US20190017974A1 - Unit for non-continuous sample fractionation and integration, and dual online multifunctional liquid chromatography device having same - Google Patents

Unit for non-continuous sample fractionation and integration, and dual online multifunctional liquid chromatography device having same Download PDF

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US20190017974A1
US20190017974A1 US16/070,100 US201616070100A US2019017974A1 US 20190017974 A1 US20190017974 A1 US 20190017974A1 US 201616070100 A US201616070100 A US 201616070100A US 2019017974 A1 US2019017974 A1 US 2019017974A1
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sample
fraction
solvent
column
inlet port
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Sang-won Lee
Hangyeore LEE
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Korea University Research and Business Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/80—Fraction collectors
    • G01N30/82—Automatic means therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/24—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the treatment of the fractions to be distributed
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/201—Injection using a sampling valve multiport valves, i.e. having more than two ports
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/202—Injection using a sampling valve rotary valves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/207—Injection using a sampling valve with metering cavity, e.g. sample loop
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional chromatography

Definitions

  • the present disclosure relates to a non-continuous sample fractionating and concatenating device and a dual online multidimensional liquid chromatography system having the same, and more particularly, a non-continuous sample fractionating and concatenating device and a dual online multidimensional liquid chromatography system having the same to achieve low sample complexity and high fraction uniformity.
  • the separation orthogonality refers to independency of each separation modes, which means that sample mixtures are separated by different physiochemical properties by each separation mode.
  • One of two-dimensional separation methods that have been developed and widely used in recent years is a method including fractionating a sample according to the degree of hydrophobicity at alkaline pH and separating the separated fractions again according to the degree of hydrophobicity under acidic pH condition (two-dimensional reversed-phase liquid chromatography-reversed-phase liquid chromatography, 2D RP-RPLC).
  • two separation modes that separate the mixture by hydrophobicity does not have separation orthogonality, but peptides under different pH conditions have different charge distributions depending on unique amino acid composition, and accordingly, even the same peptide can have different degrees of hydrophobicity. Due to this, the 2D RP-RPLC method has separation orthogonality.
  • FIG. 1 shows a distribution of identified peptides at the elution time of each separation method after RPLC separation at pH 10, RPLC separation of each fraction at pH2.6 again and analysis using a mass analyzer. Seeing FIG. 1 , it is observed that there are many areas where peptides are not found, which implies that the 2D RP-RPLC method did not exploit all separation spaces of two-dimensional separation.
  • the non-continuous concatenation after fractionation includes fractionation into 96 (1 st to 96 th ) in the order of elution of the first dimension RLPC separation under alkaline pH and non-continuous concatenation into 24 fractions.
  • Nos. 1 to 24 become 24 fractions
  • No. 25 is concatenated into the first fraction
  • No. 26 is concatenated into the second fraction.
  • Nos. 1, 25, 49 and 73 fractions are concatenated into the first fraction
  • Nos. 2, 26, 50 and 74 are concatenated into the second fraction
  • Nos. 24, 48, 72 and 96 are concatenated into the last 24th fraction.
  • 2D RP-RPLC achieved high separation orthogonality.
  • the present disclosure is directed to providing a non-continuous sample fractionating and concatenating device and a dual online multidimensional liquid chromatography system having the same for improving reproducibility of fractionation and preventing a sample loss through automation of non-continuous sample fractionation and concatenation process with low sample complexity and high fraction uniformity.
  • a non-continuous sample fractionating and concatenating device including a sample supply module which supplies a sample to be analyzed, and a sample fractionation module connected to the sample supply module, and which is continuously supplied with the sample, sets a plurality of unit sample supply times obtained by equally dividing a total sample supply time during which the sample is supplied from the sample supply module, sets a plurality of unit fractionation intervals obtained by equally dividing each of the plurality of unit sample supply times, and concatenates and stores the sample supplied during corresponding unit fractionation intervals within each unit sample supply time to acquire a plurality of fractions.
  • the sample fractionation module may include a first fractionation valve connected to the sample supply module, into which the sample is introduced, a second fractionation valve provided adjacent to the first fractionation valve, and a plurality of fraction storage loops provided corresponding to a number of the plurality of unit fractionation intervals within the unit sample supply time, each having one end connected to the first fractionation valve and the other end connected to the second fractionation valve, to concatenate and store the sample supplied during the corresponding unit fractionation intervals within each of the unit sample supply times in a sequential order.
  • the first fractionation valve may include a fraction inlet port through which the sample is introduced from the sample supply module, a plurality of first fraction storage loop connection ports provided adjacent to the fraction inlet port, each connected to one end of the plurality of fraction storage loops, and a first connecting channel connecting the fraction inlet port and one of the plurality of first fraction storage loop connection ports in communication with each other corresponding to the unit fractionation interval
  • the second fractionation valve may include a plurality of second fraction storage loop connection ports to which the other end of the plurality of fraction storage loops is each connected, a fraction outlet port provided adjacent to the plurality of second fraction storage loop connection ports, and connected to the other end of one of the plurality of fraction storage loops to discharge the fraction, and a second connecting channel connecting the fraction outlet port and one of the plurality of second fraction storage loop connection ports in communication with each other corresponding to the unit fractionation interval.
  • the sample supply module may include a first sample supply valve to which a first pump and a sample injector are connected, wherein the first pump supplies a first solvent, and a second sample supply valve connected to the first sample supply valve to receive the sample supplied from the first sample supply valve and supply the sample to the fraction inlet port.
  • the first sample supply valve may include a first sample inlet port connected to the sample injector, a first sample outlet port provided adjacent to the first sample inlet port, a first solvent inlet port connected to the first pump, a first solvent outlet port provided adjacent to the first solvent inlet port and connected to the second sample supply valve, and a first sample storage loop connection port and a second sample storage loop connection port to which two ends of a sample storage loop are each connected, and in a state that the first sample storage loop connection port and the second sample storage loop connection port are each connected to the first sample inlet port and the first sample outlet port, the sample may be stored in the sample storage loop, and in a state that the first sample storage loop connection port and the second sample storage loop connection port are each connected to the first solvent inlet port and the first solvent outlet port, the first solvent may be injected into the sample storage loop to supply the sample to the second sample supply valve.
  • the second sample supply valve may include a second sample inlet port connected to the first solvent outlet port, a second sample outlet port provided adjacent to the second sample inlet port and connected to the fraction inlet port, and a sample separation column having two ends, each connected to the second sample inlet port and the second sample outlet port, so that the sample is eluted at the second sample outlet port, and the sample eluted in the sample separation column may be supplied to the fraction inlet port.
  • a dual online multidimensional liquid chromatography system including a non-continuous sample fractionating and concatenating device which is continuously supplied with a sample to be analyzed, sets a plurality of unit sample supply times obtained by equally dividing a total sample supply time during which the sample is supplied, sets a plurality of unit fractionation intervals obtained by equally dividing each of the plurality of unit sample supply times, and supplies a plurality of fractions acquired by concatenating and storing the sample supplied during corresponding unit fractionation intervals within each unit sample supply time, a dual column valve to which a first reversed-phase liquid chromatography column and a second reversed-phase liquid chromatography column are connected, and having 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 provided between the non-continuous sample fractionating and concatenating device and the dual column valve to supply the fractions supplied
  • the non-continuous sample fractionating and concatenating device may include a sample supply module which supplies the sample, and a sample fractionation module connected to the sample supply module to continuously receive the sample supplied from the sample supply module to acquire a plurality of fractions and supply the plurality of fractions to the column selection module in a sequential order.
  • the sample fractionation module may include a first fractionation valve connected to the sample supply module, into which the sample is introduced, a second fractionation valve provided adjacent to the first fractionation valve, and a plurality of fraction storage loops provided corresponding to a number of the plurality of unit fractionation intervals within the unit sample supply time, each having one end connected to the first fractionation valve and the other end connected to the second fractionation valve, to concatenate and store the sample supplied during the corresponding unit fractionation intervals within each of the unit sample supply times in a sequential order.
  • the first fractionation valve may include a first fraction inlet port through which the sample is introduced from the sample supply module, a plurality of first fraction storage loop connection ports provided adjacent to the first fraction inlet port, to which one end of the plurality of fraction storage loops is each connected, and a first connecting channel connecting the fraction inlet port and one of the plurality of first fraction storage loop connection ports in communication with each other corresponding to the unit fractionation interval
  • the second fractionation valve may include a plurality of second fraction storage loop connection ports to which the other end of the plurality of fraction storage loops is each connected, a first fraction outlet port provided adjacent to the plurality of second fraction storage loop connection ports, the first fraction outlet port to which the other end of one of the plurality of fraction storage loops is connected to discharge the fraction, and a second connecting channel connecting the first fraction outlet port and one of the plurality of second fraction storage loop connection ports in communication with each other corresponding to the unit fractionation interval.
  • the sample supply module may include a first sample supply valve having a first sample inlet port connected to a sample injector, a first sample outlet port provided adjacent to the first sample inlet port, a first solvent inlet port connected to a first pump, a first solvent outlet port provided adjacent to the first solvent inlet port, and a first sample storage loop connection port and a second sample storage loop connection port to which two ends of a sample storage loop are each connected, and a second sample supply valve having a second sample inlet port connected to the first solvent outlet port, a second sample outlet port provided adjacent to the second sample inlet port and connected to the first fraction inlet port, and a sample separation column having two ends, each connected to the second sample inlet port and the second sample outlet port, so that the sample is eluted at the second sample outlet port.
  • the column selection module may include a column equilibration valve connected to the first fraction outlet port to provide a channel through which the plurality of fractions is supplied to the first solid phase extraction column or the second solid phase extraction column in alternating manner, and to equilibrate 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 alternating manner, and a column selection valve connected to the column equilibration valve to receive the supply of the plurality of fractions and supply the plurality of fractions to the first solid phase extraction column or the second solid phase extraction column in alternating manner, so that the fraction is eluted with the first reversed-phase liquid chromatography column in the first solid phase extraction column or the second reversed-phase liquid chromatography column in the second solid phase extraction column.
  • a column equilibration valve connected to the first fraction outlet port to provide a channel through which the plurality of fractions is supplied to the first solid phase extraction column or the second solid phase extraction column
  • the column equilibration valve may include a second fraction inlet port connected to the first fraction outlet port and a second pump which supplies a second solvent, a third solvent inlet port connected to a third pump which supplies a third solvent, through which the third solvent is introduced, a second fraction outlet port provided adjacent to the second fraction inlet port and selectively connected to the second fraction inlet port and the third solvent inlet port, and a third solvent outlet port provided adjacent to the third solvent inlet port and selectively connected to the second fraction inlet port and the third solvent inlet port, and in a state that the second fraction inlet port and the third solvent outlet port are connected, the fraction into which the second solvent is injected may be supplied to the first solid phase extraction column or the second solid phase extraction column via the column selection valve, and in a state that the third solvent inlet port and the third solvent outlet port are connected, the third solvent may equilibrate 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
  • the column selection valve may include a fraction and third solvent inlet port connected to the third solvent outlet port, through which the fraction or the third solvent is introduced, a fourth solvent inlet port connected to a fourth pump which supplies a fourth solvent, through which the fourth solvent is introduced, a fraction and third solvent outlet port provided adjacent to the fraction and third solvent inlet port and selectively connected to the fraction and third solvent inlet port and the fourth solvent inlet port, and a fourth solvent outlet port provided adjacent to the fourth solvent inlet port and selectively connected to the fraction and third solvent inlet port and the fourth solvent inlet port, and as the fraction and third solvent inlet port and the fraction and third solvent outlet port are connected, the fraction may be supplied to the first solid phase extraction column, and then in a state that the fourth solvent inlet port and the fraction and third solvent outlet port are connected, the fourth solvent may elute the fraction with the first reversed-phase liquid chromatography column in the first solid phase extraction column, and in a state that the fraction and third solvent inlet port and the fourth solvent outlet port are connected, the third solvent may
  • the dual column valve may include a first solid phase extraction column connection port and a first solid phase extraction column channel port, each connected to two ends of the first solid phase extraction column, a first solid phase extraction column inlet port connected to the fraction and third solvent outlet port, 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 connected to the first reversed-phase liquid chromatography column, and connected or disconnected to/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 connected to two ends of the second solid phase extraction column, a second solid phase extraction column inlet port connected to the fourth solvent outlet port, 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 connected to the second reversed-phase liquid chromatography column, and connected or disconnected to/from the second solid phase extraction column connection port.
  • the dual column valve may further include a first outlet port provided adjacent to the first solid phase extraction column channel port, and connected or disconnected to/from the first solid phase extraction column channel port, and a second outlet port provided adjacent to the first outlet port, and connected or disconnected to/from the second solid phase extraction column channel port.
  • the embodiment of the present disclosure is provided with a non-continuous sample fractionating and concatenating device which concatenates and stores a sample supplied during corresponding unit fractionation intervals within each unit sample supply time to automatically acquire a plurality of fractions, thereby reducing sample complexity and increasing fraction uniformity, and further, improving reproducibility of fractionation and preventing a sample loss.
  • FIG. 1 is a diagram showing a distribution of identified peptides after RPLC separation at pH 10, RPLC separation of each fraction at pH2.6 again and analysis using a mass analyzer.
  • FIG. 2 is a diagram illustrating non-continuous concatenation after sample fractionation.
  • FIG. 3 is a diagram showing an operation of acquiring a first fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.
  • FIG. 4 is a diagram showing an operation of acquiring a second fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.
  • FIG. 5 is a diagram showing an operation of acquiring a tenth fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.
  • FIG. 6 is a diagram showing an operation of 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.
  • FIG. 7 is a diagram showing an operation of supplying a first fraction to a first reversed-phase liquid chromatography column and equilibrating a second solid phase extraction column and a second reversed-phase liquid chromatography column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.
  • FIG. 8 is a diagram showing an operation of 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.
  • FIG. 9 is a diagram showing an operation of supplying a second fraction to a second reversed-phase liquid chromatography column and equilibrating a first solid phase extraction column and a first reversed-phase liquid chromatography column by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.
  • FIG. 3 is a diagram showing an operation of acquiring a first fraction by a dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 4 is a diagram showing an operation of acquiring a second fraction by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 5 is a diagram showing an operation of acquiring a tenth fraction by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 6 is a diagram showing an operation of supplying the first fraction to a first solid phase extraction column by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 6 is a diagram showing an operation of supplying the first fraction to a first solid phase extraction column by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 7 is a diagram showing an operation of supplying the first fraction to a first reversed-phase liquid chromatography column and equilibrating a second solid phase extraction column and a second reversed-phase liquid chromatography column by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 8 is a diagram showing an operation of supplying a second fraction to the second solid phase extraction column by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure
  • FIG. 9 is a diagram showing an operation of supplying the second fraction to the second reversed-phase liquid chromatography column and equilibrating the first solid phase extraction column and the first reversed-phase liquid chromatography column by the dual online multidimensional liquid chromatography system according to an embodiment of the present disclosure.
  • the dual online multidimensional liquid chromatography system 100 includes a non-continuous sample fractionating and concatenating device 200 which is continuously supplied with a sample to be analyzed to acquire a plurality of fractions and supplies the plurality of fractions, a dual column valve 400 to which a first reversed-phase liquid chromatography column (COL1) and a second reversed-phase liquid chromatography column (COL2) are connected and having 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 provided between the non-continuous sample fractionating and concatenating device 200 and the dual column valve 400 to supply the plurality of fractions supplied from the non-continuous sample fractionating and concatenating device 200 in a sequential order to the first solid phase extraction column (SPE1) and the first reversed-phase liquid
  • the non-continuous sample fractionating and concatenating device 200 plays a role in acquiring the plurality of fractions from the sample continuously supplied from a sample injector S.
  • unit sample supply times are set by equally dividing the total sample supply time during which the sample is supplied.
  • unit fractionation intervals are set by equally dividing each unit sample supply time.
  • the plurality of fractions is acquired by concatenating and storing the sample supplied during corresponding unit fractionation intervals within each unit sample supply time.
  • the unit sample supply times obtained by equally dividing the total sample supply time into five are at an interval of 20 minutes. That is, the first unit sample supply time corresponds to 0-20 minutes, and the second to fifth unit sample supply times correspond to 20-40 minutes, 40-60 minutes, 60-80 minutes, and 80-100 minutes, respectively.
  • the first to fifth unit sample supply times may have unit fractionation intervals obtained by equally dividing into ten.
  • a first unit fractionation interval corresponds to 0-2 minutes
  • second to tenth unit fractionation intervals correspond to 2-4 minutes, 4-6 minutes, 6-8 minutes, 8-10 minutes, 10-12 minutes, 12-14 minutes, 14-16 minutes, 16-18 minutes and 18-20 minutes, respectively.
  • a first unit fractionation interval corresponds to 20-22 minutes
  • second to tenth fractionation intervals correspond to 22-24 minutes, 24-26 minutes, 26-28 minutes, 28-30 minutes, 30-32 minutes, 32-34 minutes, 34-36 minutes, 36-38 minutes and 38-40 minutes, respectively.
  • the plurality of fractions are acquired by concatenating and storing the sample supplied during the corresponding first to tenth unit fractionation intervals within the first to fifth unit sample supply times.
  • the first fraction is acquired by concatenating and storing the sample supplied during the first unit fractionation intervals within the first to fifth unit sample supply times (i.e., 0-2 minutes, 20-22 minutes, 40-42 minutes, 60-62 minutes, 80-82 minutes).
  • the second to tenth fractions are obtained by the same operation as the operation of acquiring the first fraction, and its detailed description is omitted herein.
  • this embodiment sets the total sample supply time of 100 minutes and the first to fifth unit sample supply times, and sets ten unit fractionation intervals in each unit sample supply time, the scope of protection of the present disclosure is not limited thereto, and the total sample supply time, the unit sample supply time and the unit fractionation interval may be variously set.
  • the non-continuous sample fractionating and concatenating device 200 includes a sample supply module 210 which supplies a sample, and a sample fractionation module 250 connected to the sample supply module 210 to continuously receive the sample supplied from the sample supply module 210 to acquire a plurality of fractions and supply the plurality of fractions to the column selection module 300 in a sequential order.
  • the sample supply module 210 plays a role in continuously supplying the sample to the sample fractionation module 250 .
  • the sample supply module 210 includes a first sample supply valve 220 to which a first pump P 1 that supplies a first solvent and a sample injector S are connected, and a second sample supply valve 240 connected to the first sample supply valve 220 to receive the sample supplied from the first sample supply valve 220 and supply the sample to the sample fractionation module 250 .
  • the first sample supply valve 220 plays a role in receiving the sample supplied from the sample injector S, storing the sample in a sample storage loop 221 , and then receiving the first solvent supplied from the first pump P 1 and supplying the sample stored in the sample storage loop 221 to the second sample supply valve 240 .
  • the first sample supply valve 220 includes a first sample inlet port 222 connected to the sample injector S, a first sample outlet port 223 provided adjacent to the first sample inlet port 222 , a first solvent inlet port 226 connected to the first pump P 1 , a first solvent outlet port 227 provided adjacent to the first solvent inlet port 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 to which two ends of the sample storage loop 221 are each connected.
  • the sample is supplied to the first sample inlet port 222 through the sample injector S and continuously stored in the sample storage loop 221 .
  • the first solvent is injected into the first solvent inlet port 226 through the first pump P 1 and the sample stored in the sample storage loop 221 is continuously supplied to the second sample supply valve 240 .
  • the first pump P 1 supplies the first solvent to the first solvent inlet port 226 , wherein the first solvent is a mixed solution with pH 7.5 of 99% of solution A, 10 mM Triethylammonium bicarbonate (TEAB) in water, and 1% of solution B, 10 mM Triethylammonium bicarbonate (TEAB) in acetonitrile.
  • the first solvent is a mixed solution with pH 7.5 of 99% of solution A, 10 mM Triethylammonium bicarbonate (TEAB) in water, and 1% of solution B, 10 mM Triethylammonium bicarbonate (TEAB) in acetonitrile.
  • the second sample supply valve 240 plays a role in continuously injecting the sample supplied from the first sample supply valve 220 into a sample separation column 243 and continuously supplying the sample injected into the sample separation column 243 to the sample fractionation module 250 .
  • the second sample supply valve 240 includes a second sample inlet port 241 connected to the first solvent outlet port 227 , a second sample outlet port 242 provided adjacent to the second sample inlet port 241 and connected to a first fraction inlet port 261 , and a sample separation column 243 having two ends, each connected to the second sample inlet port 241 and the second sample outlet port 242 , so that the sample is separated and eluted at the second sample outlet port 242 .
  • the sample stored in the sample storage loop 221 is injected into the sample separation column 243 through the first solvent outlet port 227 and the second sample inlet port 241 by the first solvent. Additionally, the sample injected into the sample separation column 243 is separated and eluted by the first solvent supplied from the first pump P 1 and supplied to the sample fractionation module 250 described below along the second sample outlet port 242 .
  • the first pump P 1 supplies the first solvent to the first solvent inlet port 226 , wherein the first solvent is a mixed solution of the solution A, 10 mM Triethylammonium bicarbonate (TEAB) in water, reducing from 99% to 50% and the solution B, 10 mM Triethylammonium bicarbonate (TEAB) in acetonitrile, increasing from 1% to 50%.
  • the sample is easily eluted in the sample separation column 243 by gradually increasing the concentration of the solution B in the first solvent.
  • the sample fractionation module 250 plays a role in acquiring ten fractions from the sample continuously supplied from the sample supply module 210 .
  • the sample fractionation module 250 includes a first fractionation valve 260 connected to the sample supply module 210 to allow the sample to be introduced through, a second fractionation valve 280 provided adjacent to the first fractionation valve 260 , and a plurality of fraction storage loops 284 a to 284 j having one end connected to the first fractionation valve 260 and the other end connected to the second fractionation valve 280 .
  • each unit sample supply time has ten unit fractionation intervals
  • ten fraction storage loops 284 a to 284 j are provided corresponding to the number of corresponding unit fractionation intervals within the unit sample supply time.
  • the first fractionation valve 260 includes a first fraction inlet port 261 through which the sample is introduced from the second sample outlet port 242 , a plurality of first fraction storage loop connection ports 262 provided adjacent to the first fraction inlet port 261 and each connected to one end of the plurality of fraction storage loops 284 a to 284 j , and a first connecting channel 263 connecting the first fraction inlet port 261 and one of the plurality of first fraction storage loop connection ports 262 in communication with each other corresponding to the unit fractionation interval.
  • ten first fraction storage loop connection ports 262 are provided corresponding to the number of ten fraction storage loops 284 a to 284 j.
  • the second fractionation valve 280 includes a plurality of second fraction storage loop connection ports 282 to which the other end of the plurality of fraction storage loops 284 a to 284 j is each connected, a first fraction outlet port 281 provided adjacent to the plurality of second fraction storage loop connection ports 282 and connected to the other end of one of the plurality of fraction storage loops 284 a to 284 j to discharge the fraction, and a second connecting channel 283 connecting the first fraction outlet port 281 to one of the plurality of second fraction storage loop connection ports 282 in communication with each other corresponding to the unit fractionation interval.
  • ten second fraction storage loop connection ports 282 are provided corresponding to the number of ten fraction storage loops 284 a to 284 j.
  • the operation of acquiring the first fraction during the first unit fractionation interval (0-2 minutes) is as below.
  • the first connecting channel 263 is connected to the first fraction storage loop connection port 262 connected to one end of the first fraction storage loop 284 a among the plurality of first fraction storage loop connection ports 262
  • the second connecting channel 283 is connected to the second fraction storage loop connection port 282 connected to the other end of the first fraction storage loop 284 a among the plurality of second fraction storage loop connection ports 282 .
  • the first solvent is supplied from the first pump P 1 to the sample separation column 243 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 284 a via the first fraction inlet port 261 through the second sample outlet port 242 and stored in the first fraction storage loop 284 a.
  • the first connecting channel 263 is connected to the first fraction storage loop connection port 262 connected to one end of the second fraction storage loop 284 b among the plurality of first fraction storage loop connection ports 262
  • the second connecting channel 283 is connected to the second fraction storage loop connection port 282 connected to the other end of the second fraction storage loop 284 b among the plurality of second fraction storage loop connection ports 282 .
  • the first solvent is supplied from the first pump P 1 to the sample separation column 243 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 loop 284 b via the first fraction inlet port 261 through the second sample outlet port 242 and stored in the second fraction storage loop 284 b.
  • the operation of acquiring the tenth fraction during the tenth unit fractionation interval (18-20 minutes) is as below.
  • the first connecting channel 263 is connected to the first fraction storage loop connection port 262 connected to one end of the tenth fraction storage loop 284 j among the plurality of first fraction storage loop connection ports 262
  • the second connecting channel 283 is connected to the second fraction storage loop connection port 282 connected to the other end of the tenth fraction storage loop 284 j among the plurality of second fraction storage loop connection ports 282 .
  • the first solvent is supplied from the first pump P 1 to the sample separation column 243 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 284 j via the first fraction inlet port 261 through the second sample outlet port 242 and stored in the tenth fraction storage loop 284 j.
  • the first to tenth fractions may be acquired by iteratively performing the operation of acquiring the first to tenth fractions during the first to tenth unit fractionation intervals within the first unit sample supply time (0-20 minutes) among the second to fifth unit sample supply times (20-40 minutes, 40-60 minutes, 60-80 minutes, 80-100 minutes).
  • the first fraction is acquired by concatenating and storing the sample supplied during the first unit fractionation intervals (i.e., 0-2 minutes, 20-22 minutes, 40-42 minutes, 60-62 minutes, 80-82 minutes) within the first to fifth unit sample supply times.
  • the first to tenth fractions are supplied to the first reversed-phase liquid chromatography column (COL1) or the second reversed-phase liquid chromatography column (COL2).
  • the column selection module 300 plays a role in supplying the first to tenth fractions supplied from 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 dual column valve 400 described below in alternating manner.
  • the column selection module 300 plays a role in supplying the first to tenth fractions 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 performing fraction separation and analysis, and at the same time, performing an equilibration 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).
  • the column selection module 300 includes a column equilibration valve 310 connected to the first fraction outlet port 281 to provide a channel through which the plurality of fractions are supplied to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in alternating manner, and to equilibrate 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 alternating manner, and a column selection valve 330 connected to the column equilibration valve 310 to receive the supply of the plurality of fractions and supply the fractions to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in alternating manner so that the fraction is separated and eluted with the first reversed-phase liquid chromatography column (COL1) in the first solid phase extraction column (SPE1) or the second reversed-phase liquid chromatography column (COL2) in the second solid phase extraction column (S
  • the column equilibration valve 310 plays a role in providing a channel through which the first to tenth fractions are supplied to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in alternating manner.
  • the column equilibration valve 310 includes a second fraction inlet port 311 connected to the first fraction outlet port 281 and a second pump P 2 which supplies a second solvent, a third solvent inlet port 313 connected to a third pump P 3 which supplies a third solvent to allow the third solvent to be introduced through, a second fraction outlet port 312 provided adjacent to the second fraction inlet port 311 and selectively connected to the second fraction inlet port 311 and the third solvent inlet port 313 , and a third solvent outlet port 314 provided adjacent to the third solvent inlet port 313 and selectively connected to the second fraction inlet port 311 and the third solvent inlet port 313 .
  • the first fraction stored in the first fraction storage loop 284 a is supplied to the first solid phase extraction column (SPE1) of the dual selection valve described below.
  • the second fraction stored in the second fraction storage loop 284 b is supplied to the second solid phase extraction column (SPE2) of the dual section valve described below.
  • the first to tenth fractions stored in the first to tenth fraction storage loops 284 a to 284 j may be supplied to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in alternating manner in a state that the first fraction outlet port 281 , the second fraction inlet port 311 and the third solvent outlet port 314 are connected.
  • the first to tenth fractions discharged from the first to tenth fraction storage loops 284 a to 284 j are supplied with the second solvent through the second pump P 2 placed in a line connecting the first fraction outlet port 281 and the second fraction inlet port 311 .
  • the second pump P 2 supplies the second solvent, 0.2% Trifluoroacetic acid (TFA) in water.
  • the first pump P 1 supplies the first solvent to the first solvent inlet port 226 , wherein the first solvent is a mixed solution of 99% of solution A, 10 mM Triethylammonium bicarbonate (TEAB) in water, and 1% of solution B, 10 mM Triethylammonium bicarbonate (TEAB) in acetonitrile. Additionally, a ratio of amounts of the first solvent supplied from the first pump P 1 and the second solvent supplied from the second pump P 2 may be 1:9.
  • the first solvent is a mixed solution of 99% of solution A, 10 mM Triethylammonium bicarbonate (TEAB) in water, and 1% of solution B, 10 mM Triethylammonium bicarbonate (TEAB) in acetonitrile.
  • a ratio of amounts of the first solvent supplied from the first pump P 1 and the second solvent supplied from the second pump P 2 may be 1:9.
  • 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 acidize the first to tenth fractions, thereby preventing a 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.
  • the third solvent equilibrates the second solid phase extraction column (SPE2) and the second reversed-phase liquid chromatography column (COL2) via the column selection valve 330 .
  • the third solvent equilibrates the first solid phase extraction column (SPE1) and the first reversed-phase liquid chromatography column (COL1) via the column selection valve 330 .
  • the third solvent is not limited thereto and includes any solution in water 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).
  • the column selection valve 330 plays a role in receiving the plurality of fractions supplied from the column equilibration valve 310 and supplying the plurality of fractions to the first solid phase extraction column (SPE1) or the second solid phase extraction column (SPE2) in alternating manner. Additionally, the column selection valve 330 plays a role in supplying a fourth solvent to the first solid phase extraction column (SPE1) so that the fraction is eluted with the first reversed-phase liquid chromatography column (COL1) in the first solid phase extraction column (SPE1), or supplying a fourth solvent to the second solid phase extraction column (SPE2) so that the fraction is eluted with the second reversed-phase liquid chromatography column (COL2) in the second solid phase extraction column (SPE2).
  • the column selection valve 330 includes a fraction and third solvent inlet port 331 connected to the third solvent outlet port 314 to allow the fraction or the third solvent to be introduced through, a fourth solvent inlet port 333 connected to a fourth pump P 4 which supplies the fourth solvent to allow the fourth solvent to be introduced through, a fraction and third solvent outlet port 332 provided adjacent to the fraction and third solvent inlet port 331 and selectively connected to the fraction and third solvent inlet port 331 and the fourth solvent inlet port 333 , and a fourth solvent outlet port 334 provided adjacent to the fourth solvent inlet port 333 and selectively connected to the fraction and third solvent inlet port 331 and the fourth solvent inlet port 333 .
  • the fourth solvent in a state that the fourth solvent inlet port 333 and the fraction and third solvent outlet port 332 are connected, the fourth solvent elutes the first fraction with the first reversed-phase liquid chromatography column (COL1) in the first solid phase extraction column (SPE1), and in a state that the fraction and third solvent inlet port 331 , the fourth solvent outlet port 334 and the third solvent outlet port 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 equilibrate the second solid phase extraction column (SPE2) and the second reversed-phase liquid chromatography column (COL2).
  • the fourth pump P 4 supplies the fourth solvent to the fourth solvent inlet port 333 , wherein the fourth solvent is a mixed solution of solution C, 0.1% formic acid in water, reducing from 99% to 60%, and solution D, 0.1% formic acid in acetonitrile, increasing from 1% to 40%.
  • the gradually increasing concentration of the solution D in the fourth solvent allows for easy elution of the first fraction in the first solid phase extraction column (SPE1) or easy elution of the second fraction in the second solid phase extraction column (SPE2).
  • the fourth solvent in a state that the fourth solvent inlet port 333 and the fourth solvent outlet port 334 are connected, the fourth solvent elutes the second fraction with the second reversed-phase liquid chromatography column (COL2) in the second solid phase extraction column (SPE2), and in a state that the fraction and third solvent inlet port 331 and the fraction and third solvent outlet port 332 are connected, the third solvent is introduced into the first solid phase extraction column (SPE1) and the first reversed-phase liquid chromatography column (COL1) to equilibrate the first solid phase extraction column (SPE1) and the first reversed-phase liquid chromatography column (COL1).
  • the dual column valve 400 plays a role in performing separation and analysis of the fraction in one of the first reversed-phase liquid chromatography column (COL1) and the second reversed-phase liquid chromatography column (COL2), while performing equilibration on the other column.
  • the dual column valve 400 includes the first solid phase extraction column (SPE1) connected to the first reversed-phase liquid chromatography column (COL1) and the second solid phase extraction column (SPE2) connected to the second reversed-phase liquid chromatography column (COL2).
  • the fraction is supplied to the first reversed-phase liquid chromatography column (COL1) through the first solid phase extraction column (SPE1) or the second reversed-phase liquid chromatography column (COL2) through the second solid phase extraction column (SPE2).
  • the dual column valve 400 supplies the first to tenth fractions to the first reversed-phase liquid chromatography column (COL1) or the second reversed-phase liquid chromatography column (COL2) in alternating manner, and is in fluid communication with the column selection valve 330 which performs separation and analysis of the fraction in one of the first reversed-phase liquid chromatography column (COL1) and the second reversed-phase liquid chromatography column (COL2) while at the same time, performing equilibration on the other column.
  • the column selection valve 330 which performs separation and analysis of the fraction in one of the first reversed-phase liquid chromatography column (COL1) and the second reversed-phase liquid chromatography column (COL2) while at the same time, performing equilibration on the other column.
  • 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 connected to two ends of the first solid phase extraction column (SPE1), a first solid phase extraction column inlet port 411 connected to the fraction and third solvent outlet port 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 connected to the first reversed-phase liquid chromatography column (COL1) and connected or disconnected to/from the first solid phase extraction column connection port 412 , a second solid phase extraction column connection port 432 and a second solid phase extraction column channel port 433 each connected to two ends of the second solid phase extraction column (SPE2), a second solid phase extraction column inlet port 431 connected to the fourth solvent outlet port 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 4
  • the dual column valve 400 further includes a first outlet port 451 provided adjacent to the first solid phase extraction column channel port 413 and connected or disconnected to/from 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, salts are discharged through the first outlet port 451 .
  • the fourth solvent is introduced into the first solid phase extraction column (SPE1) along the fourth solvent inlet port 333 and the fraction and third solvent outlet port 332 and elutes the first fraction in the first solid phase extraction column (SPE1), and the first fraction is supplied to the first reversed-phase liquid chromatography column (COL1).
  • the third solvent is introduced into the second solid phase extraction column (SPE2) and the second reversed-phase liquid chromatography column (COL2) along the fraction and third solvent inlet port 331 and the fourth solvent outlet port 334 to equilibrate the second solid phase extraction column (SPE2) and the second reversed-phase liquid chromatography column (COL2).
  • the dual column valve 400 further includes a second outlet port 453 provided adjacent to the second solid phase extraction column channel port 433 and connected or disconnected to/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, salts are discharged through the second outlet port 453 .
  • the fourth solvent is introduced into the second solid phase extraction column (SPE2) along the fourth solvent inlet port 333 and the fourth solvent outlet port 334 and elutes the second fraction in the second solid phase extraction column (SPE2), and the second fraction is supplied to the second reversed-phase liquid chromatography column (COL2).
  • 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 port 331 and the fraction and third solvent outlet port 332 to equilibrate the first solid phase extraction column (SPE1) and the first reversed-phase liquid chromatography column (COL1).
  • the third to tenth fractions 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 alternating manner, followed by separation and analysis, and at the same time, 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.
  • the present disclosure can improve reproducibility of fractionation and prevent a sample loss through automation of non-continuous sample fractionation and concatenation process with low sample complexity and high fraction uniformity.

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