Separator for separating lipoproteins from blood, and related systems and methods of use.

Abstract

This specification discloses apparatuses, systems, and processes for separating substances from a sample using size exclusion chromatography (SEC) in a compact, configurable apparatus (referred to as a separator or separation apparatus). The separator separates substances using SEC by flowing the sample through a conduit having a length greater than the distance between the inlet and outlet of the separator.

Description

Technical Field

[0001] (Related Application) This application claims the benefit and priority of U.S. Patent Application No. 18 / 297,423, filed on April 7, 2023, "Separator for Separating Lipoproteins from Blood, and Related Systems and Methods of Use", the entire disclosure of which is incorporated herein by reference.

[0002] (Technical Field) Embodiments of the present disclosure relate to devices and methods for separating or isolating lipoproteins from blood.

Background Art

[0003] Blood contains plasma, and plasma contains lipoproteins. Lipoproteins are important samples for indicating cardiovascular and other health conditions. Lipoproteins are often measured by performing a blood test by collecting a blood sample from a subject (e.g., a human patient) and analyzing the blood sample using various methods in a laboratory. For example, conventional methods involve using centrifugation to separate lipids (e.g., cholesterol, triglycerides, lipoproteins) from other components in the blood. That is, the blood sample is rotated at a high speed for a long time to sediment lipids of various densities at different positions.

[0004] The centrifugation process may require a relatively long period, for example, compared to the analysis time required for the separated lipids. For example, although the time required for lipid separation varies, when the rotation speed is several thousand revolutions per minute, it usually takes several minutes. Also, the required time may depend on the size / amount of the sample, the specific test purpose, and other factors. Thus, lipid separation by the centrifugation process is time-consuming and may induce inaccuracies due to various factors.

Summary of the Invention

Means for Solving the Problems

[0005] This disclosure provides apparatus, systems, and processes for separating substances from a sample using size exclusion chromatography (SEC, also known as gel filtration chromatography) in a compact, configurable apparatus (referred to as a separator or separation apparatus). Unlike conventional SEC columns where each inlet and outlet are separated by a distance greater than the length of the conduit, the separator separates substances using SEC by flowing the sample through a conduit longer than the distance between the inlet and outlet of the separator.

[0006] In a first schematic embodiment, the separator of the present disclosure can receive a sample at an inlet fixed on the housing of the separator. The separator includes a conduit housed in the housing. The conduit is coupled to the inlet and filled with a resin composite, particularly agarose, dextran, etc., for separating a substance from the sample. The separator includes an outlet fixed to the housing and coupled to the conduit. The separated substance flows out from the outlet and is subsequently measured or inspected. The distance between the outlet and the inlet is shorter than the total length of the conduit.

[0007] In some embodiments, the conduit includes a plurality of parallel tubes. Each of the parallel tubes has a coupler (e.g., a cap) at each end for connecting two or more parallel tubes in series. For example, the coupler includes a fitting and a flexible tube. The fitting seals the open end of the flexible tube to one of the corresponding parallel tubes, allowing the flexible tube to carry pressurized fluid from one of the parallel tubes to another. The fitting may include threads, seals, and handles for mounting and removing. In some cases, the separator further includes a cap to surround the fitting and protect the flexible tube.

[0008] In some embodiments, the resin composite includes cross-linked agarose. The test sample contains plasma, and as the plasma is pumped through a conduit, lipoproteins are separated from the plasma by the cross-linked agarose. The absorbance values ​​of the separated lipoproteins in the plasma can be measured with an ultraviolet (UV) detector (or other spectrometer that measures the absorbance or fluorescence of lipoproteins). In some cases, the particle size of the cross-linked agarose is between 8 and 30 microns. The test sample can have a volume in the range of 50 and 200 microliters.

[0009] In another schematic embodiment, this specification discloses a system for separating substances using a SEC having a separator. The system includes a liquid chromatography pump, a liquid chromatography UV detector, and a separator between them. When in operation, the liquid chromatography pump passes the sample to the separator to separate substances in the sample. The liquid chromatography UV detector then receives the substances and measures their absorbance or fluorescence. The separator includes an inlet fixed to a housing. The inlet receives the sample pumped from the liquid chromatography pump. The separator further includes a conduit housed in the housing. The conduit is coupled to the inlet to receive the sample for SEC separation using a resin composite filling the conduit. The separator further includes an outlet fixed on the housing and coupled to the conduit, the distance between the outlet and the inlet being shorter than the total length of the conduit. The outlet then feeds the sample separated from the sample by the separator to the liquid chromatography UV detector.

[0010] In another schematic embodiment, this specification discloses a method for separating a substance using SEC. The method includes the step of supplying a sample to the inlet of a separator using a liquid chromatography pump. The method further includes the step of separating a substance from the sample in a conduit housed in the housing of the separator. The conduit is coupled to the inlet to receive the sample for SEC separation using a resin composite that fills the conduit. The method further includes the step of supplying the substance separated from the sample by the resin composite to a liquid chromatography ultraviolet (UV) detector via the outlet of the separator. The outlet is fixed on the housing and coupled to the conduit. The distance between the outlet and the inlet is shorter than the total length of the conduit.

[0011] The following describes various embodiments in detail.

[0012] The embodiments described and their advantages can be best understood by referring to the following description in conjunction with the accompanying drawings. These drawings do not limit any modifications of form and detail that a person skilled in the art may make to the embodiments described without departing from the spirit and scope of the embodiments described. [Brief explanation of the drawing]

[0013] [Figure 1] An exemplary configuration for liquid chromatography using the separator of this disclosure is shown. [Figure 2] An exploded perspective view of an exemplary separator according to a particular aspect of this disclosure is shown. [Figure 3] Figure 2 shows an exemplary front view of the separator according to a particular aspect of this disclosure. [Figure 4] Figure 2 shows an exemplary perspective view and local view of the separator according to a particular aspect of this disclosure. [Figure 5] An exemplary cap for use with the separator of Figure 2, a perspective view, and a cross-sectional view thereof are shown according to a particular aspect of this disclosure. [Figure 6] The following are fittings and tubing for use with the separator shown in Figure 2, according to a particular aspect of this disclosure. [Figure 7] Figure 2 shows a modified configuration of the inlet, outlet, and conduit length of the separator according to a specific aspect of this disclosure. [Figure 8] A flowchart illustrating how to run a system using the separator shown in Figure 2, according to a specific aspect of this disclosure, is shown. [Figure 9] Exemplary measurement results according to a particular aspect of this disclosure are shown. [Figure 10] An exploded perspective view of an exemplary separator according to a particular aspect of this disclosure is shown. [Figure 11] An exemplary cap, its perspective view, front view, and top view are shown according to a particular aspect of this disclosure. [Modes for carrying out the invention]

[0014] Similar numbers indicate similar elements.

[0015] This disclosure provides apparatus, systems, and processes for separating substances from a sample using size exclusion chromatography (SEC, also known as gel filtration chromatography) in a compact, configurable apparatus (referred to as a separator or separation device). For example, a separator can separate lipids from plasma for detection and analysis. The disclosed separator enables rapid processing of a large number of blood samples and provides high-resolution results in a compact form factor.

[0016] Traditionally, centrifugation has been used to obtain plasma, which contains various proteins, lipids, and other molecules that indicate human health. Centrifugation separates proteins and lipids (e.g., cholesterol, triglycerides, lipoproteins) by utilizing the difference in density. The time required to separate lipids in a blood sample using centrifugation varies, but typically takes several minutes (depending on rotation speed, sample size, and other factors such as temperature and container shape). After separation, the lipid sample may require further processing and quantitative measurement, which can take a considerable amount of time.

[0017] The disclosed separator eliminates the drawbacks of centrifugation by rapidly separating lipoproteins from a blood sample using SEC. For example, a pump feeds a blood sample into the inlet of the separator, and as this passes through a conduit, the lipoproteins of the blood sample exit from the outlet of the separator. By using an appropriate conduit size and SEC polymer, the separation and detection process can sometimes be much faster than the centrifugation method. Furthermore, when using SEC, the amount of blood sample required to obtain results of similar accuracy may sometimes be less than when using the centrifugation method (this speeds up the blood collection process and improves the patient experience).

[0018] Existing SEC configurations often include large and long tubes connected in series. As a result, the connected tubes often occupy a large space and are inconvenient for transportation or replacement (therefore, it is not practical for use in clinics, hospitals, etc.). Furthermore, in known SEC configurations, the material used for the stationary phase (e.g., polymer) may not prevent the passage of other proteins, so it may not be possible to separate lipoproteins from a blood sample, the separation effect decreases, and the detection / analysis accuracy decreases. The present disclosure solves these problems by packing the SEC polymer into a conduit with a zigzag-shaped length that can be reset, allowing only substances smaller than the target size to pass through and exit from the outlet for analysis.

[0019] The exemplary device has a configurable and replaceable compact form factor that enables rapid connection to common liquid chromatography devices such as liquid chromatography pumps and ultraviolet (UV) detectors, and can rapidly inspect a large number of samples without waiting for the conventional centrifugation period. The exemplary device employs SEC or ion exchange chromatography (IEC), uses cross-linked agarose as the sample filler, and blood proteins as the sample analyte. The disclosed device and method can be used for biomedical testing and lipoprotein purification. As an example, the disclosed method improves the chromatographic analysis resolution using an improved flow path to effectively reduce the installation space while providing a conduit with a full length not inferior to conventional configurations.

[0020] At a high level, liquid chromatography is a separation technique used to separate and purify compounds in chemistry, biochemistry, and other fields. In principle, a sample mixture is dissolved in a liquid (mobile phase) and then passed through a stationary phase (e.g., a medium or column). The sample components interact with the stationary phase to varying degrees and are separated as they pass through the stationary phase. In SEC, the stationary phase contains porous beads, which allow small molecules to enter the pores while large molecules are excluded and elute more rapidly.

[0021] Other examples of liquid chromatography include high-performance liquid chromatography, which uses a solid material packed into a column as the stationary phase when the mobile phase is a liquid solvent pumped through the column at high pressure. Another type of liquid chromatography includes ion-exchange chromatography, which uses charged groups in the stationary phase that attract or repel charged species in the sample. Another type of liquid chromatography includes affinity chromatography, where the stationary phase contains a ligand that specifically binds to a target molecule in the sample. Although SEC is used as an example in this disclosure, the disclosed separators and techniques can be used equally well in other types of liquid chromatography.

[0022] The basic procedure for liquid chromatography is as follows. First, prepare the sample by dissolving it in a suitable solvent and filtering to remove solid or particulate matter. Next, inject a small amount of the sample into the stationary phase (e.g., a column) and pass it through. As the sample passes through the column, the individual components are separated. As the separated components elute from the column, a detector, such as an ultraviolet-visible spectrophotometer, measures the absorbance or fluorescence of the separated components. Next, the data obtained from the detector can be analyzed to determine the identity and amount of the individual components in the sample.

[0023] Figure 1 shows an exemplary configuration 100 of liquid chromatography using the separator of this disclosure. As shown, a pump 110 transfers the sample 112 to the separator 120. The sample 112 passes through the stationary phase 125 of the separator and exits through the outlet (as the separated portion 122 of the sample 112, also referred to herein as the analyte or eluent). For example, the sample 112 may contain plasma (e.g., whole or diluted), and the separated portion 122 may contain lipids (e.g., cholesterol, triglycerides, and / or lipoproteins). The separated portion 122 is then fed to a detector 130. The detector 130 may include a UV detector for detecting and quantifying the analyte, since molecules may absorb UV light in the wavelength range between 190 and 400 nm.

[0024] Generally, a liquid chromatography system using SEC, as shown in exemplary configuration 100, includes a pump 110 configured to deliver the mobile phase of a sample 112 at a constant flow rate (may be multiple) through a separator 120 (which functions as a chromatography column). A detector 130 is located downstream of the separator 120 and is configured to detect UV radiation (e.g., absorption, reflection, or fluorescence) from the eluent 122 exiting the separator 120. In some cases, the pump 110 and detector 130 communicate with a control system (not shown) configured to regulate the flow rate of the mobile phase delivered by the pump 110 and to receive signals from the detector 130 indicating the presence and concentration of the analyte in the eluent 122. A detailed description of the separator 120 is presented below in relation to Figure 2-7, following a brief description of the pump 110 and detector 130.

[0025] In some embodiments, the pump 110 may include a high-pressure pump capable of generating high pressure and precise flow rates. The pump 110 can deliver the mobile phase (the liquid that carries the sample through the separator 120) through the stationary phase 125 of the separator 120 at a constant flow rate. The flow rate may be important to achieve reproducible separation. Examples of the pump 110 include syringe pumps, piston pumps, binary pumps, quaternary pumps, and the like.

[0026] For example, a syringe pump uses an electrically powered syringe to generate pressure and supply the mobile phase through the separator 120 at a constant flow rate. A piston pump uses a reciprocating piston and may be more reliable than a syringe pump due to the different materials used at the piston-cylinder interface. As a result, a piston pump can also generate higher pressures than a syringe pump. A peristaltic pump uses a set of rollers to compress and release a flexible tube and is a gentle pump for large quantities of samples that need to be separated. A binary or quaternary pump uses multiple pumps to deliver multiple different mobile phases at different flow rates, enabling the formation of gradient elution. In one example, pump 110 includes a piston pump for reliable operation at high pressure.

[0027] In some embodiments, the detector 130 measures the absorbance of the eluent (e.g., the separation portion 112) exiting the separator 120. In UV detection, a UV lamp is used to generate UV light that passes through the eluent and / or sample molecules. The sample molecules absorb some of the UV light, causing a decrease in the intensity of the light reaching the detector. Since the decrease in light intensity is proportional to the concentration of the analyte, its concentration can be measured. In some cases, a specific wavelength (or range) of UV light is used to measure a specific analyte. In some cases, fluorescence, reflection, or other photoresponses or reactions can be used instead of absorbance to identify and quantify the substance being measured. The detector 130 can detect a wide range of samples, including proteins, nucleic acids, and small molecules.

[0028] Figure 2 is an exploded perspective view of an exemplary separator 120 according to a particular aspect of the present disclosure. As shown in the figure, the separator includes a top cover 202, a body (or housing, interchangeable herein) 216, and a bottom cover 222. The top cover 202 may include holes for connecting tubes or connectors to reach internal conduits 218 within the body 216. In some cases, the internal conduits 218 are made of glass, metal, plastic, or a combination of various materials (e.g., plastic coated with an inert material). The internal conduits 218 and the body 216 may be a single unit or an assembled unit. In some cases, the body 216 may be a protective case or housing for the internal conduits 218. For example, the body 216 may be a hollow case that protects the internal conduits 218 and supports various components such as the top cover 202 and the bottom cover 222. The body 216 may be made of an elastic material with a lightweight geometric shape (such as a hollow pattern as shown in Figure 10). The upper cover 202 and the lower cover 222 may have female tenon mounting portions 204 that connect to corresponding male tenon mounting portions 214 on the main body 216. The upper cover 202 and the lower cover 222 protect the fittings and pipe assemblies 206 that constitute the conduits 218 of the main body 216. In some cases, each conduit 218 may have or be fitted with a threaded port 212 at each end for connection to the fittings and pipe assemblies 206 via a corresponding cap 210. Details of the structure of the cap 210 and the fittings and pipe assemblies 206 are shown in Figures 5 and 6, respectively.

[0029] Mortise joints 204 and 214 connect the upper cover 202 or lower cover 222 to the body 216 using a mortise fitting, creating a leak-free protective housing for the fitting and pipe assembly 206. The mortise joint includes a male tenon 214, which is a projection provided at one end of the body or cover, and a female tenon 204, which is a corresponding recess or hole provided at the other element. The tenons 214 and 204 fit together tightly, forming a firm, leak-free joint. Covers 202 and 222 can be made from a variety of materials, such as stainless steel or plastic, depending on the specific application and suitability for the sample and mobile phase used. The body 216 can be made from a variety of sturdy and clean materials, in particular, such as stainless steel, glass, ceramic, titanium, high-density polyethylene (HDPE), and silicone.

[0030] Once the separator 120 is assembled, the internal conduit 218 can be filled with agarose or polymer as the stationary phase for the SEC. For example, the stationary phase of the internal conduit 218 may include agarose, polyacrylamide, dextran, Sephadex, polystyrene-divinylbenzene, or other resin composites.

[0031] For example, agarose is a polysaccharide that can be used as a stationary phase in SEC. Agarose is a highly porous material that enables size-based molecular separation and is used for the separation of proteins, nucleic acids, and polysaccharides. Polyacrylamide is a polymer used as a stationary phase in gel electrophoresis and ion exchange chromatography. Polyacrylamide can be crosslinked to form gels with controlled porosity and charge density and is used for the separation of proteins and nucleic acids.

[0032] Dextran is a polysaccharide that can be used as a stationary phase in SEC. Dextran is similar to agarose in size exclusion properties but has a different chemical structure. Dextran is used for the separation of proteins, nucleic acids, and polysaccharides. Sephadex is a crosslinked polymer that can be used as a stationary phase in gel filtration chromatography. Sephadex is similar to agarose and dextran but has a different chemical structure. Sephadex is used for the separation of proteins, nucleic acids, and polysaccharides. Polystyrene-divinylbenzene (PSD) is a crosslinked polymer that can be used as a stationary phase in size exclusion chromatography and ion exchange chromatography. PSD has a high surface area and can be modified with different functional groups to confer selectivity for different types of molecules.

[0033] The performance of separator 120 may depend on various factors, particularly column efficiency (the column refers to the stationary phase in the conduit), stationary phase particle size, mobile phase flow rate, mobile phase composition, detector sensitivity, sample volume, and temperature. For example, an efficient column can effectively separate components and result in high-resolution analysis. The stationary phase particle size may determine the measurement resolution; smaller particles result in better separation, but require increased back pressure and reduced column lifetime. The mobile phase flow rate may affect the resolution by changing the time the analyte remains in the column. A slower flow rate can increase the resolution because it increases the separation time, but a slower flow rate may also increase the analysis time.

[0034] Considering other factors, the composition of the mobile phase can affect the resolution, as different solvents or additives can improve separation by altering the selectivity of the separation. The sensitivity of the detector 130 used to detect the separated analytes can also affect the resolution. A more sensitive detector can detect smaller differences in analyte concentrations, thereby increasing the resolution. The amount of sample injected into the chromatography column of the separator 120 can also affect the resolution; injecting large amounts of sample can lead to peak broadening and a decrease in resolution. In addition, high ambient temperatures can reduce the viscosity of the mobile phase and improve separation, but they can also lead to column degradation and a reduced lifespan.

[0035] In light of these factors, the separator 120 allows for various configurations that utilize any number of the multiple conduits 218 contained within the main body 216. Although Figure 2-5 shows four parallel conduit columns, the separator 120 can contain any different number of conduits 218 (e.g., four or five or more) within the main body 216, and the multiple conduits 218 can have different curvatures or shapes.

[0036] Figure 3 shows an exemplary front view 300 of the separator of Figure 2 according to a particular aspect of the present disclosure. As shown in the figure, the separator 120 includes an inlet 312 and an outlet 322, which provide ports for the incoming sample 112 and the outgoing eluent 122, respectively. The distance 310 between the inlet 312 and the outlet 322 is shorter than the length 315 of each conduit 218. In other examples, according to aspects of the present disclosure, the distance 310 is shorter than the total length of the stationary phase conduit. For example, suppose there are N conduits (N=2, 3, 4, 5, ...) and the connecting pipe length 325 is standard. According to the present disclosure, the following geometric relationship holds: Distance 310 < Conduit length 315 × N + Pipe length 325 × (N-1) Therefore, the relatively short distance 310 (compared to, for example, the conduit length 315) allows for practical handling of the separator 120 in a medical facility, provided that the separator 120 can be conveniently replaced.

[0037] In some embodiments, the conduit length 315 can be 15 cm to 25 cm. The total conduit length can be 60 cm to 100 cm, for example, 80 cm. The conduit 218 can have a diameter of 2.5-15 mm, for example, 5 mm.

[0038] Figure 4 shows exemplary perspective and local view 400 of the separator of Figure 2 according to a particular aspect of the present disclosure. As shown in the figure, the conduits 218 are packed with agarose 450 in all columns. The plasma sample 112 passes through a zigzag path created by the cap connector 430, the fitting 410, and the tube 420. Details of the cap connector 430 are shown in Figure 5. Details of the fitting 410 (and fitting and tube assembly 206) are shown in Figure 6. In Figure 4, the configuration 440 of the fitting 410 and tube 420 can vary depending on the number of conduits 218, the desired column length, and other factors. Figure 7 shows two other configurations 710 and 720 on the same body 216, but with the positions of the inlet 312 and outlet 322 changed.

[0039] In some embodiments, the agarose 450 for SEC can have a particle size of 8.6–30 microns. The sample volume is between 50 and 200 microliters. Regarding the mobile phase, the solvent can include 20 mM Tris + 0.5 mM EDTA + 0.02% NaN3. The operating temperature can be between 2 and 40°C.

[0040] Figure 5 shows an exemplary cap 430 for use with the separator 120 of Figure 2, a perspective view 500 and an enlarged section view thereof, according to a particular aspect of the present disclosure. As shown in the figure, the cap 430 includes an external engagement surface 510 for receiving torque to rotate the cap 430 relative to the body 216. The cap 430 includes a first set of threads 522 for engaging with a threaded port 212 on the body 216. The cap 430 further includes an O-ring 512 for preventing fluid leakage from the conduit 218 when installed. The cap 430 includes a frit 514 for preventing leakage of the cross-linked agarose 450 filled in the conduit 218. In some cases, the frit 514 may include filter paper.

[0041] The cap 430 further includes a second set of threads 520 for engaging with the fitting 410. Although the cap 430 and the fitting 410 are shown in the figure as two separate components (since the fitting 410 can be a standard component available as a ready-made product), in some cases the cap 430 and the fitting 410 can be integrated into a single component, and therefore do not require a threaded connection based on the second set of threads 520 (whereas the illustrated cap 430 may further have a feature for connecting to the pipe 420). Another example of the cap 430 is shown in Figure 11.

[0042] Figure 6 shows a fitting 410 and a tube 420 (of assembly 206) for use with the separator 120 of Figure 2, according to a particular aspect of the present disclosure. As shown in the figure, the fitting 410 may include threads 612 for assembly with the cap 430 via a second set of threads 520. The fitting 410 further includes a handle 610 for receiving torque from the user. The fitting 410 may optionally include a male connector 620 for receiving the tube 420, which can engage with the connector 620 by adhesive, crimp fit, and other mating means. The tube 420 can be inserted into a cavity 614 formed in the upper part of the fitting 410. In some cases, the fitting 410 is a ready-made standard 1 / 4-28 size fitting.

[0043] In some embodiments, the tube 420 is a flexible tube for transferring plasma or sample fluid. The tube 420 can be made of a material that is inert and nonreactive and has low levels of eluting compounds that may interfere with the separation or detection of the analyte. For example, the tube 420 can be made of polytetrafluoroethylene (PTFE), polyetheretherketone (PEEK), fluorinated ethylene propylene (FEP), silicone, Tygon®, and similar materials. The material selected for the tube 420 can be a highly inert material that is resistant to most solvents and acids.

[0044] The pipe 420 can be fixed to the fitting 410 using various types of connectors or adapters. In addition to the example shown in Figure 6, other examples include, in particular, compression fittings, barb fittings, luer fittings, and screw fittings.

[0045] Figure 7 shows variations 710 and 720 of the configuration of the inlet 312, outlet 322, and conduit length of the separator 120 of Figure 2, according to a particular aspect of the present disclosure. As shown in the figure, in variation 710, the outer conduit 218 can be directly connected by a single tube 725, skipping the two intermediate conduits. This configuration reduces the amount of agarose pre-packed in the body 216. In variation 720, the second and fourth conduits 218 (counting from left to right) are connected via tube 735, skipping the third conduit, and thus utilizing three of the four conduits in the body 216. Variations 710 and 720, together with the embodiment 200 shown in Figure 2, demonstrate variable configurations achievable in the body 216 that can be adjusted to specific resolution, column length, or cost requirements.

[0046] In addition to the embodiments shown in Figures 2-5 and the modified forms 710 and 720 in Figure 7, the present disclosure does not require all conduits 218 to be parallel or to open at the top and bottom of the body 216. For example, one or more conduits 218 can be connected internally without relying on the external cap 430 or the fitting / pipe assembly 206. That is, one or more conduits 218 can form a zigzag conduit inside the body 216 to provide an overall length longer than either the width or length of the body 216.

[0047] Figure 8 shows a flowchart 800 of a method for performing a system using the separator 120 of Figure 2 according to a particular aspect of this disclosure. The method of flowchart 800 can be performed by the system 100 shown in Figure 1. As shown in the figure, in 810, a liquid chromatography pump supplies the sample to the inlet of a separator (such as separator 120). In 820, the separator separates the substance from the sample in a conduit housed in the separator housing. The conduit is connected to the inlet to receive the sample for SEC separation using a resin composite that fills the conduit. In 830, the separator supplies the substance separated from the sample by the resin composite to a liquid chromatography ultraviolet (UV) detector via the outlet of the separator. The outlet is fixed to the housing and coupled to the conduit. The distance between the outlet and the inlet is shorter than the total length of the conduit.

[0048] In one embodiment, separating the test specimen within a conduit housed in a housing includes separating the test specimen within a plurality of parallel tubes. Each of the parallel tubes may have a coupler at each end for connecting two or more of the plurality of parallel tubes in series. In one instance, the method further includes sealing the open end of a flexible tube to a corresponding one of the plurality of parallel tubes using a coupler fitting. In one instance, the method further includes enclosing the coupler and the flexible tube with a rigid cap that fits snugly into the housing. For example, the coupler fitting rotates on the parallel tubes via an intermediate connector that provides a male thread for the coupler fitting and a female thread that receives the male threads of the plurality of parallel tubes.

[0049] In some cases, the method further includes the steps of filling a conduit with a resin composite having cross-linked agarose and pumping plasma of the test sample through the conduit to measure the absorbance of lipoproteins in the plasma using a liquid chromatography UV detector. The test sample may be the patient's whole blood (and plasma therein). In some cases, the test sample may be pre-treated to remove substances irrelevant to the test and to include the plasma of interest. Plasma contains various proteins (such as human serum albumin (HSA)), lipids, and other molecules in addition to lipoproteins. Lipoproteins include high-density lipoproteins (HDL) and low-density lipoproteins (LDL). Lipoproteins also include very low-density lipoproteins (VLDL).

[0050] Figure 9 shows exemplary measurement results 900 according to a particular aspect of the present disclosure. As shown in the figure, the Y-axis is the absorbance value in mAu (milli absorbance units), and the X-axis of this graph is the residence time (in minutes), which can also be expressed as a multiple of the column volume. The absorbance value is calculated using the formula: Au = -log10T%, where T% is the penetration rate. As shown in Figure 9, the separator 120 effectively separates VLDL, LDL, HDL, and HSA.

[0051] The above description provides numerous specific details, such as examples of particular systems, components, and methods, to enable a good understanding of some embodiments of the present disclosure. However, it will be apparent to those skilled in the art that at least some embodiments of the present disclosure can be implemented without these specific details. In some cases, well-known components or methods are not described in detail or are shown in simple block diagram form to avoid unnecessarily obscuring the present disclosure. Accordingly, the specific details described are merely illustrative. Certain embodiments may differ from these illustrative details and are still intended to be within the scope of the present disclosure.

[0052] In addition, some embodiments can be implemented in a distributed computing environment, where machine-readable media are stored on and / or executed by multiple computer systems. Furthermore, information transferred between computer systems can be pulled or pushed across a communication medium connecting the computer systems.

[0053] Embodiments of the subject matter described in the claims include, but are not limited to, various operations described herein. These operations can be performed by hardware components, software, firmware, or a combination thereof.

[0054] Although the operations of the methods described herein are shown and described in a specific order, the order of operations of each method can be modified so that certain operations are performed in reverse order or so that certain operations are performed at least partially simultaneously with other operations. In another embodiment, the instructions for individual operations or subordinate operations may be intermittent or alternating.

[0055] The above description of the exemplary embodiments, including those described in the abstract, is not intended to be exhaustive or to limit embodiments to the exact form disclosed. While specific configurations, embodiments, or examples are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be apparent to those skilled in the art. The terms “example” or “exemplary” are used herein to mean examples, illustrations, or illustrative purposes. Any aspect or design described herein as “example” or “exemplary” is not necessarily construed as being preferable or more advantageous than other aspects or designs. Rather, the use of the terms “example” or “exemplary” is intended to present a concept in a specific way. Where used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless otherwise specified or evident from the context, “X includes A or B” is intended to mean some specific inclusive order. In other words, if X includes A, if X includes B, or if X includes both A and B, “X includes A or B” satisfies any of the above cases. In addition, the articles “a” and “an” used in this application and the attached claims should generally be interpreted as meaning “one or more” unless otherwise specified or unless the context makes it clear that they refer to a singular form. Furthermore, throughout this specification, the use of the terms “one embodiment” or “one embodiment” or “one implementation” or “one implementation” is not intended to mean the same embodiment or embodiment unless otherwise stated. Furthermore, terms such as “first,” “second,” “third,” “fourth” used herein are intended as indicators to distinguish different elements and do not necessarily have an ordinal meaning corresponding to the number designation.

[0056] It should be understood that the features disclosed above, as well as variations or substitutions of other features and functions, may be combined in other different systems or applications. Various substitutions, modifications, variations, or improvements that are not currently foreseeable or unforeseen may subsequently be made by those skilled in the art, and these are also intended to be included in the following claims. The claims may include embodiments of hardware, software, or combinations thereof. [Explanation of symbols]

[0057] 100 configurations 110 pump 112 samples 120 Separator 122 Separation part 130 detectors

Claims

1. An apparatus for separating substances using size exclusion chromatography (SEC), An entrance fixed to the housing, A conduit housed in the housing, the conduit being connected to the inlet for receiving a test sample for SEC separation using a resin composite material filling the conduit, An outlet fixed on the housing and connected to the conduit, Equipped with, A device in which the distance between the outlet and the inlet is shorter than the total length of the conduit.

2. The apparatus according to claim 1, wherein the conduit comprises a plurality of parallel pipes, and each of the plurality of parallel pipes has a coupler at each end for connecting two or more of the plurality of parallel pipes in series.

3. The apparatus according to claim 2, wherein the coupler comprises a mounting fixture and a flexible tube, the mounting fixture seals the open end of the flexible tube to the corresponding end of the plurality of parallel tubes.

4. The apparatus according to claim 3, further comprising a cap that surrounds the coupler and protects the flexible pipe.

5. The apparatus according to claim 4, wherein the mounting fixture comprises a screw thread, a seal, and a handle for mounting and removing.

6. The apparatus according to claim 1, wherein the resin composite comprises cross-linked agarose, the test sample comprises plasma, and the apparatus is capable of measuring the absorbance value of lipoproteins in the plasma when the plasma is pumped through the conduit.

7. The apparatus according to claim 6, wherein the cross-linked agarose has a particle size between 8 and 30 microns, and the test sample has a volume in the range of 50 and 200 microliters.

8. A system for separating substances using size exclusion chromatography (SEC), Liquid chromatography pump and Liquid chromatography ultraviolet (UV) detector, Separator and, The separator is equipped with, Fixed on the housing, it has an inlet for receiving the test sample pumped from the liquid chromatography pump, A conduit housed in the housing, the conduit being connected to the inlet to receive the test sample for SEC separation using a resin composite material filling the conduit, An outlet fixed on the housing and connected to the conduit, Equipped with, The system wherein the distance between the outlet and the inlet is shorter than the total length of the conduit, and the outlet supplies the substance separated from the test sample by the separator to the liquid chromatography UV detector.

9. The system according to claim 8, wherein the conduit comprises a plurality of parallel pipes, and each of the plurality of parallel pipes has a coupler at each end for connecting two or more of the plurality of parallel pipes in series.

10. The system according to claim 9, wherein the coupler comprises a mounting fixture and a flexible tube, the mounting fixture seals the open end of the flexible tube to a corresponding one of the plurality of parallel tubes.

11. The system according to claim 10, further comprising a cap that surrounds the coupler and protects the flexible pipe.

12. The system according to claim 11, wherein the mounting fixture comprises a screw thread, a seal, and a handle for mounting and removing.

13. The system according to claim 8, wherein the resin composite comprises cross-linked agarose, the test sample comprises plasma, and the absorbance of lipoproteins in the plasma can be measured by the liquid chromatography UV detector when the plasma is pumped through the conduit.

14. The system according to claim 13, wherein the cross-linked agarose has a particle size between 8 and 30 microns, and the test sample has a volume in the range of 50 and 200 microliters.

15. A method for separating substances using size exclusion chromatography (SEC), The steps include supplying the sample to the separator inlet using a liquid chromatography pump, A step of separating a substance from the test sample in a conduit housed in the housing of the separator, wherein the conduit is coupled to the inlet to receive the test sample for SEC separation using a resin composite material filling the conduit, The steps include supplying the substance separated from the test sample by the resin composite to a liquid chromatography ultraviolet (UV) detector via the outlet of the separator, Includes, The outlet is fixed on the housing and connected to the conduit, and the distance between the outlet and the inlet is shorter than the total length of the conduit.

16. The step of separating the test sample within the conduit housed in the housing includes the step of separating the test sample into a plurality of parallel tubes, The method according to claim 15, wherein each of the plurality of parallel pipes has a coupler at each end for connecting two or more of the plurality of parallel pipes in series.

17. The method according to claim 16, further comprising the step of sealing the open end of a flexible pipe to a corresponding one of the plurality of parallel pipes using the fittings of the coupler.

18. The method according to claim 17, further comprising the step of surrounding the coupler and the flexible pipe with a rigid cap that fits securely onto the housing.

19. The method of claim 18, further comprising the step of rotating the mounting fixture of the coupler on the plurality of parallel tubes via an intermediate connector that provides a male thread on the mounting fixture of the coupler and a female thread for receiving the male threads on the plurality of parallel tubes.

20. The steps include filling the conduit with the resin composite having crosslinked agarose, The steps include pumping the plasma of the test sample through the conduit in order to measure the absorbance value of lipoproteins in the substance using the liquid chromatography UV detector, The method according to claim 15, further comprising:

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