = 3.1).

Microchip design, manufacture, and operation.

FIGS. 5A and 5B show schematic diagrams of microchip designs for CE-ESI (5A) and infusion-ESI (5B). The CE-ESI device contained four reservoirs, an injection crossover, a 46cm serpentine separation channel, an electro-osmotic (EO) pump, and an ESI pore. The reservoir label indicates the sample (S), background electrolyte (BG), sample Waste (SW), and electro-osmotic pump (EO). The infusion set consisted of two reservoirs (sample (S), sample plus E0 pump (S, EO)), a 5.5cm infusion channel and an EO pump. The channel dimensions for both devices were 10 microns deep and 70 microns wide.

Microchip ESI devices were made from B-270 (Telic Corp., valEncia, calif.) using photolithography and wet etching techniques described in detail previously. See j.s: mellors, v.gorbouunov, r.s: ramsey and j.m.ramsey in anal. Chem.,2008, 80, 6881-6887 and n.g.batz, j.s: mellors, j.p: anal. Chem. 2014, 86, 3493-3500, by Alarie and j.m.ramsey, the device was coated with APDIPES by Chemical Vapor Deposition (CVD) using a LabKote CVD system (Yield Engineering Systems, livermore, CA). The pump channel was then functionalized with a 20kDa polyethylene glycol (PEG) reagent (NanoCS, boston, MA). The PEG reagent terminates in an N-hydroxysuccinimide ester, which reacts with the primary amine on the surface of apdies to form a covalent bond between the PEG chain and the surface coating.

Both the CE-ESI and infusion designs operate by applying a voltage to the reservoir via a platinum wire electrode. The applied voltage is controlled by a custom HV power supply consisting of five independent voltage modules. The maximum output of three modules is-25 kV and the other two maximum outputs are +10kV (UltraVolt inc., ronkonkonkoma, NY). The power supply was connected to the computer through an SCB-68 junction box and a PCI-6713 channel analog card (National Instruments, austin, TX). A custom LabVIEW program was used to operate the power supply. For CE-ESI, the voltages applied to the S, B, SW and EO reservoirs were-14, -12 and +6kV, respectively. For gated injection, the voltages were switched to-14, -13 and +6kV for 0.5 seconds. This produced an electric field strength of 400V/cm, an approximate flow rate of 165nL/min. For infusion-ESI, the typical voltage for the S reservoir is +5kV, and the typical voltage for the EO reservoir is +0.5kV.

ESI-MS

Miniature mass spectrometer (ESI mass spectrometer) experiments were performed with a custom-made atmospheric interface and a differential pumping system. A schematic of a typical experimental setup is shown in fig. 1.

The microchip-ESI device (FIG. 5A/FIG. 5B CE or perfusion) is mounted on a custom x-y-z stage and is located about 5-10 mm from inlet capillary 15 (FIG. 1). A single-sided copper-clad circuit board (m.g. chemica1s, berlington, ontario, canada) is used to shield the ESI aperture from the voltage applied to the reservoir (not shown). The corners of the microfluidic device extend about 5 mm through the slits in the plate. For the CE experiments, the circuit board was kept at +1kV, for the infusion experiments, the circuit board was kept at GND.

The microchip device for capillary electrophoresis shown in FIG. 5A and the microchip device for infusion shown in FIG. 5B are glass microchips. The channels were etched to a depth of 10 μm. The reservoirs are indicated by circles and indicate the sample (S), background electrolyte (BG), sample Waste (SW) and electro-osmotic pump (P). For some experiments, the microchip has injection cross, 46cm serpentine separation channel and electroosmotic pumping channel. The infusion set (5B) has a 5.5cm channel and an electro-osmotic pump channel, and both reservoirs contain the same sample.

Ions generated during electrospray (represented by spray triangles) enter the first chamber of the mass spectrometer (about 1 torr, ambient air) from atmospheric pressure (760 torr) using a custom interface. First, the ions travel through a stainless steel capillary (2) (0.01 inch inner diameter, from Valco Instruments, houston, texas) that is applied with a voltage, typically between +100 and + 250V. The capillary tube was held in place by Swagelok UltraTorr joints (Swagelok, inc., solon, OH). The ions are then accelerated by a copper electrode (28) and focused into a trap (30) with a single 'gated' electrode (38). The tip of the capillary and the accelerating electrode were fixed at a distance of about 3 mm from the gate electrode. Ions typically accumulate for 5 milliseconds before analysis. They were then swept out of the wells and detected with an electron multiplier (Detech 2300, detector technology, inc., sturbridge, MA). A typical mass spectrum averages 30 to 1000 mass scans.

The differential pumping maintains the mass analyser and the detector at respective pressures. The electron multiplier used for detection was operated at lower pressure (< 20 mtorr). The differential pressure is provided by two sets of pumps. A dry scroll pump (SH-110 from Agilent technologies, inc., santa Clara, calif.) was used for the mass analyzer chamber (about 1 Torr), and an Agilent TPS Bench turbomolecular pump (model number TV 81M) supported by the Agilent scroll pump (SH-110) was used for the detector chamber (about 10 mTorr).

Mass analysis was performed using Towne Technologies corporation (Somerville, NJ) wet etched micro CIT electrodes. CIT dimensions r0=250 μm, z0=325 μm, and an end cap with a pore size of 200 μm. Each ring electrode contains a single trap. The trap is assembled by manual alignment using alignment pins. The electrodes were mounted on a custom plate with a 125 μm polyimide spacer between the electrodes. The driver RF waveforms were applied by Rohde and Schwarz SMB 100A signal generators and amplified using a mini-circuit TVA-R5-13 preamplifier and AR305 power amplifier alignment. The signal is resonated with the tank circuit and applied at a frequency ranging from 7 to 12 megahertz. Custom LabVIEW software was designed to monitor, control and collect data. The national instrumentation PXIe-1073 data collection chassis is used to connect electronics and LabVIEW software.

For comparison with CE separation detection, a synaptic (synapse) G2 quadrupole-ion mobility time-of-flight mass spectrometer (Waters Corporation, milford, MA) was used. Sypnapt G2 operates at a rate of 90ms induction with a 24ms (about 10 Hz) time delay between each scan. The mass range is set to 300 to 1600m/z. MassLynx software was used to collect data and was triggered by a custom LabVIEW program that was used to control the voltage applied to the microchip.

Atmospheric interface development

The interface developed for mass spectrometers has several advantages over the traditional ESI-MS interface. The mass spectrometer minimizes the complexity of the atmospheric interface. Conventional ESI-MS interfaces consist of an atmospheric inlet, multiple differential pressure regions, and complex ion optics, as required for low pressure operation of the mass analyzer. Since the mass spectrometer operates at a pressure of approximately 1 torr, the interface used introduces ions directly from the atmosphere into the mass analyser chamber through the capillary inlet. The capillary tube is held using simple fittings so the inlet can be easily removed for cleaning. Finally, because of the short distance from the ion source to the mass analyzer, minimal optics are required to maximize ion transport.

The 20 common amino acids were selected as model analytes for developing microchip to MS interface. perfusion-ESI microchips are used for the development of this interface, so there is a constant ion source. Representative infusion-ESI-MS spectra of four amino acids (arginine, histidine, glutamic acid, and proline) collected using an atmospheric interface and differential chamber setup are shown in fig. 14. Mass analysis was performed at a drive frequency of 10.2MHz, with ambient air as the buffer gas, at a pressure of 1.2 torr. Each spectrum is the average of 1000 individual mass spectral scans. The (M + H) + peak for each amino acid was clearly detected, which provides sufficient information for identifying these species. In the case of histidine and glutamic acid, some cleavage was also observed. FSI is a soft ionization technique, but operation at high pressures results in increased ion buffer gas collisions, which may transfer the energy required to induce fragmentation. These patterns of disruption may be helpful in identifying chemical species, including differentiation of isobars. The detection of twenty common amino acids indicates the ability to detect a wide variety of different analytes varying in size, polarity, and basicity over a wide range.

Mass analysis with high quality analytes was also demonstrated. Fig. 15 shows an infusion-ESI-MS profile of the small peptide, thymopentin (RKDVY, M + H) + M/z = 681). The mass analysis was performed under the following conditions: an RF drive frequency of 7.1MHz with ambient air as a buffer gas, and at a pressure of 1.3 torr. The capture and analysis of thymopentin showed that the mass range of mini-CIT can be extended to at least 681m/z. The largest peak is for the doubly protonated species, (M + 2H) 2+. Under acidic experimental conditions, this was expected due to the presence of two basic residues (R and K) in thymopentin. In addition, the signal to noise ratio (S/N) of thymopentin is significantly greater than that observed for amino acids. The smaller S/N observed for amino acids compared to peptides may be due to scattering before entering the trap resulting in less efficient capture of small molecules. This simple inlet interface is an effective way to introduce ions from atmospheric pressure into the vacuum despite differences in the signal-to-noise ratio of the analytes.

CE-ESI-MS of peptides

After demonstrating the feasibility of the atmospheric interface, the mini CIT system was evaluated as a probe for CE separation and compared to the commercial system Waters synapset G2. Fig. 16 shows the Basic Peak Intensity (BPI) electropherograms of the standard peptide mixture (methionine enkephalin, angiotensin II, bradykinin and thymopentin) detected with the mini-CIT system and Synapt G2. Fluorescein was added to the mixture as a dead time marker. The migration times are different due to slightly different field strengths.

The separation field strength was 400V/cm and the flow rate was about 165nL/min. About 7fmol of peptide mixture was injected during the 0.5 second gated injection. mini-CIT (r 0=250 μm) was operated at 1.2 torr with an RF drive frequency of 7.1 MHz. Four peptides and fluorescein were isolated and detected. The separation efficiencies calculated for these separations were: for mini-CIT, about 445,000 theoretical plates; for Synapt G2, there are 490,000 theoretical plates. Both mass spectrometers are capable of detecting these fast and efficient separations where there is a difference in computational efficiency due to differences in mass spectrometry sampling rates. The Synapt G2 collects spectra at about 10Hz, while the mini-CIT collects spectra at about 3 Hz. CIT is limited by the time required to accumulate, analyze and clear ions from the trap. As sensitivity increases, the accumulation time may be shortened and the sampling rate may be increased. It was demonstrated that fluorescein was not readily detectable with mini-CIT, but could be readily replaced with another dead time marker. Detection of these peptides after CE separation showed that: micro CIT-based mass spectrometers operating at high pressures can produce results comparable to commercial instruments. The Synapt G2 shows a slightly better S/N, but this simple comparison demonstrates the feasibility of a mass spectrometer using mini-CIT as a detector for biomolecule separations.

For mixtures like these peptides, the mini-CIT system provides a simple and inexpensive alternative to large commercial instruments such as the Synapt G2. The micro MS system can provide useful mass spectral information for label-free detection and chemical identification. Figure 17 shows sample mass spectra of bradykinin obtained during CE separation for both MS systems. Some similar features can be observed in both spectra, most notably the (M + 2H) 2+ peak at 531M/z. The most significant difference was that the observed peak width (12.0 m/z for the peak width observed with the mass spectrometer; 0.026m/z for the peak width observed with the Synapt G2) was expected to have a broader peak for the mini-CIT system due to the high pressure operation and air as the buffer gas. By increasing the operating drive frequency to 14.4MHz and operating at a lower buffer pressure, the peak width was significantly improved (< 5.0 m/z). Despite the increased peak width, mass spectrometry in combination with CE migration time provides sufficient information for the identification of many chemical species, particularly for applications where the target is the detection of a known target analyte. FIG. 18 is a graph (time versus normalized BPI, arbitrary units) illustrating the MS sampling rates of the Synapt G2 and mini-CIT/ES systems.

FIGS. 19A-19C are graphs of infusion-ESI mass spectrometry measurements of amino acids, amino acid mixtures, and peptides, respectively. Figure 19A also shows data from the mass library of amino acids (histidine) for comparison.

Figure 20 is a graph illustrating high voltage ion trap theory with operating parameters. Importantly, the resolving power of an ion trap mass spectrometer is proportional to the radio frequency drive frequency divided by the operating pressure P. Thus, as P increases, resolution can be restored by increasing accordingly. Fig. 20 also shows that the required amplitude of ion ejection is inversely proportional to the trap sizes r0 and z0. Fig. 21 is a graph showing experimental results of mass spectral resolution using different RF frequencies and r0 sizes, in normalized intensity (a.u.). The resolution varies according to the ion trap theory shown in fig. 20.

In summary, microchip electrospray ionization sources can be successfully coupled to high pressure mass spectrometers, and ions can be introduced into the mass spectrometer using the ambient pressure (e.g., atmospheric pressure) inlet and DC ion control of a metal (e.g., stainless steel) capillary. The administration and detection of the infusion of amino acids and peptides was performed using a micro cylindrical ion trap (micro CIT) based mass spectrometer operating at > 1 Torr with air as a buffer gas. Detection of thymopentin indicated that the mass range of the mini-CIT probe could be extended to at least 681m/z. Small proteins, such as cytochrome C and myoglobin, having masses of about 12kDa and 17kDa, respectively, have also been observed using the system described above.

Microchip Capillary Electrophoresis (CE) separation and mini-CIT detection were also performed and the results compared to detection using a commercial instrument (Waters Synapt G2). Comparable separation efficiencies can be observed for both mass spectrometers. Comparison of the mass spectra in the two systems revealed similar characteristics, but the peak width in the mini-CIT (shown as 12m/z, but improved to <5 m/z) was wider than the peak width in the synapse G2 (0.026 m) (as expected, due to the high pressure operation).

The foregoing is illustrative of example embodiments and is not to be construed as limiting thereof. Although a few exemplary embodiments have been described, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this disclosure. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the claims. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims (10)

1. An electrospray ionization (ESI) mass spectrometer analysis system comprising:

an ESI device having at least one emitter configured to electrospray ions; and

a mass spectrometer in fluid communication with the at least one emitter of the ESI device, comprising:

a mass analyzer held in a vacuum chamber, wherein the vacuum chamber is configured to have a high pressure of about 50 mtorr or more during operation; and

a detector in communication with the mass analyzer in a vacuum chamber having the mass analyzer,

wherein during operation, the ESI device is configured to:

(a) Electrospray ions into a spatial region at atmospheric pressure outside the vacuum chamber adjacent to an inlet device attached to the vacuum chamber, wherein the inlet device draws electrospray ions outside the vacuum chamber with the mass analyzer and discharges the ions into the vacuum chamber with the mass analyzer; or alternatively

(b) Ions are electrosprayed directly into a vacuum chamber having the mass analyzer.

2. The system of claim 1, wherein the detector is spaced apart from a mass analyzer in the vacuum chamber by a distance of between about 1 mm and about 10 mm, and wherein the high pressure in the vacuum chamber with the detector and the mass analyzer is between 50 mtorr and 100 mtorr.

3. The system of claim 1 or claim 2, wherein the ESI device is configured to electrospray ions into a spatial region external to the vacuum chamber, wherein the ESI device is positioned external to the vacuum chamber with the mass analyzer, wherein the inlet device is spaced apart from the ESI device, and wherein an end portion of the inlet device is positioned inside the vacuum chamber with the mass analyzer and is spaced apart from and a distance between 1-50 millimeters from an ion inlet of the mass analyzer.

4. The system of claim 3, wherein the inlet device is tubular with at least one inlet aperture in fluid communication with at least one longitudinally extending channel extending therethrough, and wherein the system comprises a direct current voltage input to the inlet device outside of the vacuum chamber with the mass analyzer.

5. The system of claim 1, 2 or 3, wherein the ESI device is configured to electrospray ions into a spatial region external to the vacuum chamber, wherein the inlet device comprises at least one inlet aperture and has an external end spaced apart from the ESI device, and wherein the inlet device is planar and electrically conductive and is between about 0.100 millimeters and about 5 millimeters thick.

6. The system of claim 5, further comprising: a compartment holding the ESI device in an orientation aligned with the inlet device, wherein the compartment comprises a buffer gas inlet such that during operation a buffer gas is introduced into the compartment and then transported via the inlet device into a vacuum chamber having the mass analyzer.

7. The system of claim 1, wherein the ESI device is configured to electrospray ions directly into a vacuum chamber with the mass analyzer, and wherein the ESI device is attached to a wall of the vacuum chamber such that the at least one emitter is inside the vacuum chamber and one or more containers of the ESI device are outside the vacuum chamber.

8. An electrospray ionization (ESI) mass spectrometer analysis system comprising:

an ESI device having at least one emitter configured to electrospray ions; and

a mass spectrometer in fluid communication with the at least one emitter of the ESI device, comprising:

a mass analyzer held in a vacuum chamber, wherein the vacuum chamber is configured to have a high pressure of about 50 mtorr or more during operation; and

a detector in communication with the mass analyzer,

wherein during operation, the ESI device is configured to:

(a) Electrospray ions into a spatial region at atmospheric pressure outside the vacuum chamber adjacent to an inlet device attached to the vacuum chamber, wherein the inlet device draws electrospray ions outside the vacuum chamber having the mass analyzer and discharges the ions into the vacuum chamber having the mass analyzer; or

(b) Ions are electrosprayed directly into a vacuum chamber having the mass analyzer.

9. A method of analyzing a sample, comprising:

introducing sample ions into a vacuum chamber equipped with a mass analyzer by:

(a) Electrospray ions directly from an electrospray ionization (ESI) device into a vacuum chamber having the mass analyzer, wherein a gas pressure in the mass analyzer is between 50 mtorr and 100 torr; or

(b) Electrospray ions into a spatial region outside the vacuum chamber at atmospheric pressure, the spatial region adjacent to an inlet device spaced from the ESI device, and then transport the ions through the inlet device into a vacuum chamber holding the mass analyzer, wherein a gas pressure in the mass analyzer is between 50 mtorr and 100 torr;

trapping the ions in the mass analyser;

selectively ejecting the ions from the mass analyser;

detecting an electrical signal corresponding to the ejected ions using at least one detector also in the vacuum chamber; and

generating data based on the detected electrical signals to determine information related to the sample.

10. A method of analyzing a sample, comprising:

introducing sample ions into a vacuum chamber equipped with a mass analyzer by:

(a) Electrospray ions from an electrospray ionization (ESI) device directly into a vacuum chamber having the mass analyzer, wherein a gas pressure in the mass analyzer is between 50 mtorr and 100 torr; or

(b) Electrospray ions into a spatial region outside the vacuum chamber at atmospheric pressure, the spatial region adjacent to an inlet device spaced from the ESI device, and transport the ions through the inlet device into the vacuum chamber holding the mass analyzer, wherein a gas pressure in the mass analyzer is between 50 mtorr and 100 torr;

trapping the ions in the mass analyser;

selectively ejecting the ions from the mass analyser;

detecting an electrical signal corresponding to the ejected ions using at least one detector; and

generating data based on the detected electrical signals to determine information related to the sample.

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