Mass-targeted fractionation system with asynchronous dual-dimension separation

Abstract

A fractional collector system for use in a mass spectrometry system includes a sample input, a first HPLC column, a first valve, a second valve, a second HPLC column, and a controller. The first valve is configured to receive a fluid sample from the first HPLC column and provide the fluid sample to a vessel. The second valve is configured to collect the fluid sample from the vessel. The controller operates the first valve to dispense the fluid sample to the vessel during detection of an analyte, and operates the second valve to collect the fluid sample from the vessel, thereby trapping the fluid sample prior to providing it to the second HPLC column. The first and second valves are operated asynchronously. The fractional collector system may include first and second effector arms supporting dispensing and aspirating needles, respectively, which are operated asynchronously.

Description

MASS-TARGETED FRACTIONATION SYSTEM WITH ASYNCHRONOUSDUAL-DIMENSION SEPARATIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to and all the benefits of U.S. Provisional Patent Application No. 63 / 682,917, filed on August 14, 2024, which is hereby expressly incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure generally relates to mass spectrometry systems, and in particular, to systems and methods for mass-targeted fractionation with asynchronous dualdimension separation.BACKGROUND

[0003] Mass spectrometry is a powerful analytical technique widely used in various fields, including chemistry, biology, and pharmaceutical research. It allows for the identification and quantification of molecules based on their mass-to-charge ratio. However, the complexity of biological samples often necessitates additional separation techniques to be coupled with mass spectrometry for improved analysis.

[0004] High-performance liquid chromatography (HPLC) is frequently combined with mass spectrometry to separate complex mixtures prior to mass analysis. This combination, known as LC-MS, has become an essential tool in many analytical laboratories. LC-MS systems can provide detailed information about the composition of complex samples, but they often face challenges in isolating specific compounds of interest from these mixtures.

[0005] Fraction collection is a technique used to isolate specific components from a mixture based on their retention times or other properties. Traditional fraction collectors typically operate in a single dimension, collecting fractions from a single chromatographic separation. While useful, this approach may not provide sufficient separation for highly complex samples or may otherwise be time consuming.

[0006] The integration of multiple separation techniques, such as two-dimensional liquid chromatography (2D-LC), has emerged as a powerful method to enhance the resolution and peak capacity of separations. However, implementing 2D-LC systems with mass-directed fraction collection can be challenging due to the complexity of coordinating multiple separation dimensions with mass spectrometric detection and fraction collection.

[0007] There is a growing need for advanced analytical systems that can perform targeted isolation of compounds from complex mixtures with high specificity and efficiency. Such systems could potentially improve workflows in drug discovery, metabolomics, proteomics, and other fields requiring the isolation and characterization of specific molecules from complex biological samples.BRIEF SUMMARY

[0008] According to an aspect of the present disclosure, a fractional collector system for use in a mass spectrometry system including at least one detector for detecting an analyte is provided. The fractional collector system also includes a sample input configured to receive a fluid sample comprising an analyte. The fractional collector system further includes a first HPLC column configured to fluidly connect with the sample input to receive the fluid from the sample input. The fractional collector system further includes a first valve configured to fluidly connect with the first HPLC column, wherein the first valve is configured to receive the fluid sample and provide the fluid sample to a vessel. The fractional collector system further includes a second valve configured to collect the fluid sample from the vessel. The fractional collector system further includes a second HPLC column configured to fluidly connect with the second valve to receive the fluid sample from the second valve. The fractional collector system further includes a controller configured to (i) operate the first valve to dispense the fluid sample to the vessel during detection of the analyte, and (ii) operate the second valve to collect the fluid sample from the vessel thereby trapping the fluid sample prior to providing the fluid samples to the second HPLC column. The first and second valves are operated asynchronously.

[0009] According to other aspects of the present disclosure, the fractional collector system may include one or more of the following features. The fractional collector system may include a first effector arm supporting a dispensing needle configured to fluidly connect with the first valve,wherein the dispensing needle is configured to receive the fluid and dispense the fluid to the vessel, and a second effector arm supporting an aspirating needle configured to fluidly connect with the second valve, wherein the aspirating needle is configured to collect the sample from the vessel, wherein the first and second effector arms are operated asynchronously. The first and second effector arms may be independently belt-driven along the X-axis and actuated along the Y-axis with a screw gear. The first and second effector arms may be directed along the Z-axis with a pulley movably disposed along a splined shaft. The controller may be configured to position the dispensing needle of the second effector arm and the aspirating needle of the first effector arm within a 20 millimeter (mm) distance of each other along an XY plane. The fractional collector system may include a housing, wherein the first effector arm and the second effector arm are located within the housing. The fluid sample may comprise a first partial fluid sample, and wherein the first HPLC column may be configured to receive the fluid sample and elute the first partial fluid sample to the first valve. The first valve may be configured to receive the first partial fluid sample and provide the first partial fluid sample to the vessel. The second valve may be configured to collect the first partial fluid sample from the vessel and provide the first partial fluid sample to the second HPLC column. The first partial fluid sample may comprise the second partial fluid sample, and wherein the second HPLC column may be configured to receive the first partial fluid sample from the first valve, and elute the second partial fluid sample. The fractional collector system may include a plurality of traps configured to fluidly connect to the second valve to receive the fluid sample from the second valve and trap the fluid sample. The fluid sample may comprise a first partial fluid sample, and wherein the second valve may be configured to receive the first partial fluid sample and provide the first partial fluid sample to the plurality of traps. The first partial fluid sample may comprise the second partial fluid sample, and wherein the second HPLC column may be configured to (i) fluidly connect with the plurality of traps to receive the first partial fluid sample from the plurality of traps, and (2) elute the second partial fluid sample. The fractional collector system may include a third valve configured to connect with the second HPLC column and receive the sample fluid from the second HPLC column. The fluid sample may comprise a first partial fluid sample and the first partial fluid sample may comprise the second partial fluid sample, and wherein the third valve may be configured to receive the second partial fluid sample from the second HPLC column. The fluid sample may comprise a plurality of partial fluid samples.

[0010] According to another aspect of the present disclosure, a mass spectrometry system is provided. The mass spectrometry system includes a first flow path extending through a first HPLC column for directing a fluid sample with a detector connected to one end of the first HPLC column. The mass spectrometry system includes a trap configured to retain a first partial fluid sample of the fluid sample in response to a signal output from the detector that exceeds a threshold. The mass spectrometry system includes a second flow path extending through a second HPLC column for directing the first partial fluid sample. The mass spectrometry system includes a controller configured to apply a first solvent gradient along the first flow path based on a preset method parameter to cany the fluid sample through the first HPLC column, and apply a variable second solvent gradient to carry the portion of the fluid from the trap along the second flow path. The controller is configured to automatically generate the second variable gradient based on the gradient associated with a retention time corresponding to the signal received from the detector above the threshold.

[0011] According to other aspects of the present disclosure, the mass spectrometry system may include one or more of the following features. The controller may be configured to automatically generate the second variable gradient based on the gradient associated with the retention time corresponding to the signal received from the detector, a mass of a detected analyte within the fluid, and a duration of the signal received from the detector above the threshold. The detector may be connected to one end of the second HPLC column.

[0012] According to another aspect of the present disclosure, a mass spectrometry system is provided. The mass spectrometry system includes a fractional collector system comprising a first HPLC column configured to fluidly connect with a first valve, a first effector arm supporting a dispensing needle configured to fluidly connect with the first HPLC column by first valve, a second effector arm supporting an aspirating needle configured to fluidly connect with a second valve, at least one trap configured to fluidly connect with the aspirating needle by the second valve, and a second HPLC column configured to fluidly connect with the at least one trap. The mass spectrometry system includes a first flow path extending through the first HPLC column for directing a fluid sample with a detector connected to one end of the first HPLC column. The mass spectrometry system includes a second flow path extending through the second HPLC column for directing a first partial fluid sample with the detector connected to one end of the second HPLCcolumn. The mass spectrometry system includes a controller configured to (i) apply a first solvent gradient along the first flow path based on a preset method parameter to cany the fluid sample through the first HPLC column, (ii) determine if a signal received from the detector is above a threshold, (iii) operate the first effector arm to provide fluid sample from the first valve to a vessel upon a determination that the signal is above the threshold, (iv) operate the second effector arm to collect the fluid sample from the vessel and provide the fluid to the trap, and (v) apply a second variable solvent gradient to carry the portion of the fluid sample from the trap along the second flow path. The controller is configured to automatically generate the second variable solvent gradient based on the gradient associated with a retention time corresponding to the signal received from the detector above the threshold. The controller is configured to operate the first and second effector arms asynchronously.

[0013] According to another aspect of the present disclosure a fractional collector is provided. The fractional collector includes a support frame with first and second effector arms coupled to the support frame. The first effector arm supports a dispensing needle extending from a first side of the first effector arm, with the dispensing needle configured to fluidly couple with a first HPLC column. The second effector arm is coupled to the support frame in a substantially parallel orientation to the first effector arm. The second effector arm supports an aspirating needle extending from a second side of the second effector arm, with the aspirating needle configured to fluidly connect with a second HPLC column. The fractional collector further includes at least one trap configured to fluidly connect with the aspirating needle. The first side of the first effector arm faces the second side of the second effector arm, such that the dispensing needle and the aspiration needle are adjacent.

[0014] According to other aspects of the present disclosure, the fractional collector may include one or more of the following features. The fractional collector may further include a first flow path extending through the first HPLC column for directing a fluid sample, with a detector fluidly connected to one end of the first HPLC column. The fluid sample may include a first partial fluid sample and the fractional collector may further include a second flow path extending through the second HPLC column for directing the first partial fluid sample, with the detector also fluidly connected to an end of the second HPLC column. The first and second effector arms may be independently belt-driven along the X-axis and actuated along the Y-axis with ascrew gear. The first and second effector arms may also be directed along the Z-axis with a pulley movably disposed along a splined shaft. The fractional collector may further include a controller configured to position the dispensing needle of the second effector arm and the aspirating needle of the first effector arm within a 20 millimeter (mm) distance of each other along an XY plane. The fractional collector may further include a sample input configured to receive a fluid sample including an analyte, with the fluid sample including a first partial fluid sample, and with the fractional collector further including a first valve configured to fluidly connect with the first HPLC column, with the first valve configured to receive the fluid sample from the first HPLC column and provide the fluid sample to a vessel. The fractional collector may further include a second valve configured to collect the first partial fluid sample from the vessel and provide the first partial fluid sample to the second HPLC column. The first partial fluid sample may include a second partial fluid sample and the second HPLC column may be configured to receive the first partial fluid sample from the first valve, and elute the second partial fluid sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Advantages of the present disclosure will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings. Non-limiting and non-exhaustive examples are described with reference to the following figures.

[0016] FIG. 1 illustrates a block diagram of a preparative mode chromatography system, according to aspects of the present disclosure.

[0017] FIG. 2 illustrates a block diagram of a mass spectrometry system in trapping mode, according to an embodiment.

[0018] FIG. 3 illustrates a block diagram of an analytical mode for a mass spectrometry system, in accordance with example embodiments.

[0019] FIG. 4 illustrates a block diagram of a method execution system for preparative separation, according to aspects of the present disclosure.

[0020] FIG. 5 illustrates a system diagram of an analytical method execution process for a chromatography system, according to an embodiment.

[0021] FIG. 6 illustrates a system diagram of analytical flow splitters for a mass spectrometry system, in accordance with example embodiments.

[0022] FIG. 7 illustrates a system diagram of a mass spectrometry data acquisition and control system, according to aspects of the present disclosure.

[0023] FIG. 8 illustrates an isometric view of a compact dispense / draw fraction collector, according to an embodiment.

[0024] FIG. 9 illustrates a system diagram of a fraction collector for a chromatography or mass spectrometry system, in accordance with example embodiments.

[0025] FIG. 10 is a front view of a fractional collector with two effector arms with each arm supporting a needle and the needles oriented adjacent to each other.

[0026] FIG. 11 is perspective view of the fractional collector with two effector arms with each arm supporting needles oriented adjacent to each other.DETAILED DESCRIPTION

[0027] The present disclosure provides a system for mass-targeted fractionation with asynchronous dual-dimension separation. This system is designed to enhance the isolation of specific compounds from complex mixtures, leveraging the capabilities of mass spectrometry and two-dimensional liquid chromatography. The mass-targeted fractionation system includes a fractional collector system that is integrated with a mass spectrometry system and includes a sample input, first and second high-performance liquid chromatography (HPLC) columns, first and second valves, and a controller. The controller operates the valves asynchronously, allowing for efficient and precise fraction collection. The system also includes effector arms that support dispensing and aspirating needles, which are operated asynchronously to facilitate the collection and dispensing of fluid samples. The system further includes a plurality of traps for retaining fluid samples, and a second HPLC column for directing the fluid samples. The controller is configured to apply solvent gradients along the flow paths and to automatically generate variable solvent gradients based on the gradient associated with a retention time corresponding to a signal received from a detector. This mass-targeted fractionation system offers the potential for improvedworkflows in various fields requiring the isolation and characterization of specific molecules from complex biological samples.

[0028] Referring to a fractional collector system 100 best shown in the system diagram of FIG. 1, the fractional collector system 100 for a mass spectrometry system may include several key components. In some aspects, the fractional collector system 100 may include at least two HPCL columns. In certain aspects, the fractional collector system 100 may include up to six high- performance liquid chromatography (HPLC) modules, such as modules from Knauer. These HPLC modules may include two isocratic pumps 110, one analytical flow quaternary gradient pump 120, one preparative scale binary gradient pump 130, an autosampler 140, and a multi-wavelength UV- VIS detector 150. The isocratic pumps 110 may be used to deliver a constant flow of a single solvent or a constant composition of multiple solvents to the HPLC modules. The analytical flow quaternary gradient pump 120 may be used to deliver a varying composition of up to four solvents to the HPLC modules. The preparative scale binary gradient pump 130 may be used to deliver a varying composition of two solvents to the HPLC modules at a flow rate large enough such that the rate is suitable for preparative scale separations. The autosampler 140 may be used to automatically introduce samples into the HPLC system. The multi-wavelength UV-V1S detector 150 may be used to detect the presence and quantity of analytes in the eluent from the HPLC system based on their absorption of ultraviolet or visible light.

[0029] In some cases, the fractional collector system 100 may also include five external switching valves from VICI. These valves may include one 6-port, 2-position valve 160; one 10- port, 2-position valve 170; one 4-position, common outlet stream selector valve 180; and two 12- position stream selector valves 190. The 6-port, 2-position valve 160 may be used to direct the flow of fluid between six different paths in two different configurations. The 10-port, 2-position valve 170 may be used to direct the flow of fluid between ten different paths in two different configurations. The 4-position, common outlet stream selector valve 180 may be used to select one of four different fluid paths to a common outlet. The 12-position stream selector valves 190 may be used to select one of twelve different fluid paths to a common outlet. These valves may be used to control the flow of fluid through the system, allowing for complex routing and switching of fluid paths for multi-dimensional separations.

[0030] Referring to FIG. 2, the block diagram illustrates a fractional collector system 200 in trapping mode, which may include several key components. In some aspects, the fractional collector system 200 is for use with a mass spectrometry system 255 including at least one detector for detecting an analyte. The fractional collector system 200 may include a sample input 210 configured to receive a fluid sample comprising an analyte. The fluid sample may be a complex mixture of molecules, such as a biological sample, a chemical sample, or an environmental sample, among others.

[0031] In some cases, the fractional collector system 200 may include a first HPLC column 220 configured to fluidly connect with the sample input 210 to receive the fluid from the sample input 210. The first HPLC column 220 may be used for the initial separation of the components of the fluid sample based on their different affinities for the stationary phase of the column and the mobile phase flowing through the column.

[0032] The fractional collector system 200 may also include a first valve 230 configured to fluidly connect with the first HPLC column 220. The first valve 230 may be configured to receive the fluid sample and provide the fluid sample to a vessel 240. The vessel 240 may be a part of a fraction collector, a well plate, a vial, or any other suitable container for collecting fractions of the fluid sample.

[0033] In some aspects, the fractional collector system 200 may include a second valve 250 configured to collect the fluid sample from the vessel 240. The second valve 250 may be configured to aspirate the fluid sample from the vessel 240 and provide the fluid sample to a second HPLC column 260 or a trap for further processing or analysis. The second valve 250 may be operated independently of the first valve 230, allowing for asynchronous operation of the dispensing and aspiration functions.

[0034] In some aspects, the fractional collector system 200, the fluid sample may flow from the sample input 210 to the first HPLC column 220, then to the vessel 240 via the first valve 230, and finally to the second HPLC column 260 or a trap via the second valve 250. This flow path allows for sequential processing of the fluid sample, including initial separation, fraction collection, and further separation or analysis.

[0035] Referring still to FIG. 2, the fractional collector system 200 may include a second HPLC column 260 configured to fluidly connect with the second valve 250. The second HPLC column 260 may be used to receive the fluid sample from the second valve 250. In some cases, the fluid sample may comprise a first partial fluid sample. The first HPLC column 220 may be configured to receive the fluid sample and elute the first partial fluid sample to the first valve 230. The first valve 230 may be configured to receive the first partial fluid sample and provide the first partial fluid sample to the vessel 240.

[0036] In some aspects, the second valve 250 may be configured to collect the first partial fluid sample from the vessel 240. The second valve 250 may then provide the first partial fluid sample to the second HPLC column 260. The first partial fluid sample may comprise the second partial fluid sample. In such cases, the second HPLC column 260 may be configured to receive the first partial fluid sample from the first valve 230, and elute the second partial fluid sample.

[0037] The fluid flow paths through the HPLC columns, valves, and fraction collector system 200 may be designed to facilitate the processing and transfer of partial fluid samples between components. The first and second HPLC columns 220, 260 may be used for the separation of the components of the fluid sample based on their different affinities for the stationary phase of the columns and the mobile phase flowing through the columns. The first and second valves 230, 250 may be used to control the flow of the fluid sample and the partial fluid samples between the HPLC columns, the vessel 240, and potentially other components of the system 200. The fraction collector system 200 may be used to collect and dispense fractions of the fluid sample, facilitating the isolation of specific compounds from the fluid sample.

[0038] With continued reference to FIG. 2, the system 200 may further include a plurality of traps 270 configured to fluidly connect to the second valve 250. Each of the plurality of traps 270 may be used to receive the fluid sample from the second valve 250 and trap the fluid sample. In some cases, the fluid sample may comprise the first partial fluid sample. The second valve 250 may be configured to receive the first partial fluid sample and provide the first partial fluid sample to the plurality of traps 270. The traps 270 may be used to retain the first partial fluid sample for further processing or analysis.

[0039] In some aspects, the first partial fluid sample may comprise the second partial fluid sample. The second HPLC column 260 may be configured to fluidly connect with the plurality of traps 270 to receive the first partial fluid sample from the plurality of traps 270. The second HPLC column 260 may then elute the second partial fluid sample. This configuration allows for the trapping of specific fractions or compounds of interest on the traps 270 for further analysis or purification.

[0040] The system 200 may also include a third valve 280 configured to connect with the second HPLC column 260 and receive the sample fluid from the second HPLC column 260. In some cases, the fluid sample may comprise a first partial fluid sample and the first partial fluid sample may comprise the second partial fluid sample. The third valve 280 may be configured to receive the second partial fluid sample from the second HPLC column 260. This configuration allows for the control of fluid flow from the second HPLC column 260 to other components of the system 200, such as a detector or a fraction collector.

[0041] The fluid flow paths through the HPLC columns, valves, traps, and fraction collector may be designed to facilitate the processing and transfer of partial fluid samples between components. The first and second HPLC columns 220, 260, and the plurality of traps 270 may be used for the separation and retention of the components of the fluid sample. The first, second, and third valves 230, 250, 280 may be used to control the flow of the fluid sample and the partial fluid samples between the HPLC columns, the traps 270, the vessel 240, and potentially other components of the system 200. The fraction collector may be used to collect and dispense fractions of the fluid sample, facilitating the isolation of specific compounds from the fluid sample.

[0042] Referring to FIG. 2, the system 200 may include a detector 290 connected to one end of the first HPLC column 220. The detector 290 may be configured to detect an analyte in the fluid sample as it elutes from the first HPLC column 220. In some cases, the detector 290 may be a mass spectrometer, a UV-VIS detector, or any other suitable type of detector for detecting the presence and quantity of analytes in the fluid sample. The detector 290 may output a signal in response to the detection of an analyte, and this signal may be used to trigger the trapping of a partial fluid sample.

[0043] In some aspects, the system 200 may include a trap 270 configured to retain a first partial fluid sample of the fluid sample in response to a signal output from the detector 290 that exceeds a threshold. The threshold may be a predefined minimum signal for a set of parameters defined as "targets". These targets may be mass, mobility, and retention time coordinates within which a molecule of interest lies. When the signal from the detector 290 exceeds the threshold, the solvent flow may be switched in line with the dispensing needle on the right effector arm of the fraction collector. The eluting peak may be collected until either the signal drops below the minimum again or until a predefined volume is reached.

[0044] The system 200 may also include a second flow path extending through a second HPLC column 260 for directing the first partial fluid sample. The second HPLC column 260 may be used for further separation or analysis of the first partial fluid sample. The second HPLC column 260 may be configured to receive the first partial fluid sample from the trap 270 and elute the first partial fluid sample. The first partial fluid sample may comprise a second partial fluid sample, and the second HPLC column 260 may be configured to elute the second partial fluid sample.

[0045] In some cases, the detector 290 may be connected to one end of the second HPLC column 260. The detector 290 may be used to detect an analyte in the second partial fluid sample as it elutes from the second HPLC column 260. The signal from the detector 290 may be used to monitor the elution of the second partial fluid sample and to control the collection of the second partial fluid sample. The detector 290 may output a signal in response to the detection of an analyte, and this signal may be used to trigger the collection of a second partial fluid sample. The second partial fluid sample may be collected in pre-weighed 2mL sample vials and the collected volume may be dried in a vacuum centrifuge. The yields may be determined by weighing the vials after evaporation.

[0046] Referring now to FIG. 3, the system diagram illustrates an analytical mode for a fractional collector system 300 of a mass spectrometry system 255, which may include several key components. In some aspects, the system 255 may include an isocratic pump 310, an autosampler 320, and a prep pump 330 on the left side. The autosampler 320 may be connected to a multiwavelength detector 340 and a semi-prep column 350. The prep pump 330 may also be connected to the semi-prep column 350. The autosampler 320 may be used to automatically introducesamples into the system 255. The prep pump 330 and the isocratic pump 310 may provide the mobile phase for the semi-prep column 350. The semi-prep column 350 may separate the sample components.

[0047] Referring to FIGS. 1-3, the mass spectrometry system 255 may further include a syringeless dosing pump, such as a VICI M6 syringeless dosing pump. The syringeless dosing pump may be configured to control the aspiration of collected fractions by drawing solvent through an aspirating needle into the sample loop on the 10-port valve 170 (FIG. 1). The syringeless dosing pump may be connected to the aspirating needle of a second effector arm of the fraction collector system 200. The syringeless dosing pump may be used to aspirate the dispensed peak into the sample loop on the 10-port valve 170.

[0048] In some cases, the syringeless dosing pump may be controlled by drivers written inhouse. The drivers may be integrated into a graphical user interface created using the PySide6 library, which is the official python module for the Qt for python project. The app may include five pages: main, methods, hardware, graphs, and fractions. These pages may give views of different modules’ status and provide ways to directly control discrete functions of the individual modules, such as turning on the UV-VIS lamp, switching valves, and changing pump flow and composition.

[0049] In some aspects, the syringclcss dosing pump may be used to set the flow from the analytical gradient pump and the isocratic trapping pump to a fixed flow ratio so that the trapped peak is diluted to a low enough percentage of organic modifier to retain on the trapping column. After the full volume of the aspirated peak has been trapped, JobExecutor marks the job as completed and monitors a trappingjobs directory for new jobs. This configuration allows for precise control over the aspiration of collected fractions, enhancing the efficiency and accuracy of the fraction collection process.

[0050] In the center of the diagram is a fraction collector 360, which contains the aspirating needle and a dispensing needle (described further below). These needles may be positioned above a series of collection vessels. The fraction collector 360 may be used to collect and dispense fractions of the fluid sample, facilitating the isolation of specific compounds from the fluid sample.

[0051] The multi-wavelength detector 340 may be connected to a series of valves 370. These valves may be interconnected and also connected to various other components, including a 100:1 splitter 380, multiple traps 390, and an analytical column 400. The multi-wavelength detector 340 may be used to detect the presence and quantity of analytes in the eluent from the semi-prep column 350 based on their absorption of ultraviolet or visible light.

[0052] An analytical pump 410 and another isocratic pump 420 are shown at the bottom of the diagram, connected to the valve system. The analytical column 400 is positioned between the valves and leads to a connection labeled "To MS" (to a mass spectrometer). The analytical pump 410 and the isocratic pump 420 may be used to control the flow of solvents and samples through the system 255.

[0053] The fractional collector system 300 is designed for complex sample separation and analysis. The illustrated flow paths shown in FIGS. 1-3 route the samples and solvents through the various components. The arrangement allows for sample injection, separation on the semi-prep column 350, fraction collection, further separation on traps 390 and the analytical column 400, and final analysis by mass spectrometry.

[0054] The design incorporates multiple pumps and valves, enabling precise control over sample flow and solvent composition throughout the analytical process. The fraction collector 360 with its dual-needle design allows for simultaneous dispensing and aspiration of samples, which typically increases the efficiency of the fractionation process relative to conventional systems. In some cases, the fractional collector system 300 may be connected to a mass spectrometer for detection of the analytes in the fluid sample. The mass spectrometer may be used to provide additional information about the analytes, such as their mass-to-charge ratio and abundance, which may be used to identify the analytes and quantify their concentration in the fluid sample.

[0055] Referring now to FIG. 4, the block diagram illustrates a method execution system for preparative separation. In some aspects, the system 255 may include a MethodExecutor 410 that controls the overall process, and several interconnected components and processes. The MethodExecutor 410 may be configured to direct the execution of predefined methods, including first dimension preparative scale separation and mass-directed fractionation.

[0056] The MethodExecutor 410 initiates a "Run prep gradient" process 420, represented by a graph showing a gradient profile over time. The gradient runs from t=0 to t=45, with notable changes at t=4, t=34, t=39, and t=40. This process may involve applying a first solvent gradient along a flow path based on a preset method parameter to carry a fluid sample through the first HPLC column 220.

[0057] Simultaneously, the system 255 monitors MS signals 430, depicted by a chromatogram-like graph below the gradient profile. This graph shows several peaks, indicating the detection of various compounds over time. The system 255 may include a detector, such as a mass spectrometer, configured to detect an analyte in the fluid sample as it elutes from the first HPLC column 220. The detector may output a signal in response to the detection of the analyte, and this signal may be used to trigger the trapping of a partial fluid sample including the analyte.

[0058] When specific targets are detected in the MS signals 430, the system 25 initiates a "Dispense" action 440. The diagram shows three such dispense events, each corresponding to a peak in the MS signal graph. In some cases, a controller may be configured to operate a first valve to dispense the fluid sample to a vessel during detection of the analyte. As shown in FIG. 4, each event isolates a different analyte.

[0059] Following each dispense event, a "Trapping cycle" 450 is initiated. These trapping cycles arc managed by a JobExccutor (not shown), which operates independently from the main gradient run. The JobExecutor, running in a separate thread, monitors a directory until a new job is created. This job contains the parameters necessary for the trapping of a peak of interest: well location(s), well volume(s), peak identifier, trap number. The JobExecutor initiates a trapping operation, which includes aspirating the dispensed peak into the sample loop on a valve, using a dosing pump; setting the flow from an analytical gradient pump and an isocratic trapping pump to a fixed flow ratio so that the trapped peak is diluted to a low enough percentage of organic modifier to retain on a trapping column. After the full volume of the aspirated peak has been trapped, the JobExecutor marks the job as completed and monitors the directory for new jobs.

[0060] The system 255 allows for asynchronous operation, as evidenced by the overlapping trapping cycles that continue beyond the completion of the preparative gradient run. This design enables the concentration of target compounds through repeated preparative separations. Thelayout of the diagram emphasizes the parallel and sequential nature of the various processes, illustrating how the system 255 can efficiently handle multiple targets in a single run while preparing for subsequent analytical steps.

[0061] Referring to FIG. 5, the system diagram illustrates an analytical method execution process for a chromatography system. In some aspects, the system 255 may include a Method Executor module 510 that controls three main functions: running a preparative gradient, monitoring MS signals, and recording UV signals.

[0062] With continued reference to Figure 5, the Method Executor module 510 initiates a "Run prep gradient" process 520, represented by a graph showing a gradient profile over time. The gradient runs from t=0 to t=45, with notable changes at t=4, t=34, t=39, and t=40. This process may involve applying a first solvent gradient along a flow path based on a preset method parameter to carry a fluid sample through a first HPLC column 220. The first solvent gradient may be designed to separate the components of the fluid sample based on their different affinities for the stationary phase of the column and the mobile phase flowing through the column.

[0063] Simultaneously, the system 255 monitors MS signals 530, depicted by a chromatogram-like trace showing peaks at various retention time points. The system 255 may include a detector, such as a mass spectrometer, configured to detect an analyte in the fluid sample as it elutes from the fractional collector system 200. The detector may output a signal in response to the detection of an analyte, and this signal may be used to trigger the trapping of a partial fluid sample.

[0064] When specific targets are detected in the MS signals 530, the system 255 initiates a "Dispense" action 540. The diagram shows three such dispense events, each corresponding to a peak in the MS signal graph. In some cases, a controller may be configured to operate a first valve to dispense the fluid sample to a vessel during detection of the analyte.

[0065] Following each dispense event, a "Trapping cycle" is initiated. These trapping cycles are managed by a second lobExecutor (not shown), which operates independently from the main gradient run. The second JobExecutor, running in a separate thread, monitors a directory until a new job is created. This job contains the parameters necessary for the trapping of a peak of interest:well location(s), well volume(s), peak identifier, trap number. The second JobExecutor initiates a trapping operation, which includes aspirating the dispensed peak into the sample loop on a valve, using a dosing pump; setting the flow from an analytical gradient pump and an isocratic trapping pump to a fixed flow ratio so that the trapped peak is diluted to a low enough percentage of organic modifier to retain on a trapping column. After the full volume of the aspirated peak has been trapped, the second JobExecutor marks the job as completed and monitors the directory for new jobs.

[0066] The system 255 allows for asynchronous operation, as evidenced by the overlapping trapping cycles that continue beyond the completion of the preparative gradient run. This design enables the concentration of target compounds through repeated preparative separations. The layout of the diagram emphasizes the parallel and sequential nature of the various processes, illustrating how the system 255 can efficiently handle multiple targets in a single run while preparing for subsequent analytical steps.

[0067] In some cases, the system 255 may be configured to perform two-dimensional separations, with the first dimension being preparative scale and the second dimension being analytical scale. The first dimension preparative scale separation may involve the use of the fractional collector system 200 and the first solvent gradient to separate the components of the fluid sample. The second dimension analytical scale separation may involve the use of a second HPLC column and a second solvent gradient to further separate the components of the fluid sample. The second solvent gradient may be variable and may be automatically generated by the controller based on the gradient associated with a retention time corresponding to the signal received from the detector above the threshold. The second solvent gradient may also be automatically generated based on the gradient associated with the retention time corresponding to the signal received from the detector, a mass of a detected analyte within the fluid, and a duration of the signal received from the detector above the threshold. This configuration allows for precise control over the separation and collection of specific compounds from the fluid sample.

[0068] Referring to FIG. 6, the system diagram illustrates an analytical flow splitter system for a mass spectrometry system 255. In some aspects, the system 255 may include a 6-port high- pressure valve 610. The 6-port high-pressure valve 610 may be configured to receive an analyticalflow input and a make-up flow input of 500 pL / min. The 6-port high-pressure valve 610 may be used to direct the flow of fluid between six different paths in two different configurations. This configuration allows for precise control over the flow of fluid through the system 255, enabling complex routing and switching of fluid paths for multi-dimensional separations.

[0069] The system 255 may also include an analytical column 620 with dimensions of 1000 mm length and 254 pm internal diameter, with a residence time of 5.53 seconds. The analytical column 620 may be used for the separation of the components of the fluid sample based on their different affinities for the stationary phase of the column and the mobile phase flowing through the column. The analytical column 620 may be positioned between the 6-port high-pressure valve 610 and a connection labeled "To MS" (to a mass spectrometer). This configuration allows for the separation of the components of the fluid sample prior to detection by the mass spectrometer.

[0070] With continued reference to FIG. 6, the fluid sample may flow from the 6-port high- pressure valve 610 to the analytical column 620, and then to the mass spectrometer. This flow path allows for sequential processing of the fluid sample, including initial separation, further separation on the analytical column 620, and final analysis by mass spectrometry.

[0071] In some cases, the system 255 may include a 100:1 splitter valve 630 for dividing flow between the fraction collector and other components. The 100:1 splitter valve 630 may be used to direct a portion of the fluid sample to the fraction collector for collection and a portion of the fluid sample to other components for further processing or analysis. This configuration allows for the simultaneous collection of fractions and further processing or analysis of the fluid sample.

[0072] In some aspects, the system 255 may be designed for complex sample separation, fraction collection, and analysis, with the capability to split specific compounds of interest before mass spectrometric analysis. The system 255 may be configured to perform two-dimensional separations, with the first dimension being preparative scale and the second dimension being analytical scale. The first dimension preparative scale separation may involve the use of a fractional collector system 200 and a first solvent gradient to separate the components of the fluid sample. The second dimension analytical scale separation may involve the use of the analytical column 620 and a second solvent gradient to further separate the components of the fluid sample. The second solvent gradient may be variable and may be automatically generated by a controllerbased on the gradient associated with a retention time corresponding to a signal received from a detector above a threshold. This configuration allows for precise control over the separation and collection of specific compounds from the fluid sample.

[0073] Referring to FIG. 7, the system diagram illustrates a mass spectrometry data acquisition and control system. In some aspects, the system 255 may include two main components: a WWM Computer 710 and a timsTOF Computer 720, which are interconnected to facilitate data exchange and control.

[0074] The WWM Computer 710 contains several Python modules and classes. The app.py module includes a ConnectionManager class 730 and a MainWindow class. Additional modules such as main.py, graphs.py, methods.py, hardware.py, and fractions.py are also present in the WWM Computer 710. The ConnectionManager class 730 is responsible for handling the connection of all modules, ensuring smooth communication and operation within the system 255.

[0075] Two key modules in the WWM Computer 710 are mscontrol_client.py and ms_data_catcher.py. The mscontrol_client.py module contains an MSControlClient class 740, while the ms_data_catcher.py module includes an MSDatCatcher class 750. These modules establish connections with the timsTOF Computer 720.

[0076] The timsTOF Computer 720 houses several components for instrument control and data acquisition. The instrument_control_host.py module contains an InstrumentControlHost class 760, which interfaces with the timsTOF_acq_method.m module. The recording_service.py module interacts with the timsTOF acquisition engine.

[0077] The system 255 utilizes socket connections between the WWM Computer 710 and the timsTOF Computer 720. The MSControlClient class 740 communicates with the InstrumentControlHost class 760, while the MSDatCatcher class 750 interacts with the recording_service.py module.

[0078] The timsTOF Computer 720 also includes a timsControl_timsTOF module containing a timsTOF_acqp class, which interfaces with the timsTOF acquisition engine. A data_delivery.py module is present for data transfer operations.

[0079] This system architecture allows for coordinated control of the mass spectrometer and efficient data acquisition and processing between the two computers. In some eases, the system 255 may be connected to up to two Bruker timsTOF mass spectrometers through the two different python modules: mscontrol_client.py and ms_data_catcher.py. This configuration enables the system 255 to send commands to the mass spectrometers and receive data from them, facilitating real-time control and monitoring of the mass spectrometry process.

[0080] Referring to FIG. 8, the compact dispense / draw fraction collector 800 is depicted. This device is designed to facilitate the collection and dispensing of fluid samples in a mass spectrometry system 255. The fraction collector 800 includes two effector arms, a first effector arm 810 and a second effector arm 820, each supporting a needle and capable of moving independently along three axes.

[0081] In some aspects, the first effector arm 810 supports a dispensing needle 830. The dispensing needle 830 is configured to fluidly connect with a first valve (not shown in FIG. 8), allowing it to receive fluid from the valve and dispense it into a designated vessel. This dispensing process may be controlled by a controller (not shown in FIG. 8), which operates the first valve to dispense the fluid sample to the vessel during the detection of an analyte.

[0082] The second effector arm 820, on the other hand, supports an aspirating needle 840. The aspirating needle 840 is configured to fluidly connect with a second valve (not shown in FIG. 8), enabling it to collect fluid samples from a vessel. The aspiration process may be controlled by the same or a different controller, which operates the second valve to collect the fluid sample from the vessel, thereby trapping the fluid sample prior to providing the fluid samples to a second HPLC column (not shown in FIG. 8).

[0083] In some cases, the first and second effector arms 810, 820 are operated asynchronously. This means that the dispensing and aspiration processes can occur independently of each other, allowing for efficient and precise fraction collection. This asynchronous operation can be particularly beneficial in scenarios where the timing of the dispensing and aspiration processes needs to be adjusted based on the characteristics of the fluid sample or the requirements of the analysis process.

[0084] The first and second effector arms 810, 820 are independently belt-driven along the X- axis. This belt-driven mechanism allows for precise and smooth movement of the effector arms along the X-axis, which can be important for accurately positioning the dispensing and aspirating needles over the designated vessels.

[0085] In addition to the X-axis movement, the first and second effector arms 810, 820 are actuated along the Y-axis with a screw gear. This screw gear mechanism allows for precise vertical positioning of the effector arms, which can be crucial for accurately dispensing and aspirating fluid samples from the vessels.

[0086] Furthermore, the first and second effector arms 810, 820 are directed along the Z-axis with a pulley 850 movably disposed along a splined shaft 860. This pulley and splined shaft mechanism allows for precise control over the vertical movement of the effector arms, which can be important for accurately positioning the dispensing and aspirating needles at the correct height above the vessels.

[0087] In some aspects, the compact dispense / draw fraction collector 800 may be housed within a frame structure (not shown in FIG. 8), providing support and guidance for the movement of the effector arms. This frame structure may be constructed from extruded metal profiles or any other suitable material, forming a rectangular enclosure that accommodates the effector arms and other components of the fraction collector.

[0088] Referring to FIG. 8, the compact dispense / draw fraction collector 800 may include a controller (not shown in FIG. 8) that is configured to control the operation of the first and second effector arms 810, 820. In some aspects, the controller may be configured to position the dispensing needle 830 of the second effector arm 820 and the aspirating needle 840 of the first effector arm 810 within a 20 millimeter (mm) distance of each other along an XY plane. This close positioning of the dispensing and aspirating needles may allow for efficient and precise collection and dispensing of fluid samples.

[0089] In some cases, the compact dispense / draw fraction collector 800 may be housed within a frame structure (not shown in FIG. 8). The frame structure may provide support and guidance for the movement of the effector arms 810, 820. The frame structure may be constructed fromextruded metal profiles or any other suitable material, forming a rectangular enclosure that accommodates the effector arms and other components of the fraction collector 800. The housing may protect the components of the fraction collector 800 from external interference, and may also provide a controlled environment for the collection and dispensing of fluid samples.

[0090] In some aspects, the controller may be configured to operate the first and second effector arms 810, 820 asynchronously. This means that the dispensing and aspiration processes can occur independently of each other, allowing for efficient and precise fraction collection. This asynchronous operation can be particularly beneficial in scenarios where the timing of the dispensing and aspiration processes needs to be adjusted based on the characteristics of the fluid sample or the requirements of the analysis process.

[0091] The first and second effector arms 810, 820 are independently belt-driven along the X- axis. This belt-driven mechanism allows for precise and smooth movement of the effector arms along the X-axis, which can be important for accurately positioning the dispensing and aspirating needles over the designated vessels. The Y-axes of the effector anus 810, 820 are actuated by a T8 lead screw, providing precise vertical positioning of the effector arms. The Z-axis movement of the effector arms 810, 820 is controlled by a pulley 850 that rides along a splined shaft 860. The splined shaft 860 is rotated from a motor mounted on the X-axis carnage, causing the pulley 850 to raise and lower the Z-axis carriage. This configuration allows for precise control over the vertical movement of the effector arms, which can be important for accurately positioning the dispensing and aspirating needles at the correct height above the vessels.

[0092] Referring to FIG. 9, the system diagram illustrates a fraction collector system for a chromatography or mass spectrometry system 255. In some aspects, the system 255 may include a peak parking plate 910 and an isolation vial rack 920. The peak parking plate 910 may be a grid of small circular wells arranged in rows and columns. The isolation vial rack 920 may also consist of a grid of circular openings, for holding vials. These components may be pail of a fraction collector, such as the compact dispense / draw fraction collector 800 described in relation to FIG.8.

[0093] In some cases, the system 255 may include flow paths indicated by arrows and labels. On the left side, an arrow labeled "To trap" extends from the peak parking plate 910, while anarrow labeled "From analytical" leads into the isolation vial rack 920. On the right side, two arrows labeled "To trap" and "From semi-prep" lead into the peak parking plate 910. These flow paths may be designed to facilitate the trapping and isolation of fractions from both analytical and semipreparative chromatography processes.

[0094] The diagram shows needles or probes positioned above the plates and racks. In the left setup, one needle is positioned over the peak parking plate 910, and another over the isolation vial rack 920, with one well in the isolation vial rack 920 highlighted in blue. In the right setup, two needles are positioned over adjacent wells in the peak parking plate 910, both highlighted in blue.

[0095] This configuration provides a system designed for collecting and isolating fractions from both analytical and semi-preparative chromatography processes. The peak parking plates serve as temporary storage for collected fractions, while the isolation vial racks are used for final collection or further processing of isolated compounds.

[0096] In some aspects, the system 255 may include a first effector arm 810 and a second effector arm 820, each supporting a needle and capable of moving independently along three axes. The first effector arm 810 may support a dispensing needle 830, while the second effector arm 820 may support an aspirating needle 840. The dispensing needle 830 may be configured to dispense fluid samples into the wells of the peak parking plate, while the aspirating needle 840 may be configured to aspirate fluid samples from the wells of the peak parking plate and dispense them into the vials of the isolation vial rack.

[0097] In some cases, the system 255 may be configured to perform two-dimensional separations, with the first dimension being preparative scale and the second dimension being analytical scale. The first dimension preparative scale separation may involve the use of a fractional collector system 200 and a first solvent gradient to separate the components of the fluid sample. The second dimension analytical scale separation may involve the use of a second HPLC column and a second solvent gradient to further separate the components of the fluid sample. The second solvent gradient may be variable and may be automatically generated by a controller based on the gradient associated with a retention time corresponding to a signal received from a detector above a threshold. This configuration allows for precise control over the separation and collection of specific compounds from the fluid sample. This configuration is advantageous because thesystem 255 is configured to first run the gradient with the first HPLC the fluid sample, identify the presence of analytes in the fluid sample, direct the first portion of the fluid sample to the second HPLC column, automatically configure the second solvent gradient based on data associated with the first gradient, and isolate analytes of interest from the second partial fluid sample. Additionally, the asynchronous configuration of the dispensing and aspirating needles provides for significantly quickly analytical ran times, relative to a system without asynchronous configuration.

[0098] In some aspects, the system 255 may include a software interface that provides control over the various components and processes of the system 255. The software interface may be created using the PySide6 library, which is the official python module for the Qt for Python project. The software interface may include a graphical user interface (GUI) that provides a user-friendly way to interact with the system 255. The GUI may include five pages: main, methods, hardware, graphs, and fractions. Each of these pages may provide views of different modules' status and provide ways to directly control discrete functions of the individual modules. For example, the GUI may allow a user to turn on the UV-VIS lamp, switch valves, and change pump flow and composition. This configuration allows for easy and intuitive control over the system 255, facilitating efficient and accurate operation.

[0099] In some cases, the system 255 may include a method for determining yields from collected fractions. The method may involve collecting fractions in pre-weighed 2mL sample vials during the second dimension separation. After the fractions are collected, the sample vials may be dried in a vacuum centrifuge. The drying process may remove the solvent from the collected fractions, leaving behind the isolated compounds. The sample vials may then be weighed again, and the difference in weight before and after the drying process may be used to determine the yield of the isolated compounds. This method provides a simple and accurate way to determine the yield of isolated compounds from the collected fractions.

[0100] In other aspects, the system 255 may include a different software interface or a different method for determining yields. For example, the software interface may be created using a different library or may include different pages. The method for determining yields may involve different steps or different equipment. These variations may be used to adapt the system 255 to different requirements or preferences.

[0101] According to another embodiment as best shown in FIGs. 10 and 11 , the fractional collector 800 includes a support frame 805 with first and second effector arms 810, 820 coupled to the support frame 805. The first effector aim 810 supports a dispensing needle 830 extending from a first side 825 of the first effector arm 810, with the dispensing needle 830 configured to fluidly couple with the first HPLC column. The second effector aim 820 is coupled to the support frame 805 in a substantially parallel orientation to the first effector arm 810. The second effector arm supports an aspirating needle 840 extending from a second side 835 of the second effector arm 820, with the aspirating needle 840 configured to fluidly connect with the second HPLC column. The fractional collector 800 further includes at least one trap configured to fluidly connect with the aspirating needle 840. The first side 825 of the first effector aim 810 faces the second side 835 of the second effector arm 820, such that the dispensing needle 825 and the aspiration needle 835 are adjacent. The adjacent configuration of the dispensing needle 825 and aspiration needle 835 enable the controller to operate the needles 825, 835 asynchronously in close proximity to each other. In fact, in certain embodiments, the controller is configured to position the dispensing needle 835 of the second effector arm 820 and the aspirating needle 830 of the first effector arm 810 within a 20 millimeter (mm) distance of each other along an XY plane. In other embodiments, the controller is configured to position the dispensing needle 835 of the second effector arm 820 and the aspirating needle 830 of the first effector arm 810 within a 20 millimeter (mm) distance of each other along the XY plane. The fractional collector may include one or more of the following features.

[0102] The fractional collector 800 may further include the first flow path extending through the first HPLC column for directing the fluid sample, with the detector fluidly connected to one end of the first HPLC column. As described above, the fluid sample may include the first partial fluid sample and the fractional collector 800 may further include the second flow path extending through the second HPLC column for directing the first partial fluid sample, with the detector also fluidly connected to an end of the second HPLC column. The first and second effector arms 810, 820, may be independently belt-driven along the X-axis and actuated along the Y-axis with the screw gear. The first and second effector arms 810, 830 may also be directed along the Z-axis with the pulley 850 movably disposed along the splined shaft 860. The fractional collector 800 may further include the sample input configured to receive the fluid sample including the analyte, with the fluid sample including the first partial fluid sample, and with the fractional collectorfurther including the first valve configured to fluidly connect with the first HPLC column, with the first valve configured to receive the fluid sample from the first HPLC column and provide the fluid sample to the vessel. The fractional collector 800 may further include the second valve configured to collect the first partial fluid sample from the vessel and provide the first partial fluid sample to the second HPLC column. The first partial fluid sample may include a second partial fluid sample and the second HPLC column may be configured to receive the first partial fluid sample from the first valve, and elute the second partial fluid sample.EXEMPLARY EMBODIMENTS

[0001] In one exemplary embodiment, the mass-targeted fractionation system with asynchronous dual-dimension separation includes a Knauer AZURA HPLC system as the first HPLC column. This system may comprise a P 6.1L pump capable of flow rates up to 50 mL / min, suitable for preparative scale separations. The first HPLC column may be a Phenomenex Kinetex 5pm C18 100A LC column (250 x 21.2 mm) for efficient separation of a wide range of analytes.

[0002] The system may utilize a Bruker timsTOF Pro mass spectrometer as the detector, offering high-resolution mass analysis and ion mobility separation capabilities. The timsTOF Pro can be operated in various acquisition modes, including PASEF (Parallel Accumulation Serial Fragmentation) for enhanced sensitivity and speed.

[0003] For fraction collection, the system may incorporate a custom-built fraction collector utilizing Thorlabs linear stages and Zaber rotary stages for precise XYZ positioning. The dispensing needle may be a Hamilton 22 gauge stainless steel needle, while the aspirating needle could be a Trajan Scientific 24 gauge PEEK needle for chemical resistance.

[0004] The second HPLC column in this embodiment may be an Agilent Infinity Lab Poroshell 120 EC-C18 column (4.6 x 150 mm, 2.7 pm) for high-efficiency analytical separations. This column can be coupled with an Agilent 1260 Infinity II quaternary pump system for precise gradient control.

[0005] In another exemplary embodiment, the system may utilize a Waters ACQUITY UPLC I-Class PLUS system for both the first and second dimension separations. The preparative scale separation may employ a Waters XBridge Prep C18 column (19 x 100 mm, 5 pm), while theanalytical scale separation may use a Waters ACQUTTY UPLC BEH Cl 8 column (2.1 x 100 mm, 1.7 pm).

[0006] This embodiment may incorporate a Thermo Scientific Orbitrap Exploris 480 mass spectrometer for high-resolution accurate mass analysis. The fraction collector could be a modified Gilson GX-271 ASPEC system, customized to include dual probes for simultaneous dispensing and aspiration.

[0007] For trap columns, this embodiment may use Waters Oasis HLB solid-phase extraction cartridges (30 mg sorbent per cartridge) arranged in a custom manifold. These trap columns can effectively retain a wide range of analytes for subsequent elution and analysis.

[0008] In yet another exemplary embodiment, the system may be configured for proteomics applications. This configuration may include a Thermo Scientific UltiMate 3000 RSLCnano system for both dimensions of separation. The first dimension may use a Thermo Scientific Acclaim PepMap 100 C18 LC column (75 pm x 2 cm) for trap-and-elute style separations, while the second dimension may employ a Thermo Scientific EASY-Spray PepMap RSLC Cl 8 column (75 pm x 50 cm) for high-resolution peptide separations.

[0009] This proteomics-focused embodiment may utilize a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer for advanced peptide and protein analysis. The fraction collector could be a custom-built microfluidic device fabricated using soft lithography techniques, capable of handling nanoliter-scale fractions.

[0010] In all these embodiments, the system controller may be implemented using a high- performance workstation running custom software developed in Python. The software may utilize the PySide6 library for the graphical user interface and incorporate modules for instrument control, data acquisition, and real-time data analysis. The controller may interface with commercial instrument control software, such as Bruker otofControl, Thermo Xcalibur, or Waters UNIFI, through appropriate APIs or socket connections.

[0011] These exemplary embodiments demonstrate the versatility of the mass-targeted fractionation system with asynchronous dual-dimension separation, showcasing its potentialapplications in various fields of analytical chemistry, from small molecule analysis to complex proteomics studies.

Claims

CLAIMSWhat is claimed is:

1. A fractional collector for use in a mass spectrometry system including at least one detector for detecting an analyte, the fractional collector comprising: a sample input configured to receive a fluid sample comprising an analyte; a first HPLC column configured to fluidly connect with the sample input to receive the fluid from the sample input; a first valve configured to fluidly connect with the first HPLC column, wherein the first valve is configured to receive the fluid sample and provide the fluid sample to a vessel; a second valve configured to collect the fluid sample from the vessel; a second HPLC column configured to fluidly connect with the second valve to receive the fluid sample from the second valve; and a controller configured to (i) operate the first valve to dispense the fluid sample to the vessel during detection of the analyte, and (ii) operate the second valve to collect the fluid sample from the vessel thereby trapping the fluid sample prior to providing the fluid samples to the second HPLC column; wherein the first and second valves are operated asynchronously.

2. The fractional collector of claim 1 further comprising: a first effector aim supporting a dispensing needle configured to fluidly connect with the first valve, wherein the dispensing needle is configured to receive the fluid and dispense the fluid to the vessel; and a second effector arm supporting an aspirating needle configured to fluidly connect with the second valve, wherein the aspirating needle is configured to collect the sample from the vessel; wherein the first and second effector arms are operated asynchronously.

3. The fractional collector of claim 2, wherein the first and second effector arms are independently belt-driven along the X-axis and actuated along the Y-axis with a screw gear.

4. The fractional collector of claim 3, wherein the first and second effector arms are directed along the Z-axis with a pulley movably disposed along a splined shaft.

5. The fractional collector of claim 2, wherein the controller is configured to position the dispensing needle of the second effector arm and the aspirating needle of the first effector arm within a 20 millimeter (mm) distance of each other along an XY plane.

6. The fractional collector of claim 2 further comprising a housing, wherein the first effector arm and the second effector arm are located within the housing.

7. The fractional collector of claim 1, wherein the fluid sample comprises a first partial fluid sample, and wherein the first HPLC column is configured to receive the fluid sample and elute the first partial fluid sample to the first valve.

8. The fractional collector of claim 7, wherein the first valve is configured to receive the first partial fluid sample and provide the first partial fluid sample to the vessel.

9. The fractional collector of claim 8, wherein the second valve is configured to collect the first partial fluid sample from the vessel and provide the first partial fluid sample to the second HPLC column.

10. The fractional collector of claim 9, wherein the first partial fluid sample comprises a second partial fluid sample, and wherein the second HPLC column is configured to receive the first partial fluid sample from the first valve, and elute the second partial fluid sample.

11. The fractional collector of claim 1 further comprising a plurality of traps configured to fluidly connect to the second valve to receive the fluid sample from the second valve and trap the fluid sample.

12. The fractional collector of claim 11, wherein the fluid sample comprises a first partial fluid sample, and wherein the second valve is configured to receive the first partial fluid sample and provide the first partial fluid sample to the plurality of traps.

13. The fractional collector of claim 12, wherein the first partial fluid sample comprises a second partial fluid sample, and wherein the second HPLC column is configured to (i) fluidly connect with the plurality of traps to receive the first partial fluid sample from the plurality of traps, and (2) elute the second partial fluid sample.

14. The fractional collector of claim 13 further comprising a third valve configured to connect with the second HPLC column and receive the second partial fluid sample from the second HPLC column.

15. A mass spectrometry system comprising: a first flow path extending through a first HPLC column for directing a fluid sample with a detector connected to one end of the first HPLC column; a trap configured to retain a first partial fluid sample of the fluid sample in response to a signal output from the detector that exceeds a threshold; a second flow path extending through a second HPLC column for directing the first partial fluid sample; and a controller configured to: apply a first solvent gradient along the first flow path based on a preset method parameter to carry the fluid sample through the first HPLC column, and apply a variable second solvent gradient to carry a portion of the fluid from the trap along the second flow path; wherein the controller is configured to automatically generate the second variable gradient based on the gradient associated with a retention time corresponding to the signal received from the detector above the threshold.

16. The mass spectrometry system of claim 15, wherein the controller is configured to automatically generate the second variable gradient based on the gradient associated with the retention time corresponding to the signal received from the detector, a mass of a detected analyte within the fluid, and a duration of the signal received from the detector above the threshold.

17. The mass spectrometry system of claim 15, wherein the detector is connected to one end of the second HPLC column.

18. A mass spectrometry system comprising: a fractional collector comprising: a first HPLC column configured to fluidly connect with a first valve, a first effector arm supporting a dispensing needle configured to fluidly connect with the first HPLC column by first valve, a second effector arm supporting an aspirating needle configured to fluidly connect with a second valve, at least one trap configured to fluidly connect with the aspirating needle by the second valve, and a second HPLC column configured to fluidly connect with the at least one trap; a first flow path extending through the first HPLC column for directing a fluid sample with a detector connected to one end of the first HPLC column; a second flow path extending through the second HPLC column for directing a first partial fluid sample with the detector connected to one end of the second HPLC column; and a controller configured to:(i) apply a first solvent gradient along the first flow path based on a preset method parameter to carry the fluid sample through the first HPLC column,(ii) determine if a signal received from the detector is above a threshold,(iii) operate the first effector arm to provide fluid sample from the first valve to a vessel upon a determination that the signal is above the threshold,(iv) operate the second effector arm to collect the fluid sample from the vessel and provide the fluid to the trap, and(v) apply a second variable solvent gradient to carry a portion of the fluid sample from the trap along the second flow path, wherein the controller is configured to automatically generate the second variable solvent gradient based on the gradient associated with a retention time corresponding to the signal received from the detector above the threshold, andwherein the controller is configured to operate the first and second effector arms asynchronously.

19. A fractional collector comprising: a support frame; a first effector arm coupled to the support frame and supporting a dispensing needle extending from a first side of the first effector arm, the dispensing needle configured to fluidly couple with a first HPLC column; a second effector arm coupled to the support frame in a substantially parallel orientation to the first effector arm, the second effector arm supporting an aspirating needle extending from a second side of the second effector arm, the aspirating needle configured to fluidly connect with a second HPLC column; at least one trap configured to fluidly connect with the aspirating needle; and wherein the first side of the first effector arm faces the second side of the second effector arm, such that the dispensing needle and the aspiration needle are adjacent.

20. The fractional collector of claim 19 further comprising a first flow path extending through the first HPLC column for directing a fluid sample, with a detector fluidly connected to one end of the first HPLC column.

21. The fractional collector of claim 20, wherein the fluid sample comprises a first partial fluid sample, and wherein the fractional collector further comprises a second flow path extending through the second HPLC column for directing the first partial fluid sample, and wherein the detector is also fluidly connected to an end of the second HPLC column.

22. The fractional collector of claim 19, wherein the first and second effector arms are independently belt-driven along the X-axis and actuated along the Y-axis with a screw gear.

23. The fractional collector of claim 22, wherein the first and second effector arms are directed along the Z-axis with a pulley movably disposed along a splined shaft.

24. The fractional collector of claim 22, further comprising a controller configured to position the dispensing needle of the second effector aim and the aspirating needle of the first effector ami within a 20 millimeter (mm) distance of each other along an XY plane.

25. The fractional collector of claim 24, wherein the controller is configured to position the dispensing needle of the second effector arm and the aspirating needle of the first effector arm within a 10 millimeter (mm) distance of each other along an XY plane.

26. The fractional collector of claim 19, further comprising a sample input configured to receive a fluid sample comprising an analyte, wherein the fluid sample comprises a first partial fluid sample, and wherein the fractional collector further comprises a first valve configured to fluidly connect with the first HPLC column, wherein the first valve is configured to receive the fluid sample from the first HPLC column and provide the fluid sample to a vessel.

27. The fractional collector of claim 26, further comprising a second valve configured to collect the first partial fluid sample from the vessel and provide the first partial fluid sample to the second HPLC column.

28. The fractional collector of claim 27, wherein the first partial fluid sample comprises a second partial fluid sample, and wherein the second HPLC column is configured to receive the first partial fluid sample from the first valve, and elute the second partial fluid sample.

29. The fractional collector of claim 28 further comprising a plurality of traps configured to fluidly connect to the second valve to receive the fluid sample from the second valve and trap the fluid sample.

30. The fractional collector of claim 29, wherein the fluid sample comprises a first partial fluid sample, and wherein the second valve is configured to receive the first partial fluid sample and provide the first partial fluid sample to the plurality of traps.31 . The fractional collector of claim 30, wherein the first partial fluid sample comprises the second partial fluid sample, and wherein the second HPLC column is configured to (i) fluidly connect with the plurality of traps to receive the first partial fluid sample from the plurality of traps, and (2) elute the second partial fluid sample.

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