Patent Application
20230005731
Advancements in ionization interfaces for coupling capillary electrophoresis (CE) with electrospray ionization (ESI) mass spectrometry (MS) have enabled applications of CE-ESI-MS for various chemical and biochemical analyses. Existing CE-ESI-MS uses electrospray interfaces that may be broadly categorized as either sheath flow (U.S. Pat. Nos. 5,993,633; 9,465,014; 9,234,880; and 8,613,845) or sheathless (U.S. Pat. Nos. 9,927,396; 8,754,370; 6,863,790; 10,121,645; and 5,505,832).
Sheath flow interfaces typically utilize a coaxial conducting liquid to provide electrical contact for the electrophoretic separation, modify the separation medium to be more MS-compatible, and generate electrospray for MS detection. The f sheath flow interface was developed by Smith's group and was commercialized in the 1990s (U.S. Pat. No. 5,993,633). Since then other versions of the sheath flow interface have been developed. Notably, Dovichi's group developed a sheath flow interface that uses electroosmotic nanoflow to drive the electrospray (U.S. Pat. Nos. 9,465,014 and 9,234,880). In that design, the spray emitter is a borosilicate glass pulled at the distal end to create a micro nozzle, typically with a 10 to 30 μm inner diameter. The separation capillary is inserted into the emitter filled with an MS-compatible conducting liquid supplied from the conducting liquid reservoir through a conducting liquid channel. The high ESI voltage driving the electroosmotic flow inside the emitter is delivered to the conducting liquid reservoir. While this configuration has been shown to provide good sensitivity for multiple analytes the design still has some problems. The emitter's inner diameter of 0.75 mm introduces a relatively high sheath liquid to sample dilution.
Another notable version of sheath flow interface was developed by Chen's group (U.S. Pat. No. 8,613,845). That design uses a stainless steel hollow needle with a beveled tip. The needle acts as an electrode for the CE outlet and the spray emitter for MS. Although the steel needle interface is more rugged than tapered glass interfaces, the design is typically, used with the electrospray voltage delivered directly, which often leads to bubble formation and corona discharge due to redox reaction on the metal surface. This usually limits the electrospray performance. Further, metal emitters need a mechanical pump-driven flow to maintain a stable electrospray. This requirement creates a higher flow rate than the electrokinetically pumped interface described above, which then limits its sensitivity owing to higher dilution of the analyte by the conducting liquid.
In sheathless interface designs, the separation capillary commonly serves as the emitter which eliminates sample dilution associated with sheath flow interfaces. A notable design was developed by Moini's group (U.S. Pat. No. 6,863,790) and was recently commercialized. The interface used a porous capillary to provide electrical contact to the separation buffer without the introduction of a conducting liquid. The distal end of the separation capillary is etched to a thin porous thickness, sufficiently thin to be conductive. It is placed within a metal sleeve needle filled with a conductive liquid. ESI voltage is then applied to the metal needle to drive electrospray at the capillary tip. Though the interface provides better sensitivity over sheath flow interfaces, the etched capillary is extremely fragile. Under high voltage, deterioration of the porous tip leads to degraded electrospray and decreased sensitivity. Another major drawback is insufficient flow to drive electrospray when performing separation with negligible or reversed electro-osmotic flow. The electrophoresis buffer is also the electrospray liquid, which limits the allowable separation conditions.
Due to the electrospray ionization efficiency dependence of MS detection, quantitation in CE-ESI-MS may benefit from coupling optical detection with the electrospray interface. A few attempts have been made so far to achieve this with marginal success. However, integrating optical detection with ESI in CE-ESI-MS is still a challenge due to the complexity of integrating relatively large optical components with an electrospray interface.
In existing sheath flow ESI interfaces, the use of a conducting liquid offers flexibility in separation buffer composition. In contrast, a sheathless interface may use the separation buffer without the introduction of a conducting liquid to generate electrospray. A sheathless interface has the benefit of high sensitivity due to the absence of sheath flow dilution.
An important requirement for a CE-ESI-MS interface is the stability of the electrospray. Achieving stable electrospray at a nanoliter-per-minute flow rate is a challenge with existing technologies. There is, therefore, a need for a CE-ESI-MS interface with improved electrospray stability. There is further a need for such a solution to incorporate optical detection capabilities to incorporate the benefits of such additional testing capabilities.
The disclosed solution comprises an injection subassembly that includes a nicked alignment tube, a spray needle, and a conducting liquid tube. The nicked alignment tube has a nick near one end. The spray needle is fused within the nicked end of the nicked alignment tube with an entry end of the spray needle positioned alongside the nick and an exit end of the spray needle extending out of the nicked end. The fused spray needle and nicked alignment tube are inserted into the conducting liquid tube, where the nicked alignment tube aligns the spray needle coaxially within the conducting liquid tube. The nick and the entry end of the spray needle are positioned within the conducting liquid tube and the exit end of the spray needle extends out of the conducting liquid tube. The spray needle may be a sheath flow spray needle or a sheathless spray needle. The nicked alignment tube and the conducting liquid tube fit is fluid-tight. The nick allows a conducting liquid to flow from the conducting liquid tube to within the nicked alignment tube and the spray needle.
The disclosed solution further comprises an ESI system that includes the injection subassembly disclosed above. The ESI system also includes a junction fitting with at least a first port, a second port, and a third port coaxial with the first port. The injection subassembly is connected to the third port and the conducting liquid tube and the third port fit is fluid-tight. The ESI system also includes a capillary which may be a sheath flow capillary and a sheathless capillary. The capillary runs through the first port and the third port of the junction fitting into the injection subassembly, where the nicked alignment tube aligns the capillary coaxially within the conducting liquid tube. The capillary and the nicked alignment tube fit is fluid-tight, and the capillary and the first port fit is fluid-tight. If the capillary is a sheath flow capillary, a narrow end of the sheath flow capillary extends into the sheath flow spray needle from the entry end to the exit end such that the conducting liquid and an analyte species flowing in the sheath flow capillary are mixed at the exit end of the sheath flow spray needle. If the capillary is a sheathless capillary, an adjustable gap is configured between the sheathless capillary and the entry end of the sheathless spray needle such that the conducting liquid and the analyte species within the sheathless capillary are mixed at the entry end of the sheathless spray needle. The ESI system also includes a conducting liquid reservoir holding the conducting liquid and configured with a conducting liquid channel. The conducting liquid channel is connected to the second port of the junction fitting and the conducting liquid channel, and the second port fit is fluid-tight. The ESI system further includes a high voltage source configured to electrically charge the conducting liquid within the conducting liquid reservoir. In one aspect, the ESI system further includes an optical manifold. The optical manifold includes at least one light source, at least one input-output optical port, and at least one light detector. The capillary extends from the first port of the junction fitting through the optical manifold. The ESI system and optical manifold together form a unitary optical-ESI system.
Finally, the disclosed solution includes a method for using the ESI system and unitary optical-ESI system equipped with the injection subassembly. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.
To easily identify the discussion of any particular element or act, the most significant digit or digits in a reference number refer to the figure number in which that element is first introduced.
Disclosed is a unitary optical-ESI system that generates electrospray with a spray-needle-fused, nicked alignment tube embedded in a conducting liquid tube to form an injection subassembly that may allow users to choose between sheath flow or sheathless ESI configurations based on what is most suitable for a specific application. Conventional ESI interfaces for use in CE-ESI-MS may be expensive, and conventional interface designs do not permit both sheath flow and sheathless ESI configurations.
In one embodiment, the disclosed solution integrates inline optical detection with ESI to enable orthogonal detection such as ultraviolet detection, infrared detection, laser-induced fluorescence detection, thermo-optical detection, scattering, and Raman detection, simultaneously with MS detection in nanoflow liquid separations.
The unitary optical-ESI system 100 may also include a capillary 136 configured to contain an analyte species 138. The capillary 136 may be a sheath flow capillary or a sheathless capillary. The ESI system 102 may include flow restriction valves 140 preventing flow of the conducting liquid 132 out of the first port 122 and the second port 124.
In addition to the ESI system 102, the unitary optical-ESI system 100 may further include an optical manifold 142. The optical manifold 142 may include at least one input-output optical port 144, one of which is shown here. The optical manifold 142 may further comprise the light source 202 and light detector 204 illustrated in
The injection subassembly 104 may comprise a nicked alignment tube 106 having a nick 112 near one end. The nicked alignment tube may be manufactured from at least one of polymer, metal, plastic, and ceramics. The nick may be created by at least one of micro dicing, laser cut, and mechanical excision of materials from the nicked alignment tube.
A spray needle 108 may be fused within the nicked end 114 of the nicked alignment tube 106. An entry end 116 of the spray needle 108 may be positioned alongside the nick 112 and an exit end 118 of the spray needle 108 may extend out of the nicked end 114. The spray needle may be manufactured from at least one of polymer, glass, metal, and ceramics. The tapered end of the spray needle may be formed by thermal pulling or grinding.
The fused nicked alignment tube 106 and spray needle 108 may be inserted into the conducting liquid tube 110. The conducting liquid tube may be manufactured from polymer using an extrusion tubing process.
The nicked alignment tube 106 may align the spray needle 108 coaxially within the conducting liquid tube 110. The nick 112 and the entry end 116 of the spray needle 108 may be positioned within the conducting liquid tube 110 while the exit end 118 of the spray needle 108 may extend out of the conducting liquid tube 110. The nicked alignment tube 106 may fit into the conducting liquid tube 110 such that their fit is fluid-tight. The nick 112 of the nicked alignment tube 106 allows a conducting liquid 132 to flow from the conducting liquid tube 110 to within the nicked alignment tube 106 and the spray needle 108, as shown.
The ESI system 102 of the unitary optical-ESI system 100 may further include a junction fitting 120 having at least a first port 122, a second port 124, and a third port 126 coaxial with the first port 122. The junction fitting 120 may in one embodiment be a polyether ether ketone (PEEK) tee fitting. The injection subassembly 104 may be connected to the third port 126 as illustrated. The conducting liquid tube 110 and the third port 126 may fit together in a fluid-tight manner.
A capillary 136, either a sheath flow capillary or a sheathless capillary, may be inserted through the first port 122 and the third port 126 of the junction fitting 120 and into the injection subassembly 104. The nicked alignment tube 106 may align the capillary 136 coaxially within the conducting liquid tube 110. The fit between the capillary 136 and the nicked alignment tube 106 may be fluid-tight. The capillary 136 and the first port 122 fit may also be fluid-tight.
The ESI system 102 of the unitary optical-ESI system 100 may include a conducting liquid reservoir 130 holding the conducting liquid 132 and configured with a conducting liquid channel 128. The conducting liquid channel 128 may be connected to the second port 124 of the junction fitting 120. The fit between the conducting liquid channel 128 and the second port 124 may be fluid-tight
A high voltage source 134 may be configured to electrically charge the conducting liquid 132 within the conducting liquid reservoir 130 when it is activated. The voltage provided by the high voltage source 134 may be of positive or negative polarity. In one embodiment, a voltage of 2 kV may be used. The charge differential induced across the conducting liquid 132 by the activation of the high voltage source 134 may cause the conducting liquid 132 to flow from the conducting liquid reservoir 130 through the conducting liquid channel 128 to the junction fitting 120 and into the conducting liquid tube 110. This may further induce movement and separation of the components of the analyte species 138 within the capillary 136 through the polarized or ionized nature of its constituents, including, in some embodiments, a separation buffer contained therein. For example, the separation buffer and/or the conductive liquid may be an organic-aqueous mixture containing acetic acid or formic acid.
The ESI system 102 may further include one or more flow restriction valves 140. These flow restriction valves 140 may be used to create the fluid-tight fits between the first port 122 and the capillary 136, as well as between the second port 124 and the conducting liquid channel 128. These flow restriction valves may be compression fittings, plugs, or ferrules manufactured from polymers, rubber, or adhesives.
The capillary 136 may extend from the first port 122 through an optical manifold 142 as illustrated. The ESI system 102 and the optical manifold 142 and its components, as illustrated in and described with respect to
A capillary 136 containing an analyte species 138, as described with respect to
The light sources 202 of the optical manifold 142 may be configured to produce light 206 having a wavelength on the electromagnetic spectrum suitable for use in at least one of ultraviolet detection, infrared detection, laser-induced fluorescence (LIF) detection, thermo-optical detection, scattering, and Raman detection. The light detectors 204 may be devices suitable to detect these energies. In one embodiment, at least one of photodiode array (PDA), charge-couple device (CCD), or a photon counting device may be used as a light detector 204. A gradient-index (GRIN) rod may be used to transmit the transformed light to the detector 204.
The narrow end 304 of the sheath flow capillary 302 may extend into the sheath flow spray needle 306, from its entry end 116 to its exit end 118. The nick 112 may allow the conducting liquid 132 to enter the nicked alignment tube 106 and the sheath flow spray needle 306, but because of the extension of the narrow end 304 into the sheath flow spray needle 306, the conducting liquid 132 may be prevented from mixing with the analyte species 138 before both substances are ejected as nanospray at the exit end 118 of the sheath flow spray needle 306 for analysis by a mass spectrometer 146 or similar equipment.
In a sheathless ESI configuration 400, the sheathless capillary 402 may not include a narrow end 304 meant to extend into the sheath flow spray needle 306 as shown in
Through this adjustable gap 408, the conducting liquid 132 may be in contact with the analyte species 138 within the sheathless capillary 402 as the analyte species 138 transits the gap between the meniscal tapered end 404 of the sheathless capillary 402 and the entry end 116 of the sheathless spray needle 406, and this mixture may be ejected as nanospray at the exit end 118 of the sheathless spray needle 406. In one embodiment, the adjustable gap 408 may be variable, such that setting it to a certain dimension permits a specific amount of conducting liquid 132 to be in contact with the analyte species 138. In this manner, the percentage of the conducting liquid 132 in the resulting nanospray may be selectably varied.
In some embodiments, the meniscal tapered end 404 may be configured to immediately abut the entry end 116 of the sheathless spray needle 406 with a fluid-tight fit when pressed far enough into the injection subassembly 104. In these embodiments, the adjustable gap 408 may be completely closed, such that no conducting liquid 132 is mixed with the analyte species 138. Thus the analyte species 138 alone, unmixed with conducting liquid 132 may be propelled as nanospray from the sheathless spray needle 406.
In this manner the disclosed unitary optical-ESI system 100 may take advantage of the flexibility of the injection subassembly 104 to perform a wider variety of optical, CE-ESI-MS, and similar testing without needing to purchase and house multiple largely redundant versions of expensive test equipment.
The flat substrate 502 may be manufactured from polymer, glass, plastic, ceramic, or similarly rigid materials, as are appropriate for the application in which it is utilized. The flat substrate 502 may be configured to support a capillary 136 carrying an analyte species 138 as shown. While the configuration illustrated is a sheath flow ESI configuration similar to that shown in
The flat substrate injection subassembly 500 may support miniaturization of the ESI system 102 previously described. In this manner, the flat substrate injection subassembly 500 may provide a small form-factor test sample collection device that may be used in mobile testing apparatus comparable in size to a mobile telephone or small tablet computer.
In block 604, a capillary may be inserted through the first port and the third port of the junction fitting and into the injection subassembly. The nicked alignment tube may align the capillary coaxially within the conducting liquid tube. The fits between the capillary and the first port of the junction fitting and the capillary and the nicked alignment tube may be fluid tight. The capillary may be either a sheath flow capillary or a sheathless capillary.
In block 606, a conducting liquid channel may be inserted into the second port of the junction fitting. The conducting liquid channel may be configured to convey conducting liquid from a conducting liquid reservoir. The conducting liquid channel and the second port fit may be fluid-tight.
In block 608, the free end of the capillary extending from the first port of the junction fitting may be inserted through an optical manifold, such as was introduced in
In block 614, an electric field from a high voltage source may be applied to the conducting liquid in the conducting liquid reservoir and to the inlet of the capillary. The application of this voltage may induce a charge differential in the conducting liquid, causing it to flow and to potentially propel the analyte species through the injection subassembly, to generate a nanospray at the exit end of the spray needle. This nanospray may be injected into analysis equipment such as mass spectrometers and similar devices.
In block 616, routine 600 light from at least one light source may be applied to the capillary within the optical manifold as described with respect to
The UV electropherogram results 700a for replications 702-708 are displayed with the x-axis measuring migration time 710 in minutes, and the y-axis measuring UV absorbance 712. The MS total ion electropherogram results 700b for replications 702-708 are displayed with the x-axis measuring migration time 710 and the y-axis measuring relative intensity 720. In each electropherogram, for each replication, peaks may be seen corresponding to the lysozyme 714, bovine serum albumin 716, and myoglobin 718 in the analyte species introduced for analysis.
The UV electropherogram results 700a and MS total ion electropherogram results 700b were obtained during a proof-of-concept study to demonstrate the improvements represented by a unitary optical-ESI system configured with an injection subassembly as disclosed herein. The unitary optical-ESI system was a unitary optical-ESI system 100 such as was introduced in
Unless otherwise specified, below all reagents were purchased from Sigma Aldrich Co, St. Louis, Mo. Experiments were performed on a CeMAX UV™ (GMJ Technologies, Inc., Seattle Wash.) equipped with a cartridge comprising a capillary and the optical-ESI interfaces disclosed herein. The capillary was a 50 mm i.d.×360 mm o.d.×100 cm long polyacrylamide coated silica capillary (GMJ Technologies, Inc., Seattle Wash.).
For all experiments, the separation buffer was 5% acetic acid, and the electrospray conductive liquid was 0.1% formic acid in 10% methanol. For each analysis, the separation capillary was conditioned with 0.1 M hydrochloric acid, followed by a water rinse, then the separation buffer. Each conditioning step was performed with 20 psi pressure for 5 minutes. For protein analysis, the sample was a mixture of standard proteins containing lysozyme 714, bovine serum albumin 716 (BSA), and myoglobin 718 diluted in distilled de-ionized water at 1 mg/mL concentration of each protein.
Samples were hydrodynamically injected at 5 psi for 5 seconds. In all experiments, the spray needle outlet was placed about 3 mm away from the mass spectrometer inlet. All experiments were performed with a Thermo Velos-Orbitrap mass spectrometer in positive mode using the following settings:
capillary voltage: 38 V,
capillary temperature: 275° C.,
tube lens: 200,
maximum injection: 350 ms, and
m/z range: 200 to 2000.
The CE separation voltage was +20 kV and the ESI voltage was set at +2.0 kV. UV detection was performed at 200 to 400 nm with 100 ms integration time.
As may be seen, both the UV electropherogram results 700a and MS total ion electropherogram results 700b for an ESI system using an injection subassembly disclosed herein show similar peak profiles, a base peak width of approximately 30 seconds, good sensitivity, and repeatability, as will be appreciated by one of ordinary skill in the art.
The x-axis of each graph measures migration time 908 in minutes. The y-axis shows the measured intensity 910 across the migration time 908 during testing according to the experimental conditions described with respect to
One drawback to use of conventional configurations such as those illustrated in
In comparison, an ESI system such as the ESI system 102 illustrated in
Within this disclosure, different entities (which may variously be referred to as “units,” “circuits,” other components, etc.) may be described or claimed as “configured” to perform one or more tasks or operations. This formulation—[entity] configured to [perform one or more tasks]—is used herein to refer to structure (i.e., something physical, such as an electronic circuit). More specifically, this formulation is used to indicate that this structure is arranged to perform the one or more tasks during operation. A structure may be said to be “configured to” perform some task even if the structure is not currently being operated. A “credit distribution circuit configured to distribute credits to a plurality of processor cores” is intended to cover, for example, an integrated circuit that has circuitry that performs this function during operation, even if the integrated circuit in question is not currently being used (e.g., a power supply is not connected to it). Thus, an entity described or recited as “configured to” perform some task refers to something physical, such as a device, circuit, memory storing program instructions executable to implement the task, etc. This phrase is not used herein to refer to something intangible.
The term “configured to” is not intended to mean “configurable to.” An unprogrammed FPGA, for example, would not be considered to be “configured to” perform some specific function, although it may be “configurable to” perform that function after programming.
Reciting in the appended claims that a structure is “configured to” perform one or more tasks is expressly intended not to invoke 35 U.S.C. § 112(f) for that claim element. Accordingly, claims in this application that do not otherwise include the “means for” [performing a function] construct should not be interpreted under 35 U.S.C § 112(f).
As used herein, the term “based on” is used to describe one or more factors that affect a determination. This term does not foreclose the possibility that additional factors may affect the determination. That is, a determination may be solely based on specified factors or based on the specified factors as well as other, unspecified factors. Consider the phrase “determine A based on B.” This phrase specifies that B is a factor that is used to determine A or that affects the determination of A. This phrase does not foreclose that the determination of A may also be based on some other factor, such as C. This phrase is also intended to cover an embodiment in which A is determined based solely on B. As used herein, the phrase “based on” is synonymous with the phrase “based at least in part on.”
As used herein, the phrase “in response to” describes one or more factors that trigger an effect. This phrase does not foreclose the possibility that additional factors may affect or otherwise trigger the effect. That is, an effect may be solely in response to those factors, or may be in response to the specified factors as well as other, unspecified factors. Consider the phrase “perform A in response to B.” This phrase specifies that B is a factor that triggers the performance of A. This phrase does not foreclose that performing A may also be in response to some other factor, such as C. This phrase is also intended to cover an embodiment in which A is performed solely in response to B.
As used herein, the terms “first,” “second,” etc. are used as labels for nouns that they precede, and do not imply any type of ordering (e.g., spatial, temporal, logical, etc.), unless stated otherwise. For example, in a register file having eight registers, the terms “first register” and “second register” may be used to refer to any two of the eight registers, and not, for example, just logical registers 0 and 1.
When used in the claims, the term “or” is used as an inclusive or and not as an exclusive or. For example, the phrase “at least one of x, y, or z” means any one of x, y, and z, as well as any combination thereof.
This application claims the benefit of U.S. provisional patent application Ser. No. 63/217,381, filed on Jul. 1, 2021, the contents of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63217381 | Jul 2021 | US |
