Electro spray ionization with conductive PEEK voltage application

Abstract

An integrated system for liquid separation and electrospray ionization includes an emitter capillary; an LC column; a conductive sheath; a bio-inert conductive union forming a fluidic connection between the emitter capillary and the LC column, the bio-inert conductive union electrically coupled to the conductive sheath, the bio-inert conductive union facilitating the application of high voltage to a mobile phase flowing from the LC column to the emitter capillary; and an insulating plastic material encapsulating the LC column.

Description

ELECTRO SPRAY IONIZATION WITH CONDUCTIVE PEEK VOLTAGE APPLICATION

[0001] This application claims benefit to U.S. Provisional Application S / N 63 / 642,960, filed May 6, 2024, which is incorporated by reference in its entirety.FIELD

[0002] The present disclosure generally relates to the field of liquid chromatography including a replaceable electrospray emitter system for liquid separation and electrospray ionization.INTRODUCTION

[0003] Proteomics, being the study of protein structure and function, is a research focus for decades to come as it can allow one to elucidate the fundamentals of life and the molecular basis of health and disease. Analysis of complex protein mixtures usually involves two steps: molecular separation and identification / characterization. In the context of bottom-up proteomics experiments, proteins are subject to proteolytic digestion to break down into fragments of peptides which are then separated, usually with liquid chromatography (LC), before being introduced into an ion source of a mass spectrometer. Typically, the ion source for proteomics experiments implements electrospray ionization (ESI) to ionize the peptide to form ions that can be transported among components of a mass spectrometer.

[0004] While conventional HPLC columns (i.e. columns with fittings for connecting to conventional instruments) for use with nano-liter flow rates (also referred to as nano-LC) show superior performance, incorrect assembly of fittings and fluid connections often compromises the advantages associated with conventional nano-LC columns. In other words, incorrect connections of LC transfer tubing to the LC columns may result in leaks and consequently poor sensitivity and chromatographic separation. Also, incorrect connection of a conventional nano-electrospray emitter after the LC column may give rise to undesired dead-volumes which also leads to reduced sensitivity and poor separating power.

[0005] The columns and transfer lines ordinarily used in liquid chromatography systems that employ flow rates less than 10 pL / minute most frequently have very narrow inner diameters as well as outer diameters. Consequently, such transfer lines and columns may be physically fragile. Thus, it is highly desirable to provide some means of mechanical relief from strain, pressure, bends, twists etc. such that the thin tubing components are protected and become robust enough to withstand use in everyday laboratory work.

[0006] The commonly used interface between chromatography and mass spectrometry is made up by the electrospray ion-source. In the ion source, the eluate from the LC column is passed through an emitter (also termed a needle) that is held at an electric potential that usually differs by one or more kilovolts from an opposing inlet orifice of the mass spectrometer. However, metal unions for applying the high voltage electric potential can attract and retain some low abundance sample or samples that have an affinity for metals that prevent them being ionized and detected in the mass spectrometer. The metal also tends to oxidize in the mobile phase of the LC column reducing the efficiency of voltage application to the liquid path and reducing the lifetime and robustness of the electrospray ion source. It is therefore desirable to minimize the exposure of the samples to metals within the flow path.SUMMARY

[0007] In a first aspect, an integrated system for liquid separation and electrospray ionization can include an emitter capillary; an LC column; a conductive sheath; and a bio-inert conductive union and an insulating plastic material encapsulating the LC column. The bio-inert conductive union can form a fluidic connection between the emitter capillary and the LC column. The bio-inert conductive union can be electrically coupled to the conductive sheath. The bio-inert conductive union can facilitate the application of high voltage to a mobile phase flowing from the LC column to the emitter capillary.

[0008] In exemplary embodiments of the first aspect, the bio-inert conductive union ensures a near zero dead-volume connection to the emitter.

[0009] In exemplary embodiments of the first aspect, the bio-inert conductive union can prevent sample loss due to metal interactions and can inhibit the formation of metal oxidations within the liquid junction.

[0010] In exemplary embodiments of the first aspect, the bio-inert conductive union can include a conductive PEEK material. In particular embodiments, the conductive PEEK material includes PEEK doped with carbon, such as carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof

[0011] In exemplary embodiments of the first aspect, the liquid junction formed by the conductive PEEK material can ensure the direct application of high voltage to the liquid prior to entering the emitter tip inlet.

[0012] In exemplary embodiments of the first aspect, the insulating plastic material can further encapsulate a temperature control board or an identification tag.

[0013] In a second aspect, an integrated system for liquid separation and electrospray ionization can include a combined emitter and column capillary; a conductive sheath; a bio-inert conductive union; and an insulating plastic material encapsulating the LC column. The bio-inert conductive union can form a fluidic connection between the combined emitter and column capillary and upstream LC components. The bio-inert conductive union can be electrically coupled to the conductive sheath. The bio-inert conductive union can facilitate the application of high voltage to a mobile phase flowing into the combined emitter and column capillary.

[0014] In exemplary embodiments of the second aspect, the bio-inert conductive union can ensure a near zero dead-volume connection to the emitter.

[0015] In exemplary embodiments of the second aspect, the bio-inert conductive union can prevent sample loss due to metal interactions and can inhibit the formation of metal oxidations within the liquid junction.

[0016] In exemplary embodiments of the second aspect, the bio-inert conductive union can include a conductive PEEK material.

[0017] In particular embodiments, the conductive PEEK material can include PEEK doped with carbon, such as carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof.

[0018] In exemplary embodiments of the second aspect, the liquid junction can be formed by the conductive PEEK material ensures the direct application of high voltage to the liquid prior to entering the emitter tip inlet.

[0019] In exemplary embodiments of the second aspect, the insulating plastic material can further encapsulate a temperature control board or an identification tag.

[0020] In a third aspect, a method of separation can include a) flowing a sample through an LC column and an electrospray emitter; b) utilizing a bio-inert conductive union to form a liquid junction, ensuring a near zero dead-volume connection; and c) employing the bio-inert conductive union to apply high voltage to the mobile phase upstream of the emitter.

[0021] In exemplary embodiments of the third aspect, upstream of the emitter can include at the inlet to a combined emitter and column capillary or at the union between a column capillary and an emitter capillary.

[0022] In exemplary embodiments of the third aspect, the bio-inert conductive union can include a conductive PEEK material.

[0023] In particular embodiments, the conductive PEEK material can include PEEK doped with carbon, such as carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof.

[0024] In exemplary embodiments of the third aspect, the bio-inert conductive union can reduce sample loss by minimizing interactions with metals and can inhibit metal oxidations within the liquid junction, facilitating efficient voltage application.DRAWINGS

[0025] For a more complete understanding of the principles disclosed herein, and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

[0026] Figure 1 is an external view of the conventional system for electrospray ionization, in accordance with various embodiments.

[0027] Figure 2 is a cross section views of the conventional system of Figure 1, in accordance with various embodiments.

[0028] Figure 3 is a close-up cross section view illustrating the union between an emitter and a liquid chromatography column within the conventional system of Figure 1, in accordance with various embodiments.

[0029] Figure 4 is an exploded view of the conventional system of Figure 1, in accordance with various embodiments.

[0030] Figure 5 is a cross section view illustrating an alternate system with a combined emitter and column capillary, in accordance with various embodiments.

[0031] Figures 6A and 6B are cross section views illustrating an alternate replaceable emitter system with the emitter assembly can be removed and reattached, in accordance with various embodiments.

[0032] Figures 7 is an extracted ion chromatogram (EIC) comparing a bio-inert conductive union to a metal union using PENNY peptide.

[0033] Figure 8A is a comparison of mean full width half height (FWHM) and Figure 8B is a comparison of mean peak areas for a bio-inert conductive union and a metal union using PENNY peptide.

[0034] Figure 9 is a comparison of FWHM for a bio-inert conductive union and a metal union across a range of voltages.

[0035] It is to be understood that the figures are not necessarily drawn to scale, nor are the objects in the figures necessarily drawn to scale in relationship to one another. The figures are depictions that are intended to bring clarity and understanding to various embodiments of apparatuses, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. Moreover, it should be appreciated that the drawings are not intended to limit the scope of the present teachings in any way.DESCRIPTION OF VARIOUS EMBODIMENTS

[0036] Embodiments of a replaceable electrospray emitter system for liquid separation and electrospray ionization are described herein.

[0037] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way.

[0038] In this detailed description of the various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the embodiments disclosed. One skilled in the art will appreciate, however, that these various embodiments may be practiced with or without these specific details. In other instances, structures and devices are shown in block diagram form. Furthermore, one skilled in the art can readily appreciate that the specific sequences in which methods are presented and performed are illustrative and it is contemplated that the sequences can be varied and still remain within the spirit and scope of the various embodiments disclosed herein.

[0039] All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose. Unless described otherwise, all technical and scientific terms used herein have a meaning as is commonly understood by one of ordinary skill in the art to which the various embodiments described herein belongs.

[0040] It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0041] As used herein, "a" or "an" also may refer to "at least one" or "one or more." Also, the use of “or” is inclusive, such that the phrase “A or B” is true when “A” is true, “B” is true, or both “A” and “B” are true. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0042] A “system” sets forth a set of components, real or abstract, comprising a whole where each component interacts with or is related to at least one other component within the whole.

[0043] One challenge with integrated column-emitter systems, like EASY- SPRAY™ columns, is that the liquid path must be in direct contact with a conductive union forming a liquid junction. The union has a reliable liquid junction to ensure that the high voltage is applied to the liquid upstream of the emitter tip. In various embodiments, the liquid junction can be between an emitter capillary and a column capillary. In other embodiments, the liquid junction is at an inlet end of a combined emitter and column capillary.

[0044] This liquid junction is used to ensure that the high voltage is applied directly to the liquid and sample to effectively ionize the liquid exiting the emitter. The metal union can attract and retain some low abundance samples or samples that have an affinity for metals that prevent them being ionized detected in the mass spectrometer. The metal also tends to oxidize in the mobile phase of the LC column reducing the efficiency of voltage application to the liquid path.

[0045] Replacing the metal contacting the liquid path inside the conductive union with a conductive bio-inert material, such as a conductive PEEK (Poly etheretherketone). The PEEK is doped with carbon to allow for the electricity to flow through the typically insulative PEEK. The conductive PEEK is fixed at the union using a plug-type fitting, such as a nanoViper™ fitting, to ensure a near zero dead-volume connection.

[0046] The EASY-SPRAY column format includes internal temperature control, safe application of electrospray voltage, and an insulating plastic encapsulating the LC column, temperature control board, and union for voltage application.

[0047] FIG. 1 shows an external view of an emitter system 100 for electrospray ionization. FIG. 2 shows a cross section view of the emitter system 100, and FIG. 3 shows a zoomed in view of the union between the emitter capillary and the column capillary. FIG. 4 shows an exploded view of the emitter system 100. Emitter system 100 includes a capillary column 102 and an emitter capillary 104. The capillary column 102 has an inlet end 106 and an outlet end 108. The emitter capillary 104 has an inlet end 110 and an outlet end 112 pulled to a tip to function as an electrospray emitter. The outlet end 108 of the capillary column 102 is in fluid communication with the inlet end 110 of the emitter capillary 104.

[0048] The column capillary 102 is preferably an LC column, e.g. HPLC column. The LC column may be used with various flow rates, e.g. down to as low as nano-LC flow rates, i.e. 100 nL / min or less.

[0049] The column capillary 102 is coiled into a loop 114 comprising multiple column windings to increase the separation length. This enables space saving since it allows a column to take up less space than if it were laid straight and it permits different column lengths to be used in the same design of integrated system, i.e. by changing the number of windings in the coil. Coiling the column further makes the column compact and able to fit into a small volume that may more easily be temperature controlled by a heating element than if it were laid straight and would occupy an elongated, typically long, space.

[0050] The emitter capillary 104 can include an electrically conductive capillary, such as a metal capillary or a glass capillary, e.g. glass coated with electrically conductive material. However, glass capillaries that are not conductive or coated may be used.

[0051] In various embodiments, the outlet end 112 with of the emitter capillary 104 can include a porous matrix. The porous matrix can reduce the void volume of the tip.

[0052] The capillary column 102 and an emitter capillary 104 is embedded in a molding part 116. The molding part 116 comprises a plastic material, for example, a thermoplastic material, for example, polyamide and polyurethane based MacroMeltTM. Suitable methods to embed the assembly are described in the applicant’s patent US Pat 9302415. The molding 116 provides rigidity to the system, as well as provides a shield against a user disassembling or damaging, intentionally or by accident, the fittings, HPLC column and emitter.

[0053] The inlet end 106 of the column capillary 102 is provided with fitting 118, e.g. for connection to an injector or other HPLC components. In various embodiments, the fitting 118 can include a plug type end fitting 120 with cap screw 122 and an external union with small internal diameter. In other embodiments, the fitting 118 can include a ferrule, a nut, and cap (not shown).

[0054] A protective sleeve 124 of generally cylindrical form is slidably located on the outlet end 104 of the emitter capillary 104. The sleeve has a main body 126 and a base 128 of wider diameter than the main body. The protective sleeve 124 is desirably made of a rigid material, such as a metal or polymer material. In this way the rigidity of the sleeve can protect the fragile emitter portion of the emitter-enabled capillary column 102 that it covers. Mounted about the protective sleeve 124 is an electrically conductive sheath 130, e.g. made of metal. The conductive sheath 130 has an internal diameter such as to accommodate therein the protective sleeve 124 and permit the protective sleeve 124 to slidably move in a reciprocating manner inside the sheath as further described below.

[0055] The electrically conductive sheath 130 is supported at one end by a supporting structure 132. Within the supporting structure 130, the column capillary 102 and the emitter capillary 104 can be fluidically connected by a bio-inert conductive union 134. In particular embodiments, a plug type fitting 136 at the outlet end 106 of the column capillary 102 can abut the bio-inert conductive union 134 on a first side and a plug type fitting 138 on the inlet side 110 of the emitter capillary 104 can abut the bioinert conductive union 134 on a second side. The supporting structure 132 can be configured to hold the plug type fitting 136, the bio-inert conductive union 134 and the plug-type fitting 138 in a close contact arrangement to minimize dead volume and maintain the integrity of the fluidic connection between the column capillary 102 and the emitter capillary 104.

[0056] The bio-inert conductive union 134 can be a conductive bio-inert material and in the case of our example a conductive PEEK. The PEEK can be doped with carbon to allow for the electricity to flow through the typically insulative PEEK. In particular embodiments, the PEEK can be doped with carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof. Other plastics and conductive fillers can be used. However, it is preferable to utilize non-metallic fillers and bio-inert plastics that are compatible with the solvents used in liquid chromatography. The liquid junction is formed by the conductive PEEK to ensure that the high voltage is applied to the liquid before entering the inlet 110 of the emitter capillary 104. The bio-inert PEEK high voltage bio-inert conductive union 134 prevents sample loss due to interactions of sample compounds with metals and prevents metal oxidation from forming within the liquid junction impeding voltage application.

[0057] The electrically conductive sheath 130 can be enclosed within a holder having a high-voltage contact point when the emitter system 100 is in use. The holder can be a holder located on an instrument, e.g. for mass spectrometric analysis. The electrically conductive sheath 130 provides an electrical connection to enable the emitter to receive a high voltage. The electrically conductive sheath 130 may provide an electrical connection to the liquid junction through the supporting structure 132 and the bio-inert conductive union 134. The electrically conductive sheath 130 has a recess in theform of a circumferential groove 140 on its outer surface for making an electrical contact with a high voltage contact, e.g. a contact ball.

[0058] In some embodiments, the protective sleeve 126 is fixed with respect to the outlet end 112 with of the emitter capillary 104. However, the protective sleeve 124 is most preferably retractable, i.e. with respect to the outlet end 112 with of the emitter capillary 104. Where the protective sleeve 124 is retractable, this ensures that the outlet end 112 of the emitter capillary 104 is exposed when in use and thereby the protective sleeve 124 does not interfere, for example, with gas flows and equipotential lines around the emitter tip. Moreover, a retractable protective sleeve 124, when in use, does not block visibility of the emitter tip so one can readily monitor the spray. The protective sleeve 124 is preferably slidably located around the emitter capillary 104. The protective sleeve 124 is preferably movable between an extended (or cover) position wherein it covers the emitter tip, and a retracted position wherein the emitter tip is exposed. When the emitter tip is exposed, it may be used for electrospray ionization. The emitter tip herein means the tip from which ions are produced when in use. The protective sleeve 124 thus covers and supports the emitter capillary 104 along at least a portion of its axis which includes the emitter tip.

[0059] A spring 142 is further provided inside the electrically conductive sheath 130, positioned in a space between the supporting structure 132 and the protective sleeve 124. The spring 142 acts upon the base 128 of the protective sleeve 124 to bias the protective sleeve 124 to force it out of the electrically conductive sheath 130. The length of the protective sleeve 124 and its extension out of the sheath is sufficient to cover the outlet end 112 of the emitter column 104 and act to protect it against damage. A part of the main body 126 of the protective sleeve 124 protrudes outside the sheath 130 and thereby covers the emitter. The extent of travel of the sleeve 124 out of the sheath 130 is restricted by a reduced internal diameter at the end of the sheath 130 that stops the wider diameter base 128 of the protective sleeve 124. If a force is applied to the protective sleeve 124 to push the protective sleeve 124 backwards into the sheath 130, the spring 142 becomes compressed and the tip of the emitter becomes exposed and ready for use.

[0060] The plastic molding 1 16 covers the emitter system 100 with the electrically conductive sheath 130 mounted at the front end and the protective sleeve 124 protecting the outlet end 112 of the emitter column 104. It will be appreciated from the description that the whole emitter system 100 is thus formed as a type of cartridge for use with an instrument, e.g. mass spectrometer. An emitter cap 144 can cover the electrically conductive sheath when not in use.

[0061] In order to meet the most relevant requirements in relation to the operation of the column, the integrated separation column may be equipped with one or more embedded components of: a heating and / or cooling element and a thermal sensor in close proximity or contact with the column and preferably embedded in the plastic material.

[0062] For optimum performance, channels for gas flow may also be embedded in the plastic material; the outlet of these channels being in close proximity with the apex (tip) of the emitter, whereby gas leaving the outlet assists in the desolvation of the spray cloud.

[0063] In order to facilitate identification of the integrated column, the embedded components may further comprise an identification tag, such as a radio frequency identification tag (RFID) embedded in the plastic material.

[0064] In manufacturing the emitter system 100 of the present invention, plastifying the plastic material that is used for embedding the integrated system may be achieved in various ways, preferably by heating the plastic material beyond the softening temperature for bringing it in its softening range and making it soft. In a preferred embodiment the entire column and fittings are surrounded by the plastic material. In various embodiments, the plastic material can be injection molded around the emitter system 100. In other embodiments, the plastic material can be provide as molded parts that can be assembled around the emitter system 100. For example, the plastic material can be a two piece molded part that snaps together or is other fastened together to surround the emitter system 100.

[0065] FIG. 1 illustrates the use of the bio-inert conductive union 134 in an embodiment where the emitter capillary 104 and column capillary 102 are fluidically coupled by the conductive union and fixed in place and embedded within a molding. A bio-inert conductive union can also be used in other embodiments, such as an emitter system with a combined emitter and column capillary (see FIG. 5) or an emitter system with a replaceable emitter (see FIG. 6A and 6B).

[0066] FIG. 5 shows a cross section view of the emitter system 500. Emitter system 500 includes a combined emitter and column capillary 502 with an outlet end 512 pulled to a tip to function as an electrospray emitter. The combined emitter and column capillary 502 also includes an inlet end 506. The combined emitter and column capillary 502 includes a column portion 552 and an emitter portion 554. Preferably, the column portion 552 is an LC column, e.g. HPLC column. The LC column may be used with various flow rates, e.g. down to as low as nano-LC flow rates, i.e. 100 nL / min or less.

[0067] The column portion 552 of the combined emitter and column capillary 502 is coiled into a loop 514 comprising multiple column windings to increase the separation length.

[0068] The emitter portion 554 includes an electrospray emitter. The electrospray emitter can include an electrically conductive portion of the capillary, such as a metal capillary or a glass capillary, e.g. glass coated with electrically conductive material. However, glass capillaries that are not conductive or coated may be used.

[0069] In various embodiments, the outlet end 512 with integrated emitter tip can include a porous matrix. The porous matrix can reduce the void volume of the tip. Additionally, the porous matrix can provide a defined end to the column portion 552 to ensure consistent packing of column material.

[0070] The combined emitter and column capillary 502 is embedded in a molding part 516. The molding part 516 comprises a plastic material, for example, a thermoplastic material. The molding 516 provides rigidity to the system, as well as provides a shieldagainst a user disassembling or damaging, intentionally or by accident, the fittings, HPLC column and emitter.

[0071] The inlet end 506 of the combined emitter and column capillary 502 is provided with fitting 518, e g. for connection to an injector or other HPLC components. In various embodiments, fitting 518 can incorporate a bio-inert conductive union 534, such that a fitting from the HPLC components, such as a plug-type fitting, can join with the fitting 518 and create a fluid connection between the HPLC components and the inlet end 506 of the combined emitter and column capillary 502 through the bio-inert conductive union 534. The conductive union 534 can be a conductive bio-inert material and in the case of our example a conductive PEEK. The PEEK can be doped with carbon to allow for the electricity to flow through the typically insulative PEEK. In particular embodiments, the PEEK can be doped with carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof. Other plastics and conductive fillers can be used. However, it is preferable to utilize non-metallic fillers and bio-inert plastics that are compatible with the solvents used in liquid chromatography. The liquid junction is formed by the bio-inert conductive union 534 to ensure that the high voltage is applied to the liquid before entering the inlet 506 of the combined emitter and column capillary 502. The bio-inert PEEK high voltage bio-inert conductive union 534 prevents sample loss due to interactions of sample compounds with metals and prevents metal oxidation from forming within the liquid junction impeding voltage application.

[0072] A protective sleeve 524 of generally cylindrical form is slidably located on the outlet end 512 of the combined emitter and column capillary 502. The sleeve has a main body 526 and a base 528 of wider diameter than the main body. The protective sleeve 524 is desirably made of a rigid material, such as a metal or polymer material. In this way the rigidity of the sleeve can protect the fragile emitter portion of the combined emitter and column capillary 502 that it covers. Mounted about the protective sleeve 524 is an electrically conductive sheath 530, e.g. made of metal. The conductive sheath 530 has an internal diameter such as to accommodate therein the protective sleeve 524 and permit the protective sleeve 524 to slidably move in a reciprocating manner inside the sheath as further described below.

[0073] The electrically conductive sheath 530 is supported at one end by a supporting structure 532. The combined emitter and column capillary 502 can be threaded through the supporting structure 532. Additionally, the supporting structure 532 can be in electrical contact with the bio-inert conductive union 534 by way of a conductor 556, such as a wire.

[0074] The electrically conductive sheath 530 can be enclosed within a holder having a high-voltage contact point when the emitter system 500 is in use. The holder can be a holder located on an instrument, e.g. for mass spectrometric analysis. The electrically conductive sheath 530 provides an electrical connection to enable the emitter to receive a high voltage. The electrically conductive sheath 530 may provide an electrical connection to the liquid junction through the supporting structure 532, the wire conductor 556 and the bio-inert conductive union 534. The electrically conductive sheath 530 has a recess in the form of a circumferential groove 540 in its outer surface for making an electrical contact with a high voltage contact, e.g. a contact ball.

[0075] In some embodiments, the protective sleeve 526 is fixed with respect to the outlet end 512 with of the combined emitter and column capillary 502. However, the protective sleeve 524 is most preferably retractable, i.e. with respect to the outlet end 512 with of the combined emitter and column capillary 502. Where the protective sleeve 524 is retractable, this ensures that the outlet end 512 with of the combined emitter and column capillary 502 is exposed when in use and thereby the protective sleeve 524 does not interfere, for example, with gas flows and equipotential lines around the emitter tip.

[0076] A spring 542 is further provided inside the electrically conductive sheath 530, positioned in a space between the supporting structure 532 and the protective sleeve 524. The spring 542 acts upon the base 528 of the protective sleeve 524 to bias the protective sleeve 524 to force it out of the electrically conductive sheath 530.

[0077] The plastic molding 516 covers the emitter system 500 with the electrically conductive sheath 530 mounted at the front end and the protective sleeve 524 protecting the outlet end 512 of the combined emitter and column capillary 502. It will be appreciated from the description that the whole emitter system 500 is thus formed as atype of cartridge for use with an instrument, e.g. mass spectrometer. An emitter cap 544 can cover the electrically conductive sheath when not in use.

[0078] In order to meet the most relevant requirements in relation to the operation of the column, the integrated separation column may be equipped with one or more embedded components of: a heating and / or cooling element and a thermal sensor in close proximity or contact with the column and preferably embedded in the plastic material.

[0079] For optimum performance, channels for gas flow may also be embedded in the plastic material; the outlet of these channels being in close proximity with the apex (tip) of the emitter, whereby gas leaving the outlet assists in the desolvation of the spray cloud.

[0080] In order to facilitate identification of the integrated column, the embedded components may further comprise an identification tag, such as a radio frequency identification tag (RFID) embedded in the plastic material.

[0081] FIG. 6A and 6B illustrate a finger tight replaceable emitter assembly compatible with the EASY-SPRAY column design. The emitter assembly can be removed without tools or removal of the protective insulating injection molding plastic. A pug type capillary fitting is installed on the emitter inlet to ensure a proper seal and assembly of the unit.

[0082] The replaceable emitter tip gives the customer the flexibility to change the emitter dimensions to better match the experimental parameters they are trying to reach. Emitter profiles along with the internal and external dimensions can be changed as long as they are assembled with a plug type connection.

[0083] The reliability of the column lifetime, union liquid junction and the ability to replace the emitter provides more versatility and long-term performance compared to the integrated column-emitter systems.

[0084] FIG. 6A shows a cross section view of the emitter system 600 with an attached emitter, and FIG. 6B shows a cross section view of the emitter system 600 witha detached emitter. Emitter system 600 includes a capillary column 602 and an emitter capillary 604. The capillary column 602 has an inlet end 606 and an outlet end 608. The emitter capillary 604 has an inlet end 610 and an outlet end 612 pulled to a tip to function as an electrospray emitter. The outlet end 608 of the capillary column 602 is in fluid communication with the inlet end 610 of the emitter capillary 604.

[0085] The column capillary 602 is preferably an LC column, e.g. HPLC column. The LC column may be used with various flow rates, e.g. down to as low as nano-LC flow rates, i.e. 100 nL / min or less.

[0086] The column capillary 602 is coiled into a loop 614 comprising multiple column windings to increase the separation length. This enables space saving since it allows a column to take up less space than if it were laid straight and it permits different column lengths to be used in the same design of integrated system, i.e. by changing the number of windings in the coil. Coiling the column further makes the column compact and able to fit into a small volume that may more easily be temperature controlled by a heating element than if it were laid straight and would occupy an elongated, typically long, space.

[0087] The emitter capillary 604 can include an electrically conductive capillary, such as a metal capillary or a glass capillary, e.g. glass coated with electrically conductive material. However, glass capillaries that are not conductive or coated may be used.

[0088] In various embodiments, the outlet end 612 with of the emitter capillary 604 can include a porous matrix. The porous matrix can reduce the void volume of the tip.

[0089] The capillary column 602 is embedded in a molding part 616. The molding part 616 comprises a plastic material, for example, a thermoplastic material, for example, polyamide and polyurethane based MacroMeltTM. Suitable methods to embed the assembly are described in the applicant’s patent US Pat 9302415. The molding 616 provides rigidity to the system, as well as provides a shield against a user disassembling or damaging, intentionally or by accident, the fittings, HPLC column and emitter.

[0090] The inlet end 606 of the column capillary 602 is provided with fitting 618, e.g. for connection to an injector or other HPLC components. In various embodiments, the fitting 618 can include a plug type end fitting 620 with cap screw 622 and an external union with small internal diameter. In other embodiments, the fitting 618 can include a ferrule, a nut, and cap (not shown).

[0091] A protective sleeve 624 of generally cylindrical form is slidably located on the outlet end 612 of the emitter capillary 604. The sleeve 624 has a main body 626 and a base 628 of wider diameter than the main body 626. The protective sleeve 624 is desirably made of a rigid material, such as a metal or polymer material. In this way the rigidity of the sleeve can protect the fragile emitter portion of the emitter-enabled capillary column 602 that it covers. Mounted about the protective sleeve 624 is an electrically conductive sheath 630, e g. made of metal. The conductive sheath 630 has an internal diameter such as to accommodate therein the protective sleeve 624 and permit the protective sleeve 624 to slidably move in a reciprocating manner inside the sheath 630.

[0092] The electrically conductive sheath 630 is supported at one end by a supporting structure 632A of the emitter portion. Supporting structure 632A can couple with supporting structure 632B of the column portion. Within the supporting structure 632A and 632B, the column capillary 602 and the emitter capillary 604 can be fluidically connected by a bio-inert conductive union 634. In particular embodiments, a plug type fitting 636 at the inlet end 610 of the emitter capillary 604 can abut the bio-inert conductive union 634 when the supporting structure 632A of the emitter portion couples with a supporting structure 632B of the column portion. Additionally, a plug type fitting 638 at the outlet end 608 of column capillary 602 can abut the bio-inert conductive union 634 opposite the plug type fitting 636. When coupling the emitter capillary 604 with the column capillary 602, plug-type fitting 636 can slide into an interior space of supporting structure 632B while supporting structure 632B threadingly engages with supporting structure 632A. The bio-inert conductive union 634 can include a through hole to allow liquid to flow from the capillary column 602 to the emitter capillary 604. The through hole can be sized to minimize dead volume, generally being similar to the inner diameterof the capillary column 602 and the emitter capillary 604 to avoid the accumulation of liquid in the through hole. The supporting structure 632B can be configured to hold the plug type fitting 638 and the bio-inert conductive union 634 in a fixed close contact arrangement and the plug type fitting 636 can be brought into a close contact arrangement by coupling the supporting structure 632 A and the supporting structure 632B to minimize dead volume and maintain the integrity of the fluidic connection between the column capillary 602 and the emitter capillary 604.

[0093] The bio-inert conductive union 634 can be a conductive bio-inert material and in the case of our example a conductive PEEK. The PEEK can be doped with carbon to allow for the electricity to flow through the typically insulative PEEK. In particular embodiments, the PEEK can be doped with carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof. Other plastics and conductive fillers can be used. However, it is preferable to utilize non-metallic fillers and bio-inert plastics that are compatible with the solvents used in liquid chromatography. The liquid junction is formed by the conductive PEEK to ensure that the high voltage is applied to the liquid before entering the inlet 610 of the emitter capillary 604. The high voltage bio-inert conductive union 634 prevents sample loss due to interactions of sample compounds with metals and prevents metal oxidation from forming within the liquid junction impeding voltage application.

[0094] The electrically conductive sheath 630 can be enclosed within a holder having a high-voltage contact point when the emitter system 600 is in use. The holder can be a holder located on an instrument, e.g. for mass spectrometric analysis. The electrically conductive sheath 630 provides an electrical connection to enable the emitter to receive a high voltage. The electrical path for applying the high voltage to the liquid flowing through the emitter capillary 604 can include the electrically conductive sheath 630, the supporting structure 632A, and the supporting structure 632B, and the bio-inert conductive union 634. The liquid flowing through the emitter capillary 604 can carry the high voltage to the distal end 612 to enable electrospray of the liquid. The electrically conductive sheath 630 has a recess in the form of a circumferential groove 640 in itsouter surface for making an electrical contact with a high voltage contact, e.g. a contact ball.

[0095] In some embodiments, the protective sleeve 626 is fixed with respect to the outlet end 612 with of the emitter capillary 604. However, the protective sleeve 124 is most preferably retractable, i.e. with respect to the outlet end 612 with of the emitter capillary 604. Where the protective sleeve 624 is retractable, this ensures that the outlet end 612 with of the emitter capillary 604 is exposed when in use and thereby the protective sleeve 624 does not interfere, for example, with gas flows and equipotential lines around the emitter tip. Moreover, a retractable sleeve 626, when in use, does not block visibility of the emitter tip so one can readily monitor the spray. The protective sleeve 624 is preferably slidably located around the emitter capillary 604. The protective sleeve 624 is preferably movable between an extended (or cover) position wherein it covers the emitter tip, and a retracted position wherein the emitter tip is exposed. When the emitter tip is exposed, it may be used for electrospray ionization. The emitter tip herein means the tip from which ions are produced when in use. The protective sleeve 624 thus covers and supports the emitter capillary 604 along at least a portion of its axis which includes the emitter tip.

[0096] A spring 642 is further provided inside the electrically conductive sheath 630, positioned in a space between the supporting structure 632 and the protective sleeve 624. The spring 642 acts upon the base 628 of the protective sleeve 624 to bias the protective sleeve 624 to force it out of the electrically conductive sheath 630. The length of the protective sleeve 624 and its extension out of the sheath is sufficient to cover the outlet end 612 of the emitter column 604 and act to protect it against damage. A part of the main body 626 of the protective sleeve 624 protrudes outside the sheath 630 and thereby covers the emitter. The extent of travel of the sleeve 624 out of the sheath 630 is restricted by a reduced internal diameter at the end of the sheath 630 that stops the wider diameter base 628 of the protective sleeve 624. If a force is applied to the protective sleeve 624 to push the protective sleeve 624 backwards into the sheath 630, the spring 642 becomes compressed and the tip of the emitter becomes exposed and ready for use.

[0097] The plastic molding 616 covers the emitter system 600 with the electrically conductive sheath 630 mounted at the front end and the protective sleeve 624 protecting the outlet end 612 of the emitter column 604. It will be appreciated from the description that the whole emitter system 600 is thus formed as a type of cartridge for use with an instrument, e.g. mass spectrometer. An emitter cap 644 can cover the electrically conductive sheath when not in use.

[0098] In order to meet the most relevant requirements in relation to the operation of the column, the integrated separation column may be equipped with one or more embedded components of: a heating and / or cooling element and a thermal sensor in close proximity or contact with the column and preferably embedded in the plastic material.

[0099] For optimum performance, channels for gas flow may also be embedded in the plastic material; the outlet of these channels being in close proximity with the apex (tip) of the emitter, whereby gas leaving the outlet assists in the desolvation of the spray cloud.

[0100] In order to facilitate identification of the integrated column, the embedded components may further comprise an identification tag, such as a radio frequency identification tag (RFID) embedded in the plastic material.

[0101] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0102] Further, in describing various embodiments, the specification may have presented a method and / or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed tothe method and / or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments.EXAMPLES

[0103] Performance of a conductive PEEK modified column was compared to the current standard column for separating standard NIST monoclonal antibody digests. The separation was conducted using Vanquish Neo LC coupled with Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer.

[0104] The PENNY peptide, rich for its negative charge amino acids attaching to metal surface, was selected to assess the advantages of the conductive PEEK modification. The retention times (RT) of PENNY peptide at a +3 charge state were analyzed via extracted ion chromatogram (EIC). Results depicted in Figure 7 illustrate the notably stable RTs observed with the conductive PEEK modified column, contrasting with the less stable RTs observed with the current product, with a coefficient of variation of RT at 0.23%. This stability in RTs is attributed to the mitigated attraction between PENNY peptides and conductive PEEK, facilitating the concentrated elution of PENNY peptide.

[0105] Furthermore, the full width half height (FWHM) (FIG. 8A) and peak areas (FIG. 8B) of PENNY peptide (3+) were compared, revealing a slightly larger peak width in the conductive PEEK modified column, likely due to the increased detection of analytes reflected in the enhanced peak areas. Evaluation of sequence coverage, depicted in Figure 9A for heavy chain and FIG. 9B for light chain, demonstrated comparable results between the two column types, indicating that the replacement with conductive PEEK has no discernible impact on the total number of identified peptides.

[0106] In summary, the conductive PEEK modified column exhibits more stable RTs and comparable sequence coverage across replicates, along with enhanced recovery of peptides of interest.

[0107] Beyond the analysis of single protein digested peptides, we expanded our investigation to assess the efficacy of conduction PEEK modification in proteomics applications. As illustrated in FIG. 9, varying ionization voltages were explored, revealing that the conductive PEEK modified column yielded a higher number of identified protein groups and peptide groups across a range of voltages (1.9 kV to 2.5 kV). Consistent with findings regarding the PENNY peptide, the overall median FWHM from the conductive PEEK column was slightly larger than that from the current design.

Claims

WHA T IS CLAIMED IS:

1. An integrated system for liquid separation and electrospray ionization, comprising: an emitter capillary; an LC column; a conductive sheath; a bio-inert conductive union forming a fluidic connection between the emitter capillary and the LC column, the bio-inert conductive union electrically coupled to the conductive sheath, the bio-inert conductive union facilitating the application of high voltage to a mobile phase flowing from the LC column to the emitter capillary; and an insulating plastic material encapsulating the LC column.

2. The integrated system for liquid separation and electrospray ionization of claim 1, wherein the bio-inert conductive union ensures a near zero dead-volume connection to the emitter.

3. The integrated system for liquid separation and electrospray ionization of claim 1 , wherein the bio-inert conductive union prevents sample loss due to metal interactions and inhibiting the formation of metal oxidations within the liquid junction.

4. The integrated system for liquid separation and electrospray ionization of claim 1, wherein the bio-inert conductive union includes a conductive PEEK material.

5. The integrated system for liquid separation and electrospray ionization of claim 4, wherein the conductive PEEK material includes PEEK doped with carbon.

6. The integrated system for liquid separation and electrospray ionization of claim 5, wherein the carbon includes carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof7. The integrated system for liquid separation and electrospray ionization of claim 1, wherein the liquid junction formed by the conductive PEEK material ensures the direct application of high voltage to the liquid prior to entering the emitter tip inlet.

8. The integrated system for liquid separation and electrospray ionization of claim 1, wherein the insulating plastic material further encapsulates a temperature control board or an identification tag.

9. An integrated system for liquid separation and electrospray ionization, comprising: combined emitter and column capillary; a conductive sheath; a bio-inert conductive union forming a fluidic connection between the combined emitter and column capillary and upstream LC components, the bio-inert conductive union electrically coupled to the conductive sheath, the bio-inert conductive union facilitating the application of high voltage to a mobile phase flowing into the combined emitter and column capillary; and an insulating plastic material encapsulating the LC column.

10. The integrated system for liquid separation and electrospray ionization of claim 9, wherein the bio-inert conductive union ensures a near zero dead-volume connection to the emitter.

11. The integrated system for liquid separation and electrospray ionization of claim 9, wherein the bio-inert conductive union prevents sample loss due to metal interactions and inhibiting the formation of metal oxidations within the liquid junction.

12. The integrated system for liquid separation and electrospray ionization of claim 9, wherein the bio-inert conductive union includes a conductive PEEK material.

13. The integrated system for liquid separation and electrospray ionization of claim 12, wherein the conductive PEEK material includes PEEK doped with carbon.

14. The integrated system for liquid separation and electrospray ionization of claim 13, wherein the carbon includes carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof15. The integrated system for liquid separation and electrospray ionization of claim 9, wherein the liquid junction formed by the conductive PEEK material ensures the direct application of high voltage to the liquid prior to entering the emitter tip inlet.

16. The integrated system for liquid separation and electrospray ionization of claim 9, wherein the insulating plastic material further encapsulates a temperature control board or an identification tag.

17. A method of separation comprising: a) flowing a sample through an LC column and an electrospray emitter; b) utilizing a bio-inert conductive union to form a liquid junction, ensuring a near zero dead-volume connection; c) employing the bio-inert conductive union to apply high voltage to the mobile phase upstream of the emitter.

18. The method of claim 17, wherein upstream of the emitter includes at the inlet to a combined emitter and column capillary or at the union between a column capillary and an emitter capillary.

19. The method of claim 17, wherein the bio-inert conductive union includes a conductive PEEK material.

20. The method of claim 19, wherein the conductive PEEK material includes PEEK doped with carbon.

21. The method of claim 20, wherein the carbon includes carbon nanofibers, carbon nanoparticles, graphite, or combinations thereof.

22. The method of claim 17, wherein the bio-inert conductive union reduces sample loss by minimizing interactions with metals and inhibits metal oxidations within the liquid junction, facilitating efficient voltage application.

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