Discontinuous sample fractionation and cascade device and dual-online multidimensional liquid chromatography system having the same

Abstract

Disclosed is a non-continuous sample grading and cascading device and a dual-online multidimensional liquid chromatography system having the device. The non-continuous sample grading and cascading device according to an embodiment of the present disclosure includes: a sample supply module for supplying samples to be analyzed; and a sample grading module connected to the sample supply module and continuously supplied with samples, the sample grading module sets a plurality of unit sample supply times obtained by equally dividing the total sample supply time of the sample supplied by the sample supply module, sets a plurality of unit grading intervals obtained by equally dividing each of the plurality of unit sample supply times, and cascades / stores samples supplied during the corresponding unit grading intervals in each unit sample supply time, thereby obtaining a plurality of fractions.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of October 31, 2016, application number 201680078937.8, and invention name “Discontinuous sample grading and cascade device and dual-online multifunctional liquid chromatography system having the same”. Technical Field

[0002] The present disclosure relates to a discontinuous sample fractionation and cascade device and a dual-online multidimensional liquid chromatography system having the device, and more particularly, to a discontinuous sample fractionation and cascade device for achieving low sample complexity and high fraction uniformity and a dual-online multidimensional liquid chromatography system having the device. Background Art

[0003] Liquid chromatography coupled to mass spectrometry (LC-MS / MS) has become a very important technique in proteomics for large-scale studies of the complete set of proteins expressed in the human body under specific conditions.

[0004] Among such techniques, bottom-up or shotgun proteomics techniques involving hydrolysis of proteins into small fragments, peptides, prior to analysis are widely used.

[0005] Bottom-up or shotgun proteomics approaches have proven useful but inevitably lead to sample complexity. For example, when proteins expressed by about 20,000 genomes are hydrolyzed into peptides, tens of millions of peptides are obtained. In addition, proteins in the human body have a wide dynamic range of concentrations (10 10 ). Therefore, separation methods with high resolution are required to perform efficient proteomic analysis with reduced sample complexity.

[0006] As a technology to solve this problem, two-dimensional liquid chromatography came into being. This technology involves the cascade of two different separation methods.

[0007] There are two key factors for the success of 2D-LC: the first is the separation efficiency of each separation method, and the second is the separation orthogonality.

[0008] Separation orthogonality refers to the independence of various separation modes, which means that the sample mixture is separated by different physiological and chemical properties through various separation modes.

[0009] One of the two-dimensional separation methods developed and widely used in recent years is a method comprising the steps of fractionating a sample according to the degree of hydrophobicity at alkaline pH and again separating the separated fractions according to the degree of hydrophobicity under acidic pH conditions (two-dimensional reversed-phase liquid chromatography-reverse-phase liquid chromatography, 2D RP-RPLC).

[0010] Basically, the two separation modes that separate mixtures by hydrophobicity do not have separation orthogonality, but peptides under different pH conditions have different charge distributions according to their unique amino acid compositions, and thus even the same peptide can have different degrees of hydrophobicity. Thus, the 2D RP-RPLC method has separation orthogonality.

[0011] However, 2D RP-RPLC methods do not have complete separation orthogonality. Figure 1 The distribution of identified peptides at elution times for various separation methods is shown after RPLC separation at pH 10, RPLC separation of each fraction at pH 2.6, and analysis using a mass analyzer. Figure 1 , it was observed that there were many regions where no peptides were found, implying that the 2DRP-RPLC method did not utilize all the separation space of the two-dimensional separation.

[0012] The method developed to solve this problem is to perform non-continuous cascading after fractionation. Figure 2 a, the discontinuous cascade after fractionation includes: fractionation into 96 fractions (1st fraction to 96th fraction) in the elution order of the first-dimensional RPLC separation at alkaline pH, and discontinuous cascading into 24 fractions. After No. 1 to No. 24 become 24 fractions, No. 25 is cascaded into the first fraction, and No. 26 is cascaded into the second fraction. As a result, No. 1, No. 25, No. 49 and No. 73 are cascaded into the first fraction, No. 2, No. 26, No. 50 and No. 74 are cascaded into the second fraction, and No. 24, No. 48, No. 72 and No. 96 are cascaded into the final 24th fraction. When discontinuous cascading is performed in this manner, the entire separation space of the two-dimensional separation can be fully utilized, such as Figure 2 As shown in b.

[0013] By using hierarchical and discontinuous cascade techniques, 2D RP-RPLC achieves high separation orthogonality.

[0014] However, this cascade process is performed manually by researchers, requiring a lot of manpower and time. In addition, in order to perform LC-MS / MS experiments on fractions obtained in the offline method at acidic pH, many manual steps are required to reduce the volume of the fractions and redissolve them in a solvent suitable for LC-MS / MS experiments, thereby causing sample loss in the process and affecting the reproducibility of the experiment. Therefore, it is necessary to develop a liquid chromatography system that automates the steps after sample injection.

[0015] Related Literature

[0016] [Non-patent literature]

[0017] Non-patent document 1: M Gilar et al., Anal Chem, 2005, 77, 6426-6434

[0018] Non-patent document 2: JM Park et al., Sci. Rep. 5, 18189; doi: 10.1038 / srep18189 (2015) Summary of the invention

[0019] Technical issues

[0020] Therefore, the present disclosure aims to provide a non-continuous sample fractionation and cascade device and a dual-online multidimensional liquid chromatography system having the device, which is used to improve the reproducibility of fractionation and prevent sample loss by automating the non-continuous sample fractionation and cascade process with low sample complexity and high fractionation uniformity.

[0021] Technical Solution

[0022] According to one aspect of the present disclosure, a non-continuous sample grading and cascading device is provided, which includes a sample supply module for supplying samples to be analyzed, and a sample grading module connected to the sample supply module, the sample grading module is continuously supplied with samples, a plurality of unit sample supply times obtained by equally dividing a total sample supply time supplied by the sample supply module is set, a plurality of unit grading intervals obtained by equally dividing each of the plurality of unit sample supply times are set, and samples supplied during corresponding unit grading intervals in each unit sample supply time are cascaded and stored to obtain a plurality of fractions.

[0023] The sample grading module may include a first grading valve connected to the sample supply module, the sample being introduced into the first grading valve; a second grading valve arranged near the first grading valve; and a plurality of fraction storage circuits arranged corresponding to the number of multiple unit grading intervals within a unit sample supply time, one end of each fraction storage circuit being connected to the first grading valve and the other end being connected to the second grading valve to cascade and store samples supplied in sequence during the corresponding unit grading interval within each unit sample supply time.

[0024] The first fractionation valve may include: a fraction inlet, through which a sample is introduced from a sample supply module; a plurality of first fraction storage circuit connection ports, which are arranged near the fraction inlet and are each connected to one end of the plurality of fraction storage circuits; and a first connecting channel, which connects the fraction inlet and one of the plurality of first fraction storage circuit connection ports to be connected to each other corresponding to the unit fractionation interval; and the second fractionation valve may include: a plurality of second fraction storage circuit connection ports, which are each connected to the other end of the plurality of fraction storage circuits; a fraction outlet, which is arranged near the plurality of second fraction storage circuit connection ports and is connected to the other end of one of the plurality of fraction storage circuits to discharge fractions; and a second connecting channel, which connects the fraction outlet and one of the plurality of second fraction storage circuit connection ports to be connected to each other corresponding to the unit fractionation interval.

[0025] The sample supply module may include: a first sample supply valve connected to a first pump and a sample syringe, wherein the first pump supplies a first solvent; and a second sample supply valve connected to the first sample supply valve to receive a sample supplied by the first sample supply valve and supply the sample to a fraction inlet.

[0026] The first sample supply valve may include: a first sample inlet, which is connected to the sample syringe; a first sample outlet, which is arranged near the first sample inlet; a first solvent inlet, which is connected to the first pump; a first solvent outlet, which is arranged near the first solvent inlet and connected to the second sample supply valve; and a first sample storage loop connection port and a second sample storage loop connection port, which are respectively connected to both ends of the sample storage loop; and in a case where the first sample storage loop connection port and the second sample storage loop connection port are respectively connected to the first sample inlet and the first sample outlet, the sample can be stored in the sample storage loop, and in a case where the first sample storage loop connection port and the second sample storage loop connection port are respectively connected to the first solvent inlet and the first solvent outlet, the first solvent can be injected into the sample storage loop to supply the sample to the second sample supply valve.

[0027] The second sample supply valve may include: a second sample inlet, which is connected to the first solvent outlet; a second sample outlet, which is arranged near the second sample inlet and connected to the fraction inlet; and a sample separation column having two ends, each end of which is respectively connected to the second sample inlet and the second sample outlet, so that the sample is eluted at the second sample outlet, and the sample eluted in the sample separation column can be supplied to the fraction inlet.

[0028] According to another aspect of the present disclosure, a dual-online multidimensional liquid chromatography system is provided, which includes: a non-continuous sample grading and cascading device, which is continuously supplied with samples to be analyzed, sets a plurality of unit sample supply times obtained by equally dividing the total sample supply time of the supplied samples, sets a plurality of unit grading intervals obtained by equally dividing one of the plurality of unit sample supply times, and supplies a plurality of fractions obtained by cascading and storing the samples supplied during the corresponding unit grading intervals in each unit sample supply time; a double column valve, which is connected to a first reversed phase liquid chromatography column and a second reversed phase liquid chromatography column, and has a first solid phase extraction column connected to the first reversed phase liquid chromatography column and a second solid phase extraction column connected to the second reversed phase liquid chromatography column; and a column selection module, which is arranged between the non-continuous sample grading and cascading device and the double column valve, so as to supply the fractions supplied by the non-continuous sample grading and cascading device in a sequential order to the first solid phase extraction column and the first reversed phase liquid chromatography column or the second solid phase extraction column and the second reversed phase liquid chromatography column in an alternating manner.

[0029] The non-continuous sample fractionation and cascade device may include: a sample supply module that supplies a sample; and a sample fractionation module connected to the sample supply module to continuously receive the sample supplied by the sample supply module, thereby obtaining multiple fractions and supplying the multiple fractions to the column selection module in a sequential order.

[0030] The sample fractionation module may include: a first fractionation valve, which is connected to the sample supply module and into which the sample is introduced; a second fractionation valve, which is arranged near the first fractionation valve; and a plurality of fraction storage loops, which are arranged corresponding to the number of a plurality of unit fractionation intervals within a unit sample supply time, one end of each fraction storage loop being connected to the first fractionation valve and the other end being connected to the second fractionation valve, so as to cascade and store samples supplied in a sequential order during the corresponding unit fractionation interval within each unit sample supply time.

[0031] The first fractionation valve may include: a first fraction inlet, through which a sample is introduced by a sample supply module; a plurality of first fraction storage circuit connection ports, which are arranged near the first fraction inlet and are respectively connected to one end of the plurality of fraction storage circuits; and a first connecting channel, which connects the fraction inlet and one of the plurality of first fraction storage circuit connection ports to be connected to each other corresponding to the unit fractionation interval; and the second fractionation valve may include: a plurality of second fraction storage circuit connection ports, which are respectively connected to the other end of the plurality of fraction storage circuits; a first fraction outlet, which is arranged near the plurality of second fraction storage circuit connection ports and is connected to the other end of one of the plurality of fraction storage circuits to discharge the fraction; and a second connecting channel, which connects the first fraction outlet and one of the plurality of second fraction storage circuit connection ports to be connected to each other corresponding to the unit fractionation interval.

[0032] The sample supply module may include: a first sample supply valve having a first sample inlet connected to a sample syringe; a first sample outlet, which is arranged near the first sample inlet; a first solvent inlet, which is connected to a first pump; a first solvent outlet, which is arranged near the first solvent inlet; and a first sample storage loop connection port and a second sample storage loop connection port, which are respectively connected to both ends of the sample storage loop; and a second sample supply valve having a second sample inlet connected to the first solvent outlet; a second sample outlet, which is arranged near the second sample inlet and connected to the first fraction inlet; and a sample separation column having two ends, each end of which is respectively connected to the second sample inlet and the second sample outlet, so that the sample is eluted at the second sample outlet.

[0033] The column selection module may include: a column balancing valve connected to the first fraction outlet to provide a channel for supplying multiple fractions to the first solid phase extraction column or the second solid phase extraction column in an alternating manner, and balancing the first solid phase extraction column and the first reversed phase liquid chromatography column or the second solid phase extraction column and the second reversed phase liquid chromatography column in an alternating manner; and a column selection valve connected to the column balancing valve to receive the supply of multiple fractions and supply the multiple fractions to the first solid phase extraction column or the second solid phase extraction column in an alternating manner, so that the fractions are eluted with the first reversed phase liquid chromatography column in the first solid phase extraction column or with the second reversed phase liquid chromatography column in the second solid phase extraction column.

[0034] The column balancing valve may include: a second fraction inlet, which is connected to the first fraction outlet and a second pump supplying a second solvent; a third solvent inlet, which is connected to a third pump supplying a third solvent, and the third solvent is introduced through the third solvent inlet; a second fraction outlet, which is arranged near the second fraction inlet and selectively connected to the second fraction inlet and the third solvent inlet; and a third solvent outlet, which is arranged near the third solvent inlet and selectively connected to the second fraction inlet and the third solvent inlet; when the second fraction inlet is connected to the third solvent outlet, the fraction injected with the second solvent can be supplied to the first solid phase extraction column or the second solid phase extraction column via the column selection valve, and when the third solvent inlet is connected to the third solvent outlet, the third solvent balances the first solid phase extraction column and the first reversed phase liquid chromatography column or the second solid phase extraction column and the second reversed phase liquid chromatography column in an alternating manner via the column selection valve.

[0035] The column selection valve may include: a fraction and third solvent inlet, which is connected to the third solvent outlet, through which the fraction or the third solvent is introduced; a fourth solvent inlet, which is connected to a fourth pump supplying a fourth solvent, through which the fourth solvent is introduced; a fraction and third solvent outlet, which is arranged near the fraction and third solvent inlet and selectively connected to the fraction and third solvent inlet and the fourth solvent inlet; and a fourth solvent outlet, which is arranged near the fourth solvent inlet and selectively connected to the fraction and third solvent inlet and the fourth solvent inlet; when the fraction and third solvent inlet is connected to the fraction and third solvent outlet, the fraction can be supplied to the first solid phase extraction column, and then connected to the fraction and third solvent inlet at the fourth solvent inlet. When the third solvent outlet is connected, the fourth solvent can elute the fraction with the first reversed phase liquid chromatography column in the first solid phase extraction column, and when the fraction and the third solvent inlet are connected to the fourth solvent outlet, the third solvent can balance the second solid phase extraction column and the second reversed phase liquid chromatography column; and when the fraction and the third solvent inlet are connected to the fourth solvent outlet, the fraction can be supplied to the second solid phase extraction column, and then when the fourth solvent inlet is connected to the fourth solvent outlet, the fourth solvent can elute the fraction with the second reversed phase liquid chromatography column in the second solid phase extraction column, and when the fraction and the third solvent inlet are connected to the fraction and the third solvent outlet, the third solvent can balance the first solid phase extraction column and the first reversed phase liquid chromatography column.

[0036] The double-column valve may include a first solid-phase extraction column connection port and a first solid-phase extraction column channel port, each of which is connected to the two ends of the first solid-phase extraction column; a first solid-phase extraction column inlet, which is connected to the fraction and the third solvent outlet, and selectively connected to the first solid-phase extraction column connection port and the first solid-phase extraction column channel port; a first reversed-phase liquid chromatography column port, which is connected to the first reversed-phase liquid chromatography column, and connected or disconnected from the first solid-phase extraction column connection port; a second solid-phase extraction column connection port and a second solid-phase extraction column channel port, each of which is connected to the two ends of the second solid-phase extraction column; a second solid-phase extraction column inlet, which is connected to the fourth solvent outlet, and selectively connected to the second solid-phase extraction column connection port and the second solid-phase extraction column channel port; and a second reversed-phase liquid chromatography column port, which is connected to the second reversed-phase liquid chromatography column, and connected or disconnected from the second solid-phase extraction column connection port.

[0037] The double-column valve may also include: a first outlet, which is arranged near the first solid phase extraction column channel port and connected to or disconnected from the first solid phase extraction column channel port; and a second outlet, which is arranged near the first outlet and connected to or disconnected from the second solid phase extraction column channel port.

[0038] Beneficial Effects

[0039] An embodiment of the present disclosure provides a non-continuous sample fractionation and cascade device, which cascades and stores samples supplied during corresponding unit fractionation intervals within each unit sample supply time to automatically obtain multiple fractions, thereby reducing sample complexity and increasing fraction uniformity, and further improving fractionation reproducibility and preventing sample loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing the distribution of peptides identified after RPLC separation at pH 10, RPLC separation of each fraction again at pH 2.6, and analysis using a mass analyzer.

[0041] Figure 2 is a diagram showing a discontinuous cascade after sample fractionation.

[0042] Figure 3 is a diagram showing an operation of obtaining a first fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0043] Figure 4 is a diagram showing an operation of obtaining a second fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0044] Figure 5 is an operation diagram showing acquisition of the tenth fraction by the dual on-line multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0045] Figure 6 is an operation diagram showing supplying a first fraction to a first solid phase extraction column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0046] Figure 7 is a diagram showing the operation of supplying a first fraction to a first reverse phase liquid chromatography column and equilibrating a second solid phase extraction column and a second reverse phase liquid chromatography column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0047] Figure 8 is a diagram showing the operation of supplying a second fraction to a second solid phase extraction column by a dual on-line multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0048] Fig. 9 is a diagram showing the operation of supplying a second fraction to a second reverse phase liquid chromatography column and equilibrating a first solid phase extraction column and the first reverse phase liquid chromatography column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0049] For a fuller understanding of the present disclosure, its operating advantages and objects attained by its practice, reference is made to the accompanying drawings in which preferred embodiments of the present disclosure and statements therein are illustrated.

[0050] Hereinafter, the present disclosure will be described in detail by explaining preferred embodiments of the present disclosure with reference to the accompanying drawings. Like reference numerals present in each figure represent like elements.

[0051] Figure 3 is an operation diagram showing obtaining a first fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure; Figure 4 is an operation diagram showing obtaining a second fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure; Figure 5 is an operation diagram showing acquisition of a tenth fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure; Figure 6 is an operation diagram showing supplying a first fraction to a first solid phase extraction column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure; Figure 7 is an operation diagram showing supplying a first fraction to a first reverse phase liquid chromatography column and balancing a second solid phase extraction column and a second reverse phase liquid chromatography column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure; Figure 8 is an operation diagram showing supplying a second fraction to a second solid phase extraction column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure; and Fig. 9 is a diagram showing the operation of supplying a second fraction to a second reverse phase liquid chromatography column and equilibrating a first solid phase extraction column and the first reverse phase liquid chromatography column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.

[0052] Reference Figures 3 to 5 , a dual-online multidimensional liquid chromatography system 100 according to an embodiment of the present disclosure includes: a non-continuous sample fractionation and cascade device 200, which is continuously supplied with a sample to be analyzed to obtain a plurality of fractions, and supplies the plurality of fractions; a dual-column valve 400, which is connected to a first reversed-phase liquid chromatography column (COL1) and a second reversed-phase liquid chromatography column (COL2), and has a first solid phase extraction column (SPE1) connected to the first reversed-phase liquid chromatography column (COL1) and a second solid phase extraction column (SPE2) connected to the second reversed-phase liquid chromatography column (COL2); and a column selection module 300, which is disposed between the non-continuous sample fractionation and cascade device 200 and the dual-column valve 400, so as to supply the plurality of fractions supplied by the non-continuous sample fractionation and cascade device 200 in a sequential order to the first solid phase extraction column (SPE1) and the first reversed-phase liquid chromatography column (COL1) or the second solid phase extraction column (SPE2) and the second reversed-phase liquid chromatography column (COL2) in an alternating manner.

[0053] Reference Figures 3 to 5 The function of the discontinuous sample fractionation and cascade device 200 according to this embodiment is to obtain a plurality of fractions from a sample continuously supplied by a sample injector S.

[0054] A plurality of fractions according to this embodiment will be described below.

[0055] First, a unit sample supply time is set by equally dividing the total sample supply time of the supplied sample. In addition, a unit fractionation interval is set by equally dividing each unit sample supply time. In addition, a plurality of fractions are obtained by cascading and storing samples supplied during corresponding unit fractionation intervals within each unit sample supply time.

[0056] For example, when the total sample supply time is 100 minutes, the unit sample supply time obtained by dividing the total sample supply time into five equal parts is an interval of 20 minutes. That is, the first unit sample supply time corresponds to 0 to 20 minutes, and the second to fifth unit sample supply times correspond to 20 minutes to 40 minutes, 40 minutes to 60 minutes, 60 minutes to 80 minutes, and 80 minutes to 100 minutes, respectively.

[0057] In addition, the first unit sample supply time to the fifth unit sample supply time may have a unit graded interval obtained by being equally divided into ten. For example, during the first unit sample supply time, the first unit graded interval corresponds to 0 to 2 minutes, and the second unit graded interval to the tenth unit graded interval correspond to 2 minutes to 4 minutes, 4 minutes to 6 minutes, 6 minutes to 8 minutes, 8 minutes to 10 minutes, 10 minutes to 12 minutes, 12 minutes to 14 minutes, 14 minutes to 16 minutes, 16 minutes to 18 minutes, and 18 minutes to 20 minutes, respectively. In addition, during the second sample supply time, the first unit graded interval corresponds to 20 to 22 minutes, and the second graded interval to the tenth graded interval correspond to 22 minutes to 24 minutes, 24 minutes to 26 minutes, 26 minutes to 28 minutes, 28 minutes to 30 minutes, 30 minutes to 32 minutes, 32 minutes to 34 minutes, 34 minutes to 36 minutes, 36 minutes to 38 minutes, and 38 minutes to 40 minutes, respectively.

[0058] In this embodiment, a plurality of fractions are obtained by concatenating and storing samples supplied during the first unit grading interval to the tenth unit grading interval corresponding to the first unit sample supply time to the fifth unit sample supply time. For example, the first fraction is obtained by concatenating and storing samples supplied during the first unit grading interval (i.e., 0 to 2 minutes, 20 minutes to 22 minutes, 40 minutes to 42 minutes, 60 minutes to 62 minutes, 80 minutes to 82 minutes) during the first unit sample supply time to the fifth unit sample supply time. The second to tenth fractions are obtained by the same operation as that of obtaining the first fraction, and their detailed description is omitted here.

[0059] In addition, although this embodiment sets the total sample supply time to 100 minutes and the first unit sample supply time to the fifth unit sample supply time, and sets ten unit grading intervals in each unit sample supply time, the protection scope of the present disclosure is not limited to this, and the total sample supply time, unit sample supply time and unit grading interval may be set differently.

[0060] The non-continuous sample fractionation and cascade device 200 according to this embodiment includes a sample supply module 210 for supplying samples; and a sample fractionation module 250, which is connected to the sample supply module 210 to continuously receive the sample supplied by the sample supply module 210, thereby obtaining multiple fractions and supplying the multiple fractions to the column selection module 300 in a sequential order.

[0061] The sample supply module 210 according to this embodiment functions to continuously supply samples to the sample classification module 250 .

[0062] The sample supply module 210 includes a first sample supply valve 220 connected to a first pump P1 supplying a first solvent and a sample syringe S; and a second sample supply valve 240 connected to the first sample supply valve 220 to receive a sample supplied by the first sample supply valve 220 and supply the sample to the sample classification module 250.

[0063] The first sample supply valve 220 receives a sample supplied from the sample syringe S, stores the sample in the sample storage circuit 221 , then receives a first solvent supplied from the first pump P1 and supplies the sample stored in the sample storage circuit 221 to the second sample supply valve 240 .

[0064] To this end, the first sample supply valve 220 includes: a first sample inlet 222, which is connected to the sample syringe S; a first sample outlet 223, which is arranged near the first sample inlet 222; a first solvent inlet 226, which is connected to the first pump P1; a first solvent outlet 227, which is arranged near the first solvent inlet 226 and connected to the second sample supply valve 240; and a first sample storage loop connection port 224 and a second sample storage loop connection port 225, which are respectively connected to both ends of the sample storage loop 221.

[0065] Although not shown, when the first sample storage circuit connection port 224 and the second sample storage circuit connection port 225 respectively connected to both ends of the sample storage circuit 221 are connected to the first sample inlet 222 and the first sample outlet 223, respectively, the sample is supplied to the first sample inlet 222 through the sample syringe S and is continuously stored in the sample storage circuit 221.

[0066] Furthermore, in the case where the first sample storage circuit connection port 224 and the second sample storage circuit connection port 225 are respectively connected to the first solvent inlet 226 and the first solvent outlet 227, the first solvent is injected into the first solvent inlet 226 by the first pump P1, and the sample stored in the sample storage circuit 221 is continuously supplied to the second sample supply valve 240. In this case, the first pump P1 supplies the first solvent to the first solvent inlet 226, wherein the first solvent is a mixed solution of 99% solution A (10 mM triethylammonium bicarbonate (TEAB) aqueous solution) and 1% solution B (10 mM triethylammonium bicarbonate (TEAB) acetonitrile solution) at pH 7.5.

[0067] In addition, the second sample supply valve 240 functions to continuously inject the sample supplied by the first sample supply valve 220 into the sample separation column 243 , and continuously supply the sample injected into the sample separation column 243 to the sample classification module 250 .

[0068] The second sample supply valve 240 includes: a second sample inlet 241, which is connected to the first solvent outlet 227; a second sample outlet 242, which is arranged near the second sample inlet 241 and connected to the first fraction inlet 261; and a sample separation column 243, which has two ends, which are respectively connected to the second sample inlet 241 and the second sample outlet 242, so that the sample is separated and eluted at the second sample outlet 242.

[0069] The sample stored in the sample storage loop 221 is injected into the sample separation column 243 through the first solvent, the first solvent outlet 227 and the second sample inlet 241. In addition, the sample injected into the sample separation column 243 is separated and eluted by the first solvent supplied by the first pump P1, and is supplied to the sample classification module 250 described below along the second sample outlet 242. In this case, the first pump P1 supplies the first solvent to the first solvent inlet 226, wherein the first solvent is a mixed solution of solution A (10 mM triethylammonium bicarbonate (TEAB) aqueous solution) decreased from 99% to 50% and solution B (10 mM triethylammonium bicarbonate (TEAB) acetonitrile solution) increased from 1% to 50%. As described above, the sample is easily eluted in the sample separation column 243 by gradually increasing the concentration of solution B in the first solvent.

[0070] The sample fractionation module 250 according to this embodiment functions to obtain ten fractions from the sample continuously supplied from the sample supply module 210 .

[0071] The sample fractionation module 250 includes: a first fractionation valve 260, which is connected to the sample supply module 210 to allow a sample to be introduced therethrough; a second fractionation valve 280, which is disposed near the first fractionation valve 260; and a plurality of fraction storage loops 284a to 284j, one end of which is connected to the first fractionation valve 260 and the other end of which is connected to the second fractionation valve 280.

[0072] In this embodiment, since each unit sample supply time has ten unit classification intervals, ten fraction storage circuits 284a to 284j are provided corresponding to the respective numbers of unit classification intervals in the unit sample supply time.

[0073] In addition, the first fractionation valve 260 includes: a first fraction inlet 261 through which a sample is introduced from the second sample outlet 242; a plurality of first fraction storage circuit connection ports 262, which are provided near the first fraction inlet 261 and are respectively connected to one end of the plurality of fraction storage circuits 284a to 284j; and a first connection channel 263, which connects the first fraction inlet 261 with one of the plurality of first fraction storage circuit connection ports 262 so as to communicate with each other corresponding to the unit fractionation interval. In this embodiment, ten first fraction storage circuit connection ports 262 are provided corresponding to the number of ten fraction storage circuits 284a to 284j.

[0074] In addition, the second fractionation valve 280 includes: a plurality of second fraction storage circuit connection ports 282, which are respectively connected to the other ends of the plurality of fraction storage circuits 284a to 284j; a first fraction outlet 281, which is provided near the plurality of second fraction storage circuit connection ports 282 and connected to the other end of one of the plurality of fraction storage circuits 284a to 284j to discharge the fraction; and a second connection channel 283, which connects the first fraction outlet 281 to one of the plurality of second fraction storage circuit connection ports 282 so as to be communicated with each other corresponding to the unit fractionation interval. In this embodiment, ten second fraction storage circuit connection ports 282 are provided corresponding to the number of ten fraction storage circuits 284a to 284j.

[0075] First, the operation of acquiring the first to tenth fractions during the first unit classification interval to the tenth unit classification interval within the first unit sample supply time (0 to 20 minutes) will be described below.

[0076] like Figure 3 As shown, the operation of acquiring the first fraction during the first unit classification interval (0 to 2 minutes) is as follows.

[0077] Among the multiple first-fraction storage circuit connection ports 262, the first connection channel 263 is connected to the first-fraction storage circuit connection port 262 connected to one end of the first-fraction storage circuit 284a, and among the multiple second-fraction storage circuit connection ports 282, the second connection channel 283 is connected to the second-fraction storage circuit connection port 282 connected to the other end of the first-fraction storage circuit 284a.

[0078] In addition, when the first solvent outlet 227 and the second sample inlet 241 are connected, the first solvent is supplied to the sample separation column 243 by the first pump P1, so that the sample is separated and eluted in the sample separation column 243, and the sample separated and eluted in the sample separation column 243 is supplied to the first fraction storage loop 284a through the second sample outlet 242 via the first fraction inlet 261 and stored in the first fraction storage loop 284a.

[0079] In addition, if Figure 4 As shown, the operation of acquiring the second fraction during the second unit classification interval (2 minutes to 4 minutes) is as follows.

[0080] Among the multiple first-fraction storage circuit connection ports 262, the first connection channel 263 is connected to the first-fraction storage circuit connection port 262 connected to one end of the second-fraction storage circuit 284b, and among the multiple second-fraction storage circuit connection ports 282, the second connection channel 283 is connected to the second-fraction storage circuit connection port 282 connected to the other end of the second-fraction storage circuit 284b.

[0081] In addition, when the first solvent outlet 227 and the second sample inlet 241 are connected, the first solvent is supplied to the sample separation column 243 by the first pump P1, so that the sample is separated and eluted in the sample separation column 243, and the sample separated and eluted in the sample separation column 243 is supplied to the second fraction storage circuit 284b through the second sample outlet 242 via the first fraction inlet 261 and stored in the second fraction storage circuit 284b.

[0082] In addition, if Figure 5 As shown, the operation of acquiring the tenth fraction during the tenth unit fractionation interval (18 to 20 minutes) is as follows.

[0083] Among the multiple first-level storage circuit connection ports 262, the first connection channel 263 is connected to the first-level storage circuit connection port 262 connected to one end of the tenth-level storage circuit 284j, and among the multiple second-level storage circuit connection ports 282, the second connection channel 283 is connected to the second-level storage circuit connection port 282 connected to the other end of the tenth-level storage circuit 284j.

[0084] In addition, when the first solvent outlet 227 and the second sample inlet 241 are connected, the first solvent is supplied to the sample separation column 243 by the first pump P1, so that the sample is separated and eluted in the sample separation column 243, and the sample separated and eluted in the sample separation column 243 is supplied to the tenth fraction storage loop 284j through the second sample outlet 242 via the first fraction inlet 261 and stored in the tenth fraction storage loop 284j.

[0085] Furthermore, within the second unit sample supply time to the fifth unit sample supply time (20 minutes to 40 minutes, 40 minutes to 60 minutes, 60 minutes to 80 minutes, 80 minutes to 100 minutes), the first to tenth fractions may be obtained by repeatedly performing the operation of obtaining the first to tenth fractions during the first unit classification interval to the tenth unit classification interval within the first unit sample supply time (0 to 20 minutes). For example, the first fraction may be obtained by concatenating and storing samples supplied during the first unit classification interval (i.e., 0 to 2 minutes, 20 minutes to 22 minutes, 40 minutes to 42 minutes, 60 minutes to 62 minutes, 80 minutes to 82 minutes) within the first unit sample supply time to the fifth unit sample supply time.

[0086] Meanwhile, in order to separate and analyze the first to tenth fractions obtained by the sample fractionation module 250 , the first to tenth fractions are supplied to the first reverse phase liquid chromatography column ( COL1 ) or the second reverse phase liquid chromatography column ( COL2 ).

[0087] To this end, the column selection module 300 according to this embodiment functions to supply the first fraction to the tenth fraction supplied by the sample fractionation module 250 in a sequential order to the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1), or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) provided in the following dual column valve 400 in an alternating manner.

[0088] In addition, the column selection module 300 functions to supply the first fraction to the tenth fraction to one of the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2), and perform fraction separation and analysis, and at the same time perform a balancing operation on the other of the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2).

[0089] The column selection module 300 includes: a column balancing valve 310, which is connected to the first fraction outlet 281 to provide a channel for supplying multiple fractions to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in an alternating manner, and balancing the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) in an alternating manner; and a column selection valve 330, which is connected to the column balancing valve 310 to receive the supply of multiple fractions and supply the fractions to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in an alternating manner, so as to separate and elute the fractions using the first reversed phase liquid chromatography column (COL1) in the first solid phase extraction column (SPE1) or using the second reversed phase liquid chromatography column (COL2) in the second solid phase extraction column (SPE2).

[0090] The column balancing valve 310 according to this embodiment functions to provide a channel for supplying the first fraction to the tenth fraction to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in an alternating manner.

[0091] The column balancing valve 310 includes: a second fraction inlet 311, which is connected to the first fraction outlet 281 and the second pump P2 supplying the second solvent; a third solvent inlet 313, which is connected to the third pump P3 supplying the third solvent to allow the third solvent to be introduced; a second fraction outlet 312, which is arranged near the second fraction inlet 311 and selectively connected to the second fraction inlet 311 and the third solvent inlet 313; and a third solvent outlet 314, which is arranged near the third solvent inlet 313 and selectively connected to the second fraction inlet 311 and the third solvent inlet 313.

[0092] Reference Figure 6 In the case where the second fraction inlet 311 is connected to the first fraction outlet 281 and the second fraction inlet 311 is connected to the third solvent outlet 314, the first fraction stored in the first fraction storage loop 284a is supplied to the first solid phase extraction column (SPE1) of the double selector valve described below. Figure 8 , when the second fraction inlet 311 is connected to the first fraction outlet 281 and the second fraction inlet 311 is connected to the third solvent outlet 314, the second fraction stored in the second fraction storage loop 284b is supplied to the second solid phase extraction column (SPE2) of the dual selection valve described below.

[0093] As described above, when the first fraction outlet 281, the second fraction inlet 311 and the third solvent outlet 314 are connected, the first to tenth fractions stored in the first to tenth fraction storage loops 284a to 284j can be supplied to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in an alternating manner.

[0094] In this case, the second solvent is supplied to the first to tenth fractions discharged from the first fraction storage loop 284a to the tenth fraction storage loop 284j by the second pump P2 placed in the pipeline connecting the first fraction outlet 281 and the second fraction inlet 311. Here, the second pump P2 supplies the second solvent, i.e., a 0.2% trifluoroacetic acid (TFA) aqueous solution. In addition, the first pump P1 supplies the first solvent to the first solvent inlet 226, wherein the first solvent is a mixed solution of 99% solution A (10 mM triethylammonium bicarbonate (TEAB) aqueous solution) and 1% solution B (10 mM triethylammonium bicarbonate (TEAB) acetonitrile solution). In addition, the ratio of the amount of the first solvent supplied by the first pump P1 to the amount of the second solvent supplied by the second pump P2 can be 1:9.

[0095] This is because the second solvent is injected into the first to tenth fractions supplied to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) to dilute and acidify the first to tenth fractions, thereby preventing the loss of the first to tenth fractions in the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) and reducing the composition of the organic solvent.

[0096] In addition, refer to Figure 7 , during the separation and analysis operation of the first fraction stored in the first reversed phase liquid chromatography column (COL1), when the third solvent inlet 313 and the third solvent outlet 314 are connected, the third solvent is balanced between the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) via the column selection valve 330. In addition, referring to Fig. 9During the separation and analysis operation of the second fraction stored in the second reversed-phase liquid chromatography column (COL2), when the third solvent inlet 313 and the third solvent outlet 314 are connected, the third solvent balances the first solid phase extraction column (SPE1) and the first reversed-phase liquid chromatography column (COL1) via the column selection valve 330.

[0097] Although a 0.1% formic acid aqueous solution is used as the third solvent in this embodiment, the third solvent is not limited thereto and includes any aqueous solution capable of equilibrating the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1), or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2).

[0098] The column selection valve 330 according to this embodiment functions to receive a plurality of fractions supplied by the column balancing valve 310 and to supply the plurality of fractions to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in an alternating manner. In addition, the column selection valve 330 functions to supply a fourth solvent to the first solid phase extraction column (SPE1) so as to elute the fractions with the first reverse phase liquid chromatography column (COL1) in the first solid phase extraction column (SPE1), or to supply a fourth solvent to the second solid phase extraction column (SPE2) so as to elute the fractions with the second reverse phase liquid chromatography column (COL2) in the second solid phase extraction column (SPE2).

[0099] The column selection valve 330 includes: a fraction and third solvent inlet 331, which is connected to the third solvent outlet 314 so that a fraction or a third solvent can be introduced therethrough; a fourth solvent inlet 333, which is connected to a fourth pump P4 for supplying a fourth solvent so that a fourth solvent can be introduced therethrough; a fraction and third solvent outlet 332, which is arranged near the fraction and third solvent inlet 331 and is selectively connected to the fraction and third solvent inlet 331 and the fourth solvent inlet 333; and a fourth solvent outlet 334, which is arranged near the fourth solvent inlet 333 and is selectively connected to the fraction and third solvent inlet 331 and the fourth solvent inlet 333.

[0100] like Figure 6 As shown, the operation of balancing the second solid phase extraction column (SPE2) and the second reverse phase liquid chromatography column (COL2) is described, and the first fraction is supplied to the first solid phase extraction column (SPE1) when the fraction and third solvent outlet 332, the fraction and third solvent inlet 331, and the third solvent outlet 314 and the second fraction inlet 311 are connected. In addition, as Figure 7As shown, when the fourth solvent inlet 333 is connected to the fraction and the third solvent outlet 332, the fourth solvent is used to elute the first fraction in the first solid phase extraction column (SPE1) using the first reversed phase liquid chromatography column (COL1), and when the fraction and the third solvent inlet 331, the fourth solvent outlet 334 and the third solvent outlet 314 are connected, the third solvent is introduced into the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) to balance the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2).

[0101] In this embodiment, the fourth pump P4 supplies the fourth solvent to the fourth solvent inlet 333, wherein the fourth solvent is a mixed solution of solution C (0.1% formic acid in water) decreased from 99% to 60% and solution D (0.1% formic acid in acetonitrile) increased from 1% to 40%. As described above, the gradual increase in the concentration of solution D in the fourth solvent enables the first fraction to be easily eluted in the first solid phase extraction column (SPE1), or enables the second fraction to be easily eluted in the second solid phase extraction column (SPE2).

[0102] In addition, if Figure 8 As shown, the operation of balancing the first solid phase extraction column (SPE1) and the first reverse phase liquid chromatography column (COL1) is described, and the second fraction is supplied to the second solid phase extraction column (SPE2) when the fourth solvent outlet 334, the fraction and the third solvent inlet 331, the third solvent outlet 314 and the second fraction inlet 311 are connected; then as Fig. 9 As shown, when the fourth solvent inlet 333 is connected to the fourth solvent outlet 334, the fourth solvent is used to elute the second fraction in the second solid phase extraction column (SPE2) using the second reversed phase liquid chromatography column (COL2), and when the fraction and third solvent inlet 331 is connected to the fraction and third solvent outlet 332, the third solvent is introduced into the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) to balance the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1).

[0103] Meanwhile, the dual column valve 400 according to this embodiment functions to separate and analyze fractions in one of the first reverse phase liquid chromatography column (COL1) and the second reverse phase liquid chromatography column (COL2) while balancing the other column.

[0104] The dual column valve 400 includes a first solid phase extraction column (SPE1) connected to a first reverse phase liquid chromatography column (COL1) and a second solid phase extraction column (SPE2) connected to a second reverse phase liquid chromatography column (COL2). The fraction is supplied to the first reverse phase liquid chromatography column (COL1) through the first solid phase extraction column (SPE1) or supplied to the second reverse phase liquid chromatography column (COL2) through the second solid phase extraction column (SPE2).

[0105] In addition, the dual-column valve 400 supplies the first fraction to the tenth fraction to the first reversed-phase liquid chromatography column (COL1) or the second reversed-phase liquid chromatography column (COL2) in an alternating manner, and the dual-column valve 400 is fluidically connected to the column selection valve 330, which separates and analyzes the fractions in one of the first reversed-phase liquid chromatography column (COL1) and the second reversed-phase liquid chromatography column (COL2) while balancing the other column.

[0106] To this end, the dual column valve 400 includes: a first solid phase extraction column connection port 412 and a first solid phase extraction column channel port 413, each of which is connected to both ends of the first solid phase extraction column (SPE1); a first solid phase extraction column inlet 411, which is connected to the fraction and third solvent outlet 332 and selectively connected to the first solid phase extraction column connection port 412 and the first solid phase extraction column channel port 413; a first reversed phase liquid chromatography column port 414, which is connected to the first reversed phase liquid chromatography column (COL1) and connected to the first solid phase extraction column connection port 411; 2 connected or disconnected; a second solid phase extraction column connection port 432 and a second solid phase extraction column channel port 433, each of which is connected to both ends of the second solid phase extraction column (SPE2); a second solid phase extraction column inlet 431, which is connected to the fourth solvent outlet 334 and selectively connected to the second solid phase extraction column connection port 432 and the second solid phase extraction column channel port 433; and a second reversed phase liquid chromatography column port 434, which is connected to the second reversed phase liquid chromatography column (COL2) and connected or disconnected to the second solid phase extraction column connection port 432.

[0107] Reference Figure 6 , describes the operation of supplying the first fraction to the first solid phase extraction column (SPE1), and the first fraction is supplied to the first solid phase extraction column (SPE1) when the first solid phase extraction column connection port 412, the first solid phase extraction column inlet 411, the fraction and the third solvent outlet 332, the fraction and the third solvent inlet 331, and the third solvent outlet 314 are connected. In addition, the dual column valve 400 further includes a first outlet 451, which is disposed near the first solid phase extraction column channel port 413 and connected or disconnected to the first solid phase extraction column channel port 413, and when the first fraction is supplied to the first solid phase extraction column (SPE1) and concentrated, the salt is discharged through the first outlet 451.

[0108] In addition, refer to Figure 7 In order to inject the first fraction into the first reversed phase liquid chromatography column (COL1) and separate and analyze the first fraction, when the first reversed phase liquid chromatography column port 414, the first solid phase extraction column connecting port 412, the first solid phase extraction column channel port 413 and the first solid phase extraction column inlet 411 are connected, the fourth solvent is introduced into the first solid phase extraction column (SPE1) along the fourth solvent inlet 333 and the fraction and third solvent outlet 332, and the first fraction is eluted in the first solid phase extraction column (SPE1), and the first fraction is supplied to the first reversed phase liquid chromatography column (COL1). In addition, in order to simultaneously balance the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) and separate and analyze the first fraction, when the second reversed phase liquid chromatography column port 434, the second solid phase extraction column connecting port 432, the second solid phase extraction column channel port 433 and the second solid phase extraction column inlet 431 are connected, the third solvent is introduced into the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) along the fractions and the third solvent inlet 331 and the fourth solvent outlet 334 to balance the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2).

[0109] In addition, refer to Figure 8 , describes the operation of supplying the second fraction to the second solid phase extraction column (SPE2), and the second fraction is supplied to the second solid phase extraction column (SPE2) when the second solid phase extraction column connection port 432, the second solid phase extraction column inlet 431, the fourth solvent outlet 334, the fraction and the third solvent inlet 331, and the third solvent outlet 314 are connected. In addition, the dual column valve 400 further includes a second outlet 453 disposed near the second solid phase extraction column channel port 433 and connected to or disconnected from the second solid phase extraction column channel port 433, and when the second fraction is supplied to the second solid phase extraction column (SPE2) and concentrated, the salt is discharged through the second outlet 453.

[0110] In addition, refer to Fig. 9 In order to inject the second fraction into the second reversed phase liquid chromatography column (COL2) and separate and analyze the second fraction, when the second reversed phase liquid chromatography column port 434, the second solid phase extraction column connecting port 432, the second solid phase extraction column channel port 433 and the second solid phase extraction column inlet 431 are connected, the fourth solvent is introduced into the second solid phase extraction column (SPE2) along the fourth solvent inlet 333 and the fourth solvent outlet 334, and the second fraction is eluted in the second solid phase extraction column (SPE2), and the second fraction is supplied to the second reversed phase liquid chromatography column (COL2).

[0111] In addition, in order to simultaneously balance the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) and separate and analyze the second fraction, when the first reversed phase liquid chromatography column port 414, the first solid phase extraction column connecting port 412, the first solid phase extraction column channel port 413 and the first solid phase extraction column inlet 411 are connected, the third solvent is introduced into the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) along the fraction and third solvent inlet 331 and the fraction and third solvent outlet 332 to balance the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1).

[0112] In the aforementioned scheme, the first fraction to the tenth fraction are supplied to the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) in an alternating manner, and then separated and analyzed, and the first solid phase extraction column (SPE1) and the first reversed phase liquid chromatography column (COL1) or the second solid phase extraction column (SPE2) and the second reversed phase liquid chromatography column (COL2) are equilibrated at the same time.

[0113] It is obvious to those skilled in the art that the present disclosure is not limited to the disclosed embodiments, and various modifications and changes can be made thereto without departing from the spirit and scope of the present disclosure. Therefore, such modifications or changes fall within the appended claims.

[0114] Industrial Applicability

[0115] The present disclosure can improve fractionation reproducibility and prevent sample loss by automating discontinuous sample fractionation and cascade processes with low sample complexity and high fraction uniformity.

Claims

1. A method for temporally grading and concatenating samples based on unit grading intervals, comprising the following steps: - receiving a sample to be analyzed and storing the sample in a sample storage circuit of a first sample supply valve, wherein the sample is supplied from a sample syringe of a sample supply module, wherein the sample supply module comprises: A first sample supply valve connected to the first pump and the sample syringe; and a second sample supply valve connected to the first sample supply valve; The first sample supply valve comprises: A first sample inlet, connected to the sample syringe; a first sample outlet, which is disposed near the first sample inlet; a first solvent inlet connected to the first pump; a first solvent outlet disposed adjacent to the first solvent inlet and connected to the second sample supply valve; and A first sample storage loop connection port and a second sample storage loop connection port, which are connected to two ends of the sample storage loop respectively; wherein the sample is stored in the sample storage loop so as to be in a state where the first sample storage loop connection port and the second sample storage loop connection port are connected to the first sample inlet and the first sample outlet, respectively; The second sample supply valve comprises: a second sample inlet connected to the first solvent outlet; A second sample outlet, which is disposed near the second sample inlet and connected to the sample classification module; and a sample separation column having a first end and a second end, wherein the first end is connected to the second sample inlet, and the second end is connected to the second sample outlet; - supplying a first solvent from the first pump and injecting a sample stored in a sample storage loop into a sample separation column of a second sample supply valve; wherein the first solvent is injected into the sample storage circuit and the sample is injected into the sample separation column in a state where the first sample storage circuit connection port and the second sample storage circuit connection port are connected to the first solvent inlet and the first solvent outlet, respectively; - supplying a first solvent from the first pump and separating and eluting the sample injected into the sample separation column; - continuously introducing the separated and eluted samples into a sample fractionation module; and - During the unit sample supply time, injecting the sample separated and eluted in the sample fractionation module into each of a plurality of fraction storage loops for a unit fractionation interval, and discontinuously fractionating, cascading and storing the sample by repeating the unit sample supply time multiple times; The sample classification module includes a plurality of fraction storage loops, which are arranged corresponding to the number of unit classification intervals; wherein the unit fractionation intervals are set in the same order in the unit sample supply time of the total sample supply time to obtain the plurality of fractions; wherein the total sample supply time is the entire duration during which the sample separated and eluted in the sample separation column is supplied from the sample separation column of the second supply valve to the sample fractionation module and stored in the plurality of fraction storage loops; Wherein the unit sample supply time is the chronologically averaged division of the total sample supply time; and The unit grading interval is the average division of the unit sample supply time into the number of final grades in chronological order.

2. The method according to claim 1, wherein the sample classification module further comprises: a first fractionation valve connected to the second sample supply valve to receive the sample from the second sample outlet in the step of continuously introducing the separated and eluted sample into the sample separation column; A second grading valve disposed adjacent to the first grading valve; and a plurality of fraction storage circuits provided corresponding to the number of unit fractionation intervals, one end of each of the fraction storage circuits being connected to the first fractionation valve and the other end being connected to the second fractionation valve, so as to inject the sample in the sample fractionation module into each of the plurality of fraction storage circuits for the unit fractionation interval during a unit sample supply time, and to cascade and store the separated samples supplied by the sample separation column in the unit fractionation intervals provided in the same time sequence in each unit sample supply time during the total sample supply time by repeating the steps of non-continuously fractionating, cascading and storing the samples for the unit sample supply time a plurality of times, and The steps of injecting the sample in the sample classification module into each of the plurality of fraction storage loops for a unit classification interval during the unit sample supply time, and discontinuously classifying, cascading and storing the sample by repeating the unit sample supply time multiple times include: simultaneously switching the first fractionation valve and the second fractionation valve to inject the sample separated and eluted in the sample separation column into each of the plurality of fraction storage loops corresponding to unit fractionation intervals sequentially arranged during the unit sample supply time; and The step of switching is repeated per unit sample supply time to discontinuously fractionate, cascade, and store the sample separated and eluted in the sample separation column in a plurality of fraction storage loops.

3. The method of claim 2, wherein the first grading valve comprises: a fraction inlet through which the sample separated and eluted in the sample separation column is continuously introduced by the second sample supply valve; a plurality of first fraction storage circuit connection ports, which are arranged near the fraction inlet, each of which is connected to one end of the plurality of fraction storage circuits; and a first connection channel that connects the fraction inlet and one of the plurality of first fraction storage circuit connection ports so as to communicate with each other corresponding to the unit fractionation interval; as well as The second grading valve comprises: a plurality of second fraction storage circuit connection ports, which are respectively connected to the other ends of the plurality of fraction storage circuits; a fraction outlet disposed near the plurality of second fraction storage circuit connection ports and connected to the other end of one of the plurality of fraction storage circuits to discharge a fraction; and A second connection passage connects the fraction outlet and one of the plurality of second fraction storage circuit connection ports so as to communicate with each other corresponding to the unit fractionation interval.

4. The method according to claim 1, further comprising the steps of: -Set the total sample supply time; -Set unit sample supply time; and -Set the unit grade interval.

5. A method for controlling a dual online multidimensional liquid chromatography system, comprising: -By a non-continuous sample fractionation and cascade device, samples are fractionated and cascaded in time sequence based on a unit fractionation interval to obtain a plurality of sample fractions, the non-continuous sample fractionation and cascade device comprising a sample supply module and a sample fractionation module, the sample supply module comprising: a first sample supply valve connected to a first pump and a sample syringe; a first pump supplying a first solvent; and a second sample supply valve connected to the first sample supply valve to receive a sample supplied by the first sample supply valve; the second sample supply valve is used to separate and elute the sample injected into a sample separation column; the sample fractionation module is connected to the second sample supply valve to receive the separated and eluted sample, and the sample fractionation module separates the separated and eluted sample during a unit sample supply time. and the eluted samples are non-continuously fractionated, cascaded and stored in each of a plurality of fraction storage circuits for a continuous unit fractionation interval; wherein the sample fractionation module comprises a plurality of fraction storage circuits, which are arranged corresponding to the number of unit fractionation intervals; wherein the unit fractionation intervals are arranged in the same order in a unit sample supply time of a total sample supply time to obtain a plurality of fractions; wherein the total sample supply time is the entire duration during which the sample separated and eluted in the sample separation column is supplied from the sample separation column of the second supply valve to the sample fractionation module and stored in the plurality of fraction storage circuits; wherein the unit sample supply time is an average division of the total sample supply time in a time sequence; and wherein the unit fractionation interval is an average division of the final fraction number of the unit sample supply time in a time sequence; - supplying the fractions to the column selection module in sequential order; - supplying the fractions from the column selection module to the dual column valve in sequential order; The double-column valve comprises a first reversed-phase liquid chromatography column, a second reversed-phase liquid chromatography column, a first solid-phase extraction column and a second solid-phase extraction column, wherein the first solid-phase extraction column is connected to the first reversed-phase liquid chromatography column, and the second solid-phase extraction column is connected to the second reversed-phase liquid chromatography column; The step of supplying the fractions from the column selection module in a sequential order supplies the fractions to the first solid phase extraction column and the first reverse phase liquid chromatography column or the second solid phase extraction column and the second reverse phase liquid chromatography column in an alternating manner.

6. The method according to claim 5, wherein the step of temporally fractionating and concatenating samples based on a unit fractionation interval to obtain a plurality of sample fractions comprises: - receiving a sample to be analyzed and storing the sample in a sample storage circuit of a first sample supply valve, wherein the sample is supplied from a sample syringe, The first sample supply valve comprises: A first sample inlet, connected to the sample syringe; a first sample outlet, which is disposed near the first sample inlet; a first solvent inlet connected to the first pump; a first solvent outlet disposed adjacent to the first solvent inlet and connected to the second sample supply valve; and A first sample storage loop connection port and a second sample storage loop connection port, which are connected to two ends of the sample storage loop respectively; wherein the sample is stored in the sample storage loop so as to be in a state where the first sample storage loop connection port and the second sample storage loop connection port are connected to the first sample inlet and the first sample outlet, respectively; The second sample supply valve comprises: a second sample inlet connected to the first solvent outlet; A second sample outlet, which is disposed near the second sample inlet and connected to the sample classification module; and a sample separation column having a first end and a second end, wherein the first end is connected to the second sample inlet, and the second end is connected to the second sample outlet; - supplying a first solvent from the first pump and injecting a sample stored in a sample storage loop into a sample separation column of a second sample supply valve; wherein the first solvent is injected into the sample storage circuit and the sample is injected into the sample separation column in a state where the first sample storage circuit connection port and the second sample storage circuit connection port are connected to the first solvent inlet and the first solvent outlet, respectively; - supplying a first solvent from the first pump and separating and eluting the sample injected into the sample separation column; - continuously introducing the separated and eluted samples into a sample fractionation module; and - During the unit sample supply time, the separated and eluted sample is injected into each of a plurality of fraction storage loops for a unit fractionation interval, and the sample is discontinuously fractionated, cascaded, and stored by repeating the unit sample supply time multiple times.

7. The method according to claim 6, wherein the sample classification module comprises: a first fractionation valve connected to the second sample supply valve to receive the sample from the second sample outlet in the step of continuously introducing the separated and eluted sample into the sample separation column; A second grading valve disposed adjacent to the first grading valve; and a plurality of fraction storage circuits provided corresponding to the number of unit fractionation intervals, one end of each of the fraction storage circuits being connected to the first fractionation valve and the other end being connected to the second fractionation valve, so as to inject the sample in the sample fractionation module into each of the plurality of fraction storage circuits for the unit fractionation interval during a unit sample supply time, and to cascade and store the separated samples supplied by the sample separation column in the unit fractionation intervals provided in the same time sequence in each unit sample supply time during the total sample supply time by repeating the steps of non-continuously fractionating, cascading and storing the samples for the unit sample supply time a plurality of times, and The steps of injecting the sample in the sample classification module into each of the plurality of fraction storage loops for a unit classification interval during the unit sample supply time, and discontinuously classifying, cascading and storing the sample by repeating the unit sample supply time multiple times include: simultaneously switching the first fractionation valve and the second fractionation valve to inject the sample separated and eluted in the sample separation column into each of the plurality of fraction storage loops corresponding to unit fractionation intervals sequentially arranged during the unit sample supply time; and The step of switching is repeated per unit sample supply time to discontinuously fractionate, cascade, and store the sample separated and eluted in the sample separation column in a plurality of fraction storage loops.

8. The method of claim 7, wherein the first grading valve comprises: a fraction inlet through which the sample separated and eluted in the sample separation column is continuously introduced from the second sample supply valve; A plurality of first fraction storage circuit connection ports, which are arranged near the fraction inlet, each of which is connected to one end of the plurality of fraction storage circuits; and a first connection channel that connects the fraction inlet and one of the plurality of first fraction storage circuit connection ports so as to communicate with each other corresponding to the unit fractionation interval; as well as The second grading valve comprises: a plurality of second fraction storage circuit connection ports, each connected to the other end of the plurality of fraction storage circuits; a fraction outlet disposed near the plurality of second fraction storage circuit connection ports and connected to the other end of one of the plurality of fraction storage circuits to discharge the fraction; and A second connection passage connects the fraction outlet and one of the plurality of second fraction storage circuit connection ports so as to communicate with each other corresponding to the unit fractionation interval.

9. The method according to claim 8, further comprising the step of equilibrating the first solid phase extraction column and the first reverse phase liquid chromatography column or the second solid phase extraction column and the second reverse phase liquid chromatography column in an alternating manner.

10. The method according to claim 9, wherein the column selection module comprises: a column balancing valve connected to the first fraction outlet to provide a passage through which the steps of supplying the plurality of fractions to the first solid phase extraction column or the second solid phase extraction column in an alternating manner and balancing the first solid phase extraction column and the first reverse phase liquid chromatography column or the second solid phase extraction column and the second reverse phase liquid chromatography column in an alternating manner occur; and a column selection valve connected to the column balancing valve to receive the supply of the plurality of fractions and to supply the plurality of fractions to the first solid phase extraction column or the second solid phase extraction column in an alternating manner so as to elute the fractions in the first solid phase extraction column with the first reverse phase liquid chromatography column or in the second solid phase extraction column with the second reverse phase liquid chromatography column.

11. The method according to claim 10, further comprising the steps of: - supplying a second solvent at the column balancing valve via a second fraction inlet connected to the first fraction outlet and a second pump; - introducing a third solvent into the column balancing valve via a third solvent inlet connected to a third pump; wherein the column balancing valve comprises: a second fraction inlet connected to the first fraction outlet and a second pump for supplying a second solvent; a third solvent inlet connected to a third pump for introducing a third solvent; a second fraction outlet disposed near the second fraction inlet and selectively connected to the second fraction inlet and the third solvent inlet; and a third solvent outlet disposed near the third solvent inlet and selectively connected to the second fraction inlet and the third solvent inlet; In a case where the second fraction inlet is connected to the third solvent outlet, the fraction injected with the second solvent is supplied to the first solid phase extraction column or the second solid phase extraction column via the column selection valve; and When the third solvent inlet is connected to the third solvent outlet, the third solvent balances the first solid phase extraction column and the first reverse phase liquid chromatography column or the second solid phase extraction column and the second reverse phase liquid chromatography column in an alternating manner via the column selection valve.

12. The method according to claim 11, further comprising the step of introducing a fourth solvent at the column selection valve; Wherein the column selection valve comprises: a fraction and third solvent inlet connected to the third solvent outlet for introducing the fraction or the third solvent; a fourth solvent inlet connected to a fourth pump and used for introducing the fourth solvent; a fraction and third solvent outlet disposed near the fraction and third solvent inlet and selectively connected to the fraction and third solvent inlet and the fourth solvent inlet; and a fourth solvent outlet, which is disposed near the fourth solvent inlet and selectively connected to the fraction and the third solvent inlet and the fourth solvent inlet, When the fraction and third solvent inlet is connected to the fraction and third solvent outlet, the fraction is supplied to the first solid phase extraction column, and then the fourth solvent elutes the fraction with the first reverse phase liquid chromatography column in the first solid phase extraction column in the case where the fourth solvent inlet is connected to the fraction and third solvent outlet, and the third solvent equilibrates the second solid phase extraction column and the second reverse phase liquid chromatography column in the case where the fraction and third solvent inlet is connected to the fourth solvent outlet, and When the fraction and third solvent inlet is connected to the fourth solvent outlet, the fraction is supplied to the second solid phase extraction column, and then the fourth solvent elutes the fraction with the second reverse phase liquid chromatography column in the second solid phase extraction column in the case where the fourth solvent inlet is connected to the fourth solvent outlet, and the third solvent equilibrates the first solid phase extraction column and the first reverse phase liquid chromatography column in the case where the fraction and third solvent inlet is connected to the fraction and third solvent outlet.

13. The method according to claim 12, wherein the dual column valve comprises: A first solid phase extraction column connection port and a first solid phase extraction column channel port, each of which is connected to two ends of the first solid phase extraction column; a first solid phase extraction column inlet connected to the fraction and the third solvent outlet, and selectively connected to the first solid phase extraction column connection port and the first solid phase extraction column channel port; A first reversed phase liquid chromatography column port, which is connected to the first reversed phase liquid chromatography column and connected to or disconnected from the first solid phase extraction column connection port; A second solid phase extraction column connection port and a second solid phase extraction column channel port, each of which is connected to two ends of the second solid phase extraction column; a second solid phase extraction column inlet, connected to the fourth solvent outlet, and selectively connected to the second solid phase extraction column connection port and the second solid phase extraction column channel port; and The second reversed phase liquid chromatography column port is connected to the second reversed phase liquid chromatography column and is connected to or disconnected from the second solid phase extraction column connection port.

14. The method according to claim 13, wherein the dual column valve further comprises: A first outlet, which is arranged near the first solid phase extraction column channel port and is connected to or disconnected from the first solid phase extraction column channel port; and The second outlet is arranged near the first outlet and is connected to or disconnected from the second solid phase extraction column channel port.

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