CN108562678B - Three-dimensional liquid chromatographic separation system based on full online detection of same detector - Google Patents
Three-dimensional liquid chromatographic separation system based on full online detection of same detector Download PDFInfo
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- CN108562678B CN108562678B CN201810147433.8A CN201810147433A CN108562678B CN 108562678 B CN108562678 B CN 108562678B CN 201810147433 A CN201810147433 A CN 201810147433A CN 108562678 B CN108562678 B CN 108562678B
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
- G01N30/20—Injection using a sampling valve
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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
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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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
- G01N30/04—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
Abstract
The invention discloses a three-dimensional liquid chromatography separation system based on full online detection of the same detector, which belongs to the technical field of high performance liquid chromatography separation. The liquid chromatographic separation column array is used for selecting each dimension of chromatographic separation column, and the full online monitoring and control of one-dimension, two-dimension and three-dimension chromatographic separation modes are realized based on the same gradient elution system and the same detector, so that the cleanness degree of the enrichment column and the separation column is controllable. The dimensions of the separation are connected by an enrichment column and the enrichment or trapping of the compounds is assisted using a diluent pump. According to the invention, by selecting different chromatographic stationary phases and mobile phase combinations, efficient separation and compound preparation of complex system samples can be realized; the separation of high-content components can be realized, and the separation and detection of trace components and impurities can be realized; not only can realize the separation of all components, but also can realize the separation and detection of target components.
Description
Technical Field
The invention belongs to the technical field of high performance liquid chromatography separation, and relates to a full online detection three-dimensional high performance liquid chromatography separation system based on the same detector.
Background
The limited resolution and peak capacity provided by a common one-dimensional chromatographic separation system can hardly meet the requirements of full-component separation and analysis of a sample of a polar complex system. The multidimensional chromatographic technology can greatly improve the peak capacity and separation selectivity of the whole system, has unique and irreplaceable effects in solving the problems of complex components, uneven content, serious interference and unknown components of samples, and has rapidly become one of the hot spots in the field of chromatographic science research.
Due to technical limitations, the most common multi-dimensional liquid chromatography at present is two-dimensional liquid chromatography. The two-dimensional liquid chromatography is a chromatographic instrument developed on the basis of the common one-dimensional liquid chromatography, improves the separation capacity by increasing the chromatographic separation stages and the number of chromatographic columns, and is generally responsible for the concentration and primary separation of samples, and the second one-dimensional liquid chromatography is responsible for the further separation and detection of the samples, so that the samples can be processed on line, and target components can be separated from complex matrixes.
There are two ways of two-dimensional chromatography construction. The first is conventional two-dimensional chromatography using the "centre cut" technique. The second is an all-two-dimensional chromatography developed on this basis. Compared with the traditional center cutting technology, the full-two-dimensional liquid chromatography mode enables all products eluted in the first dimension to enter the second dimension on line for continuous analysis, information of all components of the sample can be obtained, and separation and analysis time is shortened. In the full two-dimensional liquid chromatography system, all components in the sample system are subjected to chromatographic separation in two different modes, and the application range is wider, so that the full two-dimensional liquid chromatography system becomes a main form of a multi-dimensional chromatographic separation system.
The most common two-dimensional liquid chromatography interface techniques are 3: interface technology based on sample ring; interface technology based on enrichment columns (also called trapping columns); interface technology based on stay mode. In an all-two-dimensional liquid chromatography system based on enrichment column interface technology, a sample separated in a first dimension can be separated into a plurality of components by using a plurality of enrichment columns, and then the components enriched in each enrichment column are separated again in sequence in a second dimension separation process. The number of the enrichment columns and the effective utilization are key basic indexes of the separation capacity of the full two-dimensional liquid chromatography system.
At present, many two-dimensional liquid chromatography systems can only detect and control the last-dimensional separation process, and the whole process monitoring and control of the separation of each dimension are not realized, so that the cleanliness of all the enrichment columns or separation columns is difficult to monitor, and the requirements of preparing samples are difficult to rapidly repeat and meet. In some two-dimensional liquid chromatography systems, each one-dimensional separation adopts an independent detector and a gradient elution system, and the whole chromatographic separation system has high cost. The cost of the multidimensional liquid chromatography system is reduced, the separation capacity of the multidimensional liquid chromatography system is further improved, and the repeatability, accuracy and automation degree of chromatographic separation are improved, so that the method is a key for constructing the multidimensional liquid chromatography separation system.
Disclosure of Invention
The invention aims to provide a three-dimensional high-performance liquid chromatography device with a low cost and a full online detection control in a separation process, aiming at some problems existing in the prior art of the two-dimensional liquid chromatography separation. Through the full on-line monitoring and control technology, the cleaning degree of the enrichment column and the separation column is controllable, and further the repeated separation preparation is realized. The complex sample can be pre-separated into several fractions with similar properties by using a stay mode or a fraction cutting technology, and the efficient repeated separation of the complex sample components is realized by combining the subsequent multidimensional liquid chromatographic separation. In addition, the enrichment efficiency of the compound on the enrichment column is improved by introducing a diluent.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the three-dimensional liquid chromatography separation system based on the full online detection of the same detector comprises a high-performance liquid chromatography pump A, a high-performance liquid chromatography pump B, a gradient mixer A, a gradient mixer B, a sample injection valve, a two-position six-way switching valve A, a two-position six-way switching valve B, an enrichment column array A, an enrichment column array B, a liquid chromatography separation column array, a detector, a high-performance liquid dilution pump, a collector and a connecting pipeline.
The (1) bit, (2) bit, (3) bit, (4) bit, (5) bit and (6) bit of the two-bit six-way switching valve A only represent an adjacent relation and do not need to correspond to the physical mark of the two-bit six-way switching valve A; the (1) bit, (2) bit, (3) bit, (4) bit, (5) bit and (6) bit of the two-bit six-way switching valve B only represent adjacent relations and do not need to correspond to physical marks of the two-bit six-way switching valve B; the detector is used for detecting chromatographic signals in the separation process; the sample injection valve is used for sample injection.
The high performance liquid chromatography gradient pump A and the high performance liquid chromatography gradient pump B are connected with the inlet of the gradient mixer A, the outlet of the gradient mixer A is connected with the inlet of the sample injection valve, the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A, the (6) position of the two-position six-way valve A is connected with the inlet of the liquid chromatography separation column array, the outlet of the liquid chromatography separation column array is connected with the inlet of the detector, the outlet of the detector is connected with the inlet of the mixer B, the diluent pump is connected with the inlet of the gradient mixer B, the outlet of the gradient mixer B is connected with the (3) position of the two-position six-way valve A, the (2) position of the two-position six-way valve A is connected with the inlet of the enrichment column array A, the (3) position of the two-position six-way valve B is connected with the inlet of the enrichment column array B, the outlet of the two-position six-way valve B is connected with the (6) position of the two-position six-way valve B, the (1) position six-way valve B is connected with the two-position six-way valve A and the two-position six-way valve B is connected with the inlet of the two-position six-way valve B (4).
The liquid chromatographic separation column array is formed by connecting a plurality of chromatographic separation columns in parallel through a multi-position selection valve, and only one chromatographic separation column is conducted at the same time; a fixed inlet and a fixed outlet are arranged outside the column, and at least one straight-through bypass is arranged, and the bypass and the separation column are connected in parallel through a multi-position selection valve; the other chromatographic separation columns will not be conducting when the bypass is conducting and the bypass will not be conducting when the other chromatographic separation columns are conducting; the number of chromatographic separation columns is determined as desired.
The enrichment column array A and the enrichment column array B are formed by connecting a plurality of chromatographic enrichment columns in parallel through a multi-position selection valve, and only one enrichment column can be conducted at the same time; there is a fixed inlet and a fixed outlet to the outside, and there is at least one bypass, the bypass and enriching column are connected in parallel through the multi-position selector valve; the other enrichment columns will not conduct when the bypass is conducting and the bypass will not conduct when the other enrichment columns are conducting; the number of enrichment columns is determined as needed. The multiple enrichment column arrays can be connected in series to form a multi-stage enrichment column array, the operation control is consistent with that of the single-stage enrichment column array, and only one enrichment column can be conducted at the same time; when the multi-stage enriching column array is in a bypass conducting state, the enriching column array of each stage is in bypass conducting state.
Based on the pipeline connection mode of the three-dimensional liquid chromatographic separation system, the system is converted from the upper dimension separation state to the lower dimension separation state by controlling the states of the two-position six-way valve A and the two-position six-way valve B, and the chromatographic separation function of full online detection of at most three dimensions can be completed.
The sample injection valve is a sample injection device, and can be a two-position six-way switching sample injection valve or a sample injector; other multi-position switching loading valves for liquid or solid loading are possible; or a chromatographic column for realizing solid-state loading.
The high performance liquid chromatography gradient pump A and the high performance liquid chromatography gradient pump B are composed of two unit pumps or a multi-element gradient pump. The diluent pump is a high-efficiency liquid-phase diluent pump, a unit pump or a multi-element pump. The diluents of the high performance liquid chromatography gradient pump A, the high performance liquid chromatography gradient pump B and the diluent pump are water, salt solution, methanol, acetonitrile, acetone, ethanol or normal alkane solvents, and the eluents of the high performance liquid chromatography gradient pump A, the high performance liquid chromatography gradient pump B and the diluent pump are common organic solvents such as methanol, acetonitrile, ethanol, water, mixtures thereof, normal alkane and the like.
The detector is a variety of means for detecting chromatographic signals in a separation process including, but not limited to, an ultraviolet detector, a diode array detector, an evaporative light scattering detector or a mass spectrometry detector, and may be a combination of one or more detectors.
The chromatographic columns of the separation column array, the enrichment column array A and the enrichment column array B can be the same or different fillers, wherein the fillers are silica gel, reverse phase silica gel matrix fillers with C18, xion, C8, CN groups or amino groups or fillers such as various macroporous adsorption resins, ion exchange resins and the like.
Multi-position switching valves are but one implementation of a column array; the other pillars in the pillar array and the bypass will not conduct when one pillar in the pillar array is conducting and the other pillars in the pillar array will not conduct when the pillar array bypass is conducting.
Compared with the prior art, the invention has the beneficial effects that:
(1) The separation process of the three-dimensional liquid chromatography system constructed by the invention is controllable in whole course, and the cleanliness of the enrichment column and the separation column can be detected. Before complex samples or fractions enter the next-dimension system for separation, a proper chromatographic column is selected to divide the complex samples or fractions into a plurality of fractions according to the properties and detection signals, and then a next-dimension separation method is established pertinently according to the properties of each fraction, so that the resolution ratio of the method can be further improved. The three-dimensional chromatographic system interface adopts an enrichment column technology, the fraction obtained by chromatographic separation is enriched by the enrichment technology, and the diluent is introduced before the compound enters the enrichment column to improve the enrichment efficiency.
(2) The three-dimensional liquid chromatography system constructed by the invention not only can utilize all the enrichment columns to the greatest extent in a two-dimensional full-component separation mode, but also can provide the three-dimensional separation capability of focusing target components, and is suitable for repeated analysis, separation and preparation of complex sample systems with high difficulty. Different chromatographic column combinations are selected according to different analyzed samples or fractions, the system is flexible and convenient to use and control, and can operate in various modes, such as a one-dimensional separation mode, a two-dimensional separation mode, a three-dimensional separation mode and the like; the system has low cost and strong expansibility, and has very wide application prospect in the high-difficulty separation analysis of complex system samples.
Drawings
Fig. 1a and fig. 1B are a pipeline connection structure diagram of a first dimension separation state of the three-dimensional liquid chromatography separation system provided by the invention, wherein a two-position six-way valve a in fig. 1a and fig. 1B is in an a state, a two-position six-way valve B in fig. 1a is in an a state, and a two-position six-way valve B in fig. 1B is in a B state.
Fig. 2 is a diagram showing a pipeline connection structure of a first dimension or a second dimension separation state of the three-dimensional liquid chromatography separation system provided by the invention. Wherein, two six-way valve A is the B state, and two six-way valve B is the B state.
Fig. 3 is a piping connection diagram of the chromatographic separation state in the first dimension, the second dimension or the third dimension, wherein the two-position six-way valve A is in the B state, and the two-position six-way valve B is in the A state.
Fig. 4 is a diagram showing the structure of piping connection of the enrichment column array a, the enrichment column array B, and the liquid chromatography separation column array.
FIG. 5a is a diagram of a two-position six-way sample valve sample loading state (LOAD state, A state) plumbing connection where sample is loaded into the dosing ring, where (4) is defined as the inlet of the sample valve and (5) is defined as the outlet of the sample valve;
FIG. 5B is a diagram showing a sample loading state (INJECT state, B state) pipeline connection structure of a two-position six-way sample injection valve, in which samples are injected from a quantitative loop into a flow path of a separation system for separation, wherein (4) is defined as an inlet of the sample injection valve, and (5) is defined as an outlet of the sample injection valve.
Fig. 6 is a block diagram of a three-dimensional hplc separation system according to an embodiment of the present invention.
Detailed Description
The following examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention, as it is defined in the claims, and variations and modifications of the inventive arrangements can be made by those skilled in the art without departing from the spirit of the invention.
The three-dimensional liquid chromatography separation system based on the full online detection of the same detector comprises a high performance liquid chromatography gradient pump A, a high performance liquid chromatography gradient pump B, a dilution liquid pump, a gradient mixer A, a gradient mixer B, a sample injection valve, an enrichment column array A, an enrichment column array B, a fraction collector, a liquid chromatography separation column array, a detector, a two-position six-way valve A, a two-position six-way valve B and a connecting pipeline. Wherein the diluent pump is a high-efficiency liquid-phase diluent pump.
The high performance liquid chromatography gradient pump A and the high performance liquid chromatography gradient pump B are connected with the inlet of the gradient mixer A, the outlet of the gradient mixer A is connected with the inlet of the sample injection valve, the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A, the (6) position of the two-position six-way valve A is connected with the inlet of the liquid chromatography separation column array, the outlet of the liquid chromatography separation column array is connected with the inlet of the detector, the outlet of the detector is connected with the inlet of the mixer B, the diluent pump is connected with the inlet of the gradient mixer B, the outlet of the gradient mixer B is connected with the (3) position of the two-position six-way valve A, the (2) position of the two-position six-way valve A is connected with the inlet of the enrichment column array A, the (3) position of the two-position six-way valve B is connected with the inlet of the enrichment column array B, the outlet of the two-position six-way valve B is connected with the (6) position of the two-position six-way valve B, the (1) position six-way valve B is connected with the two-position six-way valve A and the two-position six-way valve B is connected with the inlet of the two-position six-way valve B (4).
Further description is provided below with reference to the accompanying drawings.
In fig. 1a, the two-position six-way valve a is in the state a, and the two-position six-way valve B is in the state a. At this time, a chromatographic separation gradient elution mobile phase supply system is formed by the high performance liquid chromatography gradient pump A, the high performance liquid chromatography gradient pump B and the gradient mixer A, the outlet of the gradient mixer A is connected with a sample injection valve, and the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A; the (1) position of the two-position six-way valve A is communicated with the (6) position of the two-position six-way valve A and is connected with the inlet of the separation column array, the outlet of the separation column array is connected with the inlet of the detector, the detector detects chromatographic signals, the outlet of the detector is connected with the inlet of the gradient mixer B, the diluent pump is connected with the inlet of the gradient mixer B, the sample flows out after passing through the diluent column of the gradient mixer B, and the outlet of the diluent pump is connected with the (3) position of the two-position six-way valve A; the (3) position of the two-position six-way valve A is communicated with the (2) position of the two-position six-way valve A, the (2) position of the two-position six-way valve A is connected with the inlet of the enrichment column array A, and the outlet of the enrichment column array A is connected with the (2) position of the two-position six-way valve B, so that the enrichment of the sample is realized; the (2) position of the two-position six-way valve B is communicated with the (3) position of the two-position six-way valve B, the (3) position of the two-position six-way valve B is connected with the inlet of the enrichment column array B, and the outlet of the enrichment column array B is connected with the (6) position of the two-position six-way valve B; the (6) position of the two-position six-way valve B is communicated with the (1) position of the two-position six-way valve B, the (1) position of the two-position six-way valve B is connected with the (5) position of the two-position six-way valve A, the (5) position of the two-position six-way valve A is communicated with the (4) position of the two-position six-way valve A, the (4) position of the two-position six-way valve A is connected with the (4) position of the two-position six-way valve B, the (4) position of the two-position six-way valve B is communicated with the (5) position of the two-position six-way valve B, and the (5) position of the two-position six-way valve B is connected with the inlet of the fraction collector, so that sample collection is realized.
In fig. 1B, the two-position six-way valve a is in the state a, and the two-position six-way valve B is in the state B. At this time, a chromatographic separation gradient elution mobile phase supply system is formed by the high performance liquid chromatography gradient pump A, the high performance liquid chromatography gradient pump B and the gradient mixer A, the outlet of the gradient mixer A is connected with a sample injection valve, and the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A; the (1) position of the two-position six-way valve A is communicated with the (6) position of the two-position six-way valve A and is connected with the inlet of the separation column array, the outlet of the separation column array is connected with the inlet of the detector, the detector detects chromatographic signals, the outlet of the detector is connected with the inlet of the gradient mixer B, the diluent pump is connected with the inlet of the gradient mixer B, the sample flows out after passing through the diluent column of the gradient mixer B, and the outlet of the diluent pump is connected with the (3) position of the two-position six-way valve A; the (3) position of the two-position six-way valve A is communicated with the (2) position of the two-position six-way valve A, the (2) position of the two-position six-way valve A is connected with the inlet of the enrichment column array A, and the outlet of the enrichment column array A is connected with the (2) position of the two-position six-way valve B, so that the enrichment of the sample is realized; the (2) bit of the two-position six-way valve B is communicated with the (1) bit of the two-position six-way valve B, the (1) bit of the two-position six-way valve B is connected with the (5) bit of the two-position six-way valve A, the (5) bit of the two-position six-way valve A is communicated with the (4) bit of the two-position six-way valve A, and the (4) bit of the two-position six-way valve A is connected with the (4) bit of the two-position six-way valve B; the (4) bit of the two-bit six-way valve B is communicated with the (3) bit of the two-bit six-way valve B, the (3) bit of the two-bit six-way valve B is connected with the inlet of the enrichment column array B, and the outlet of the enrichment column array B is connected with the (6) bit of the two-bit six-way valve B; the (6) position of the two-position six-way valve B is communicated with the (5) position of the two-position six-way valve B, and the (5) position of the two-position six-way valve B is connected with the inlet of the fraction collector to realize sample collection.
In fig. 2, the two-position six-way valve a is in the B state, and the two-position six-way valve B is in the B state. At this time, a chromatographic separation gradient elution mobile phase supply system is formed by the high performance liquid chromatography gradient pump A, the high performance liquid chromatography gradient pump B and the gradient mixer A, the outlet of the gradient mixer A is connected with a sample injection valve, and the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A; the (1) bit of the two-bit six-way valve A is communicated with the (2) bit of the two-bit six-way valve A and is connected with the inlet of the enrichment column array A, and the outlet of the enrichment column array A is connected with the (2) bit of the two-bit six-way valve B; the (2) position of the two-position six-way valve B is communicated with the (1) position of the two-position six-way valve B, and the (1) position of the two-position six-way valve B is connected with the (5) position of the two-position six-way valve A; the (5) position of the two-position six-way valve A is communicated with the (6) position of the two-position six-way valve A, the (6) position of the two-position six-way valve A is connected with the inlet of the separation column array, the outlet of the separation column array is connected with the inlet of the detector, the detector detects chromatographic signals, the outlet of the detector is connected with the inlet of the gradient mixer B, the diluent pump is connected with the inlet of the gradient mixer B, the sample flows out after passing through the diluent column of the gradient mixer B, and the outlet of the diluent pump is connected with the (3) position of the two-position six-way valve A; the (3) position of the two-position six-way valve A is communicated with the (4) position of the two-position six-way valve A, and the (4) position of the two-position six-way valve A is connected with the (4) position of the two-position six-way valve B; the (4) bit of the two-bit six-way valve B is communicated with the (3) bit of the two-bit six-way valve B, the (3) bit of the two-bit six-way valve B is connected with the inlet of the enrichment column array B, and the outlet of the enrichment column array B is connected with the (6) bit of the two-bit six-way valve B; the (6) position of the two-position six-way valve B is communicated with the (5) position of the two-position six-way valve B, and the (5) position of the two-position six-way valve B is connected with the inlet of the fraction collector to realize sample collection.
In fig. 3, the two-position six-way valve a is in the B state, and the two-position six-way valve B is in the a state. At this time, a chromatographic separation gradient elution mobile phase supply system is formed by the high performance liquid chromatography gradient pump A, the high performance liquid chromatography gradient pump B and the gradient mixer A, the outlet of the gradient mixer A is connected with a sample injection valve, and the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A; the (1) bit of the two-bit six-way valve A is communicated with the (2) bit of the two-bit six-way valve A and is connected with the inlet of the enrichment column array A, and the outlet of the enrichment column array A is connected with the (2) bit of the two-bit six-way valve B; the (2) position of the two-position six-way valve B is communicated with the (3) position of the two-position six-way valve B, the (3) position of the two-position six-way valve B is connected with the inlet of the enrichment column array B, and the outlet of the enrichment column array B is connected with the (6) position of the two-position six-way valve B; the (6) position of the two-position six-way valve B is communicated with the (1) position of the two-position six-way valve B, and the (1) position of the two-position six-way valve B is connected with the (5) position of the two-position six-way valve A; the (5) position of the two-position six-way valve A is connected and communicated with the (6) position of the two-position six-way valve A, the (6) position of the two-position six-way valve A is connected with the inlet of the separation column array, the outlet of the separation column array is connected with the inlet of the detector, the detector detects chromatographic signals, the outlet of the detector is connected with the inlet of the gradient mixer B, the diluent pump is connected with the inlet of the gradient mixer B, a sample flows out after passing through the diluent column of the gradient mixer B, and the outlet of the diluent pump is connected with the (3) position of the two-position six-way valve A; the (3) position of the two-position six-way valve A is communicated with the (4) position of the two-position six-way valve A, and the (4) position of the two-position six-way valve A is connected with the (4) position of the two-position six-way valve B; the (4) position of the two-position six-way valve B is communicated with the (5) position of the two-position six-way valve B, and the (5) position of the two-position six-way valve B is connected with the inlet of the fraction collector to realize sample collection.
Embodiment a three-dimensional high performance liquid chromatography separation system structure
In this embodiment, the enriching column array a is a two-stage enriching column array, and each stage of enriching column array has 9 enriching columns, that is, the enriching column array a is 18 enriching columns, the 1 st enriching column, the 2 nd enriching column, and the like of the enriching column array a are numbered in sequence, and the last 18 th enriching column of the enriching column array a is numbered; the enriching column array B is provided with 9 enriching columns, namely a 1 st enriching column, a 2 nd enriching column and the like of the enriching column array B in sequence, and the last enriching column is a 9 th enriching column of the enriching column array B; the liquid chromatographic separation column array is provided with 5 separation columns, which are sequentially numbered as a 1 st separation column, a 2 nd separation column, and the like, and the last separation column is a 5 th separation column;
the following is an operation description of the structure of the three-dimensional high performance liquid chromatography separation system:
the main operation modes of the three-dimensional high performance liquid chromatography separation system are an enrichment mode and a separation mode, and the combination of the modes. In practical application, the selection is as required. The following is a brief description.
1. Enrichment mode
Based on selective adsorption and gradient elution of the enrichment column packing, the enrichment mode can retain main components and remove impurity components, and is mainly used for separation and analysis of target components; the method can also retain trace, remove main components, reduce the detection limit of trace components, and improve the detection level of trace components, and is mainly used for target impurity identification analysis and unknown impurity discovery.
The enrichment mode includes a single enrichment mode and a dual enrichment mode. The single enrichment mode is to repeatedly sample through a sample valve for multiple times, and realize enrichment of one type of target components by utilizing selective adsorption of an enrichment column array A or an enrichment column array B, and the enrichment mode is shown in fig. 2 and 3. The dual enrichment mode is to repeatedly sample through a sample valve for multiple times, and realize enrichment of two types of target components by utilizing selective adsorption of an enrichment column array A and an enrichment column array B respectively, and refer to fig. 2 and 3.
2. One-dimensional separation system operation mode
The one-dimensional separation system operation modes comprise a separation mode, an enrichment-separation-enrichment mode, a separation-enrichment mode and the like.
The separation mode is to sample through a sample valve, perform chromatographic separation by using one separation column in a chromatographic separation column array, and collect target components by using a collector, see fig. 1a and 1b. Wherein, enrichment post array A and enrichment post array B are in bypass conduction state.
The enrichment-separation mode is to firstly enrich target components by using an enrichment column array A, then to carry out chromatographic separation by using one separation column in the chromatographic separation column array, and to cut and collect the target components by using a collector, wherein the enrichment column array B is in a bypass conduction state. See fig. 2.
The enrichment-separation-enrichment mode is to enrich the target component by using the enrichment column array A, then to perform chromatographic separation by using one separation column in the chromatographic separation column array, and finally to enrich the target component by using the enrichment column array B. See fig. 2.
The separation-enrichment mode is to repeatedly sample the sample for a plurality of times by using a sample valve, perform chromatographic separation by using one separation column in the chromatographic separation column array, and then perform enrichment of the target component by using the enrichment column array A or the enrichment column array B. See fig. 1a and 1b. This mode can enrich up to 27 components.
3. Two-dimensional separation system operation mode
The two-dimensional separation system operating modes include a separation-enrichment-separation mode and an enrichment-separation-enrichment-separation mode.
The separation-enrichment-separation mode is that firstly, sampling is repeatedly performed for a plurality of times by using a sampling valve, chromatographic separation is performed by using one separation column in a chromatographic separation column array, and enrichment of target components is sequentially performed by using an enrichment column array A and an enrichment column array B, so that 27 components can be enriched at most, and the method is shown in fig. 1a and 1B; then, 1 enrichment column in the enrichment column array a or the enrichment column array B is selected as a sample column, and when the sample column is conducted, a second-dimension chromatographic separation is performed by using the other separation column in the separation column array, and a desired component is collected by using a collector, see fig. 3. The separation-enrichment-separation mode may also be developed into a separation-enrichment-separation-enrichment mode, in which case the first separation enrichment can be performed with an enrichment column array a of up to 18 components and the second separation enrichment can be performed with an enrichment column array B of up to 9 components.
The enrichment-separation-enrichment-separation mode is that a sample injection valve is used for repeatedly injecting sample for a plurality of times, and the target component is enriched by an enrichment column array A, wherein the chromatographic separation column array and the enrichment column array B are in a bypass state at the moment, and the chromatographic separation column array and the enrichment column array B are in a bypass state, as shown in fig. 2; then 1 enrichment column in the enrichment column array A is selected as a sample column, when the sample column is conducted, one separation column in the chromatographic separation column array is utilized to carry out first-dimension chromatographic separation, and the required components are sequentially enriched by the enrichment column array B, so that 9 components can be enriched in total, and the method is shown in figure 2; finally, 1 enrichment column in the enrichment column array B is selected as a sample column, and when the sample column is conducted, another separation column in the separation column array is used for second-dimension chromatographic separation, and the required components are collected by a collector, wherein the enrichment column array A is in a bypass conduction state, and the enrichment column array A is shown in fig. 3.
4. Three-dimensional separation mode
The three-dimensional separation mode refers to a separation-enrichment-separation mode. Firstly, repeatedly sampling for a plurality of times through a sampling valve, performing first-dimension chromatographic separation by utilizing one separation column, such as a 1 st separation column, in a chromatographic separation column array, and sequentially enriching fractions into 18 enrichment columns of an enrichment column array A according to requirements, wherein an enrichment column array B is in a bypass conduction state at the moment, as shown in fig. 1a and 1B; then, the two-position six-way valve A is switched to a B state, 1 enrichment column in the enrichment column array A is selected as a sample column, second-dimension chromatographic separation is started when the sample column is conducted, and fractions are sequentially enriched into 9 enrichment columns in the enrichment column array B according to requirements, and the two-position six-way valve A is shown in FIG. 2; finally, the two-position six-way valve B is turned to the state A, referring to FIG. 3, the enrichment column array A is in the bypass conduction state, 1 enrichment column in the enrichment column array B is selected as a sample column, and when the sample column is conducted, the third-dimension chromatographic separation is started, and fractions are collected by a collector sequentially according to the requirement.
Claims (3)
1. A three-dimensional liquid chromatography separation system based on all online detection of same detector, its characterized in that: the three-dimensional liquid chromatography separation system comprises a high-performance liquid chromatography pump A, a high-performance liquid chromatography pump B, a gradient mixer A, a gradient mixer B, a sample injection valve, a two-position six-way switching valve A, a two-position six-way switching valve B, an enrichment column array A, an enrichment column array B, a liquid chromatography separation column array, a detector, a high-performance liquid dilution pump, a fraction collector and a connecting pipeline;
the (1) bit, (2) bit, (3) bit, (4) bit, (5) bit and (6) bit of the two-bit six-way switching valve A represent adjacent relations; the (1) bit, (2) bit, (3) bit, (4) bit, (5) bit and (6) bit of the two-bit six-way switching valve B represent adjacent relations; the detector is used for detecting chromatographic signals in the separation process; the sample injection valve is used for sample injection;
the high performance liquid chromatography A and the high performance liquid chromatography B are connected with the inlet of the gradient mixer A, the outlet of the gradient mixer A is connected with the inlet of the sample injection valve, the outlet of the sample injection valve is connected with the (1) position of the two-position six-way valve A, the (6) position of the two-position six-way valve A is connected with the inlet of the liquid chromatography separation column array, the outlet of the liquid chromatography separation column array is connected with the inlet of the detector, the outlet of the detector is connected with the inlet of the mixer B, the diluent pump is connected with the inlet of the gradient mixer B, the outlet of the gradient mixer B is connected with the (3) position of the two-position six-way valve A, the (2) position of the two-position six-way valve A is connected with the inlet of the enrichment column array A, the outlet of the two-position six-way valve B is connected with the (2) position of the two-position six-way valve B, the outlet of the two-position six-way valve B is connected with the (6) position of the detector, the outlet of the two-position six-way valve B is connected with the (1) position six-way valve A and the two-position six-way valve B is connected with the two-position six-way valve B, and the two-position six-way valve B is connected with the inlet of the two-position six-way valve B (4) A is connected with the two-position six-position valve B;
the liquid chromatographic separation column array is formed by connecting a plurality of chromatographic separation columns in parallel through a multi-position selection valve, and only one chromatographic separation column is conducted at the same time; a fixed inlet and a fixed outlet are arranged outside the column, and at least one straight-through bypass is arranged, and the bypass and the separation column are connected in parallel through a multi-position selection valve; the other chromatographic separation columns will not be conducting when the bypass is conducting and the bypass will not be conducting when the other chromatographic separation columns are conducting; the number of the chromatographic separation columns is determined according to the requirement;
the enrichment column array A and the enrichment column array B are formed by connecting a plurality of chromatographic enrichment columns in parallel through a multi-position selection valve, and only one enrichment column can be conducted at the same time; there is a fixed inlet and a fixed outlet to the outside, and there is at least one bypass, the bypass and enriching column are connected in parallel through the multi-position selector valve; the other enrichment columns will not conduct when the bypass is conducting and the bypass will not conduct when the other enrichment columns are conducting; the number of the enrichment columns is determined according to the requirement;
the system is switched from the upper one-dimensional separation state to the lower one-dimensional separation state by controlling the states of the two-position six-way valve A and the two-position six-way valve B, and the chromatographic separation function of the maximum three-dimensional full online detection can be realized.
2. The three-dimensional liquid chromatography separation system based on the full online detection of the same detector as defined in claim 1, wherein the plurality of enrichment column arrays can be connected in series to form a multi-stage enrichment column array, the operation control is consistent with that of a single-stage enrichment column array, and only one enrichment column can be conducted at the same time; when the multi-stage enriching column array is in a bypass conducting state, the enriching column array of each stage is in bypass conducting state.
3. A three-dimensional liquid chromatography separation system based on all-in-line detection of the same detector according to claim 1 or 2, wherein the detector is an ultraviolet detector, a diode array detector, an evaporative light scattering detector or a mass spectrum detector.
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| CN109406692A (en) * | 2018-12-28 | 2019-03-01 | 苏州天蓝分析仪器有限公司 | Continuous quadratic enrichment resolver that is a kind of multistage and running in turn |
| CN109765323B (en) * | 2019-01-31 | 2023-12-29 | 广西电网有限责任公司电力科学研究院 | Pretreatment device and method for sample for liquid chromatography of transformer oil |
| DE102019123377A1 (en) * | 2019-08-30 | 2021-03-04 | Dionex Softron Gmbh | Procedure and system for chromatography |
| WO2023119944A1 (en) * | 2021-12-21 | 2023-06-29 | 株式会社島津製作所 | Liquid chromatograph |
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