Hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system and analysis method thereof
阅读说明：本技术 亲水作用色谱-反相色谱组合二维液相系统及其分析方法 (Hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system and analysis method thereof ) 是由 邝江濛 钟启升 刘佳琪 黄海宏 于 2020-05-29 设计创作，主要内容包括：本发明涉及一种亲水作用色谱-反相色谱组合二维液相系统及其分析方法,该系统包括亲水作用色谱柱、反相色谱柱、检测器、自动进样器、第一泵组件、第二色谱泵、第三色谱泵、混合器、样品环和切换阀；所述第一泵组件与所述亲水作用色谱柱接通,所述第二色谱泵和第三色谱泵分别与所述混合器接通,所述混合器与所述反相色谱柱接通；所述切换阀至少具备以下2种连接状态：连接状态1：将检测器的入口与亲水作用色谱柱的出口接通,并将样品环的两个端口分别与第二色谱泵、混合器接通；连接状态2：将检测器与反相色谱柱接通,并将样品环的两个端口分别与亲水作用色谱柱和系统外接通。该系统可实现对极性差异较大的多组分目标物的一次性分析。(The invention relates to a hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system and an analysis method thereof, wherein the system comprises a hydrophilic interaction chromatography column, a reversed phase chromatography column, a detector, an automatic sample injector, a first pump assembly, a second chromatography pump, a third chromatography pump, a mixer, a sample ring and a switching valve; the first pump assembly is communicated with the hydrophilic interaction chromatographic column, the second chromatographic pump and the third chromatographic pump are respectively communicated with the mixer, and the mixer is communicated with the reverse phase chromatographic column; the switching valve has at least the following 2 connection states: connection state 1: connecting the inlet of the detector with the outlet of the hydrophilic interaction chromatographic column, and connecting two ports of the sample ring with the second chromatographic pump and the mixer respectively; connection state 2: the detector was connected to the reverse phase column and the two ports of the sample loop were connected to the hydrophilic interaction column and outside the system, respectively. The system can realize one-time analysis of multi-component targets with large polarity differences.)
1. A hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system is characterized in that:
comprises a hydrophilic interaction chromatographic column, a reverse phase chromatographic column, a detector, an automatic sample injector, a first pump assembly, a second chromatographic pump, a third chromatographic pump, a mixer, a sample ring and a switching valve;
the first pump assembly is used for pushing a sample carrying solution and carrying out gradient elution on the hydrophilic interaction chromatographic column, an outlet of the first pump assembly is communicated with an inlet of the hydrophilic interaction chromatographic column, and the automatic sample injector is arranged on a connecting pipeline between the first pump assembly and the hydrophilic interaction chromatographic column;
the second chromatographic pump and the third chromatographic pump are used for gradient elution of the reversed-phase chromatographic column, the outlet of the second chromatographic pump is communicated with the inlet of the mixer through the switching valve, the outlet of the third chromatographic pump is communicated with the inlet of the mixer, and the outlet of the mixer is communicated with the inlet of the reversed-phase chromatographic column;
the outlet of the hydrophilic interaction chromatographic column, the outlet of the reverse phase chromatographic column, the inlet of the detector and the two ports of the sample ring are respectively connected with the switching valve;
the switching valve has at least the following 2 connection states by switching its position:
connection state 1: the switching valve connects the inlet of the detector with the outlet of the hydrophilic interaction chromatography column and connects one port of the sample loop with the outlet of the second chromatography pump and the other port with the inlet of the mixer;
connection state 2: the switching valve connects the inlet of the detector with the outlet of the reverse phase chromatography column and connects one port of the sample loop with the outlet of the hydrophilic interaction chromatography column and the other port to the outside of the system.
2. The system of claim 1, wherein: the switching valve is formed by connecting two six-way valves.
3. The system of claim 1, wherein: the switching valve is a ten-way valve.
4. The system according to any one of claims 1-3, wherein: the two ports of the sample ring are respectively a first port and a second port; in the connection state 1, the switching valve connects a first port of the sample loop with the outlet of the second chromatographic pump and a second port of the sample loop with the inlet of the mixer; in the connection state 2, the switching valve connects the second port of the sample loop with the outlet of the hydrophilic interaction chromatography column and connects the first port of the sample loop to the outside of the system.
5. The system according to any one of claims 1-3, wherein: the mixer is a three-way mixer and is provided with three ports, wherein two ports are inlets of the mixer and are respectively communicated with an outlet of the third chromatographic pump and the switching valve through pipelines, and the other port is an outlet of the mixer and is communicated with an inlet of the reversed-phase chromatographic column through a pipeline.
6. The system according to any one of claims 1-3, wherein: the first pump assembly comprises a first chromatographic pump and a low-pressure gradient proportional valve arranged in the first chromatographic pump; alternatively, the first pump assembly is a binary high pressure gradient system comprising two chromatography pumps.
7. A method of analyzing the system of claim 1, wherein: the method comprises the following steps:
1) loading a hydrophilic interaction chromatographic column, balancing a reversed phase chromatographic column:
the switching valve is adjusted to a state that an inlet of the detector is communicated with an outlet of the hydrophilic interaction chromatographic column, then the automatic sample injector introduces the sample into the system, the first pump assembly outputs a high proportion organic solvent to push the sample to enter the hydrophilic interaction chromatographic column, so that the sample is retained on a column head of the hydrophilic interaction chromatographic column, and the high proportion organic solvent enters the detector through the switching valve; meanwhile, the second chromatographic pump and the third chromatographic pump respectively convey the aqueous phase solvent to enter a mixer for mixing to form a high-proportion aqueous phase mixed solvent to enter the reversed-phase chromatographic column;
2) collecting weak polar substances:
the first pump assembly is used for conveying the solvent, the proportion of the water phase solvent in the solvent is increased, the weak polar substances in the sample are eluted from the hydrophilic interaction chromatographic column, the switching valve is switched to a connection state 2, and the eluted weak polar substances are led into the sample ring through the switching valve; meanwhile, the second chromatographic pump and the third chromatographic pump keep the state of the step 1);
3) separating strong polar substances, focusing weak polar substances:
the switching valve is switched to a connection state 1, the proportion of the water phase solvent in the solvent conveyed by the first pump assembly is continuously increased, the strong polar substance retained on the hydrophilic interaction chromatographic column is subjected to gradient elution, and the strong polar substance enters the detector through the switching valve for detection; meanwhile, the second chromatographic pump outputs an organic phase solvent to push the weak polar substances in the sample ring to the mixer through the switching valve, the third chromatographic pump transmits a water phase solvent to the mixer, a high proportion water phase mixed solvent carrying the weak polar substances is formed in the mixer and enters the reversed phase chromatographic column, the weak polar substances are focused on the column head of the reversed phase chromatographic column, and the high proportion water phase mixed solvent is discharged out of the system through the switching valve;
4) balancing a hydrophilic interaction chromatographic column, and separating weak polar substances:
after the separation and detection of the strong polar substances on the hydrophilic interaction chromatographic column are finished, the switching valve is switched to a connection state 2, the first chromatographic pump conveys a high proportion of organic phase solvent into the hydrophilic interaction chromatographic column, and the solvent which is discharged from the hydrophilic interaction chromatographic column is discharged out of the system through the sample ring and the switching valve; simultaneously, the second chromatographic pump and the third chromatographic pump respectively convey the aqueous phase solvent to enter a mixer for mixing, the formed high-proportion aqueous phase mixed solvent enters an inverse chromatographic column, the proportion of the organic phase solvent in the mixed solvent is increased along with the lapse of time, the weak polar substances focused on the column head of the inverse chromatographic column are eluted one by one, and the weak polar substances enter a detector for detection through a switching valve;
5) and returning to the initial state:
after all the substances on the reverse phase chromatographic column are eluted to the detector, the system is restored to the state of the step 1) and waits for the next sample injection.
8. The analytical method of claim 6, wherein: step 2), the switching valve switches to the connection state 2 before the first component flows out of the hydrophilic interaction chromatography column; step 3), the switching valve switches to connection state 1 before the sample loop is filled with the effluent of the hydrophilic interaction chromatography column.
9. The analytical method of claim 6, wherein: in the steps 1) and 4), the volume ratio of the organic phase solvent in the high-ratio organic solvent delivered by the first pump assembly is more than 95%; in steps 1) to 3), the volume proportion of the aqueous phase solvent in the high-proportion aqueous phase mixed solvent formed in the mixer is always greater than 90%, and in step 4), the volume proportion gradient of the organic phase solvent in the mixed solvent formed in the mixer is increased to be greater than 95%.
10. The analytical method of claim 6, wherein: step 2) is opposite to the flow direction of the liquid phase in the sample ring in step 3).
Technical Field
The invention relates to the field of instrument analysis, in particular to a hydrophilic interaction chromatography-reverse phase chromatography combined two-dimensional liquid phase system and an analysis method thereof.
Background
In fields such as metabonomics, environment, and pesticide residue analysis, a plurality of target substances are generally required to be analyzed, and when the polarity difference of each target substance is too large, the analysis of all the substances is difficult to be completed by one method. In the face of this problem, two kinds of analysis methods, two kinds of chromatography columns (e.g., a hydrophilic interaction chromatography column and a reverse phase chromatography column) are generally used to perform the analysis of the strongly polar substance and the weakly polar substance, respectively. Such a processing method is time-consuming and labor-consuming, and can generate twice more redundant data, which is not favorable for improving the analysis efficiency.
In an on-line two-dimensional liquid chromatography system, chromatographic columns with different characteristics are connected together through pipeline connection and valve switching, so that substances which cannot be separated in a first dimension are introduced into a second dimension for further analysis, and the separation degree and the peak capacity are improved. Based on the difference of principle and connection mode, two-dimensional liquid chromatography systems are various in types. As the technology has matured, two-dimensional liquid chromatography systems have been increasingly used for analysis of complex samples.
Disclosure of Invention
The invention provides a hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system, which takes a switching valve as a connecting center and connects the hydrophilic interaction chromatography and the reversed phase chromatography to realize one-time analysis of a multi-component target object with larger polarity difference.
The technical scheme adopted by the invention is as follows:
a hydrophilic interaction chromatography-reverse phase chromatography combined two-dimensional liquid phase system comprises a hydrophilic interaction chromatography column, a reverse phase chromatography column, a detector, an automatic sample injector, a first pump assembly, a second chromatography pump, a third chromatography pump, a mixer, a sample ring and a switching valve;
the first pump assembly is used for pushing a sample carrying solution and carrying out gradient elution on the hydrophilic interaction chromatographic column, an outlet of the first pump assembly is communicated with an inlet of the hydrophilic interaction chromatographic column, and the automatic sample injector is arranged on a connecting pipeline between the first pump assembly and the hydrophilic interaction chromatographic column;
the second chromatographic pump and the third chromatographic pump are used for gradient elution of the reversed-phase chromatographic column, the outlet of the second chromatographic pump is communicated with the inlet of the mixer through the switching valve, the outlet of the third chromatographic pump is communicated with the inlet of the mixer, and the outlet of the mixer is communicated with the inlet of the reversed-phase chromatographic column;
the outlet of the hydrophilic interaction chromatographic column, the outlet of the reverse phase chromatographic column, the inlet of the detector and the two ports of the sample ring are respectively connected with the switching valve;
the switching valve has at least the following 2 connection states by switching its position:
connection state 1: the switching valve connects the inlet of the detector with the outlet of the hydrophilic interaction chromatography column and connects one port of the sample loop with the outlet of the second chromatography pump and the other port with the inlet of the mixer;
connection state 2: the switching valve connects the inlet of the detector with the outlet of the reverse phase chromatography column and connects one port of the sample loop with the outlet of the hydrophilic interaction chromatography column and the other port to the outside of the system.
By switching the switching valve, a hydrophilic interaction chromatographic mode (a first dimension) and a reversed phase chromatographic mode (a second dimension) can be realized in sequence; when the switching valve is switched to the connection state 1, the flow path of the system can complete the process of loading a hydrophilic chromatographic column or separating and detecting a strong polar substance, and when the switching valve is switched to the
The hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system takes the switching valve as a connecting center, and connects the hydrophilic interaction chromatography column and the reversed phase chromatography column, wherein the hydrophilic interaction chromatography column has the characteristic of retaining and separating strong polar substances, and the reversed phase chromatography column has the characteristic of retaining and separating weak polar substances. The system is obtained by using simple hardware equipment and a connecting flow path design, realizes the separation of substances with weak polarity to strong polarity by one injection, and can be used for analysis by being combined with an ultraviolet detector or a mass spectrum detection technology.
Specifically, the switching valve is composed of two six-way valve connections.
Specifically, the switching valve is a ten-way valve, and the system is simpler to build.
Specifically, the two ports of the sample loop are a first port and a second port respectively; in the connection state 1, the switching valve connects a first port of the sample loop with the outlet of the second chromatographic pump and a second port of the sample loop with the inlet of the mixer; in the
Through the configuration, the first chromatographic pump in the hydrophilic interaction chromatographic mode delivers the solvent to elute the component to be detected in the hydrophilic interaction chromatographic column to the direction of the sample ring, and the flow direction of the organic solvent delivered by the second chromatographic pump in the subsequent reversed-phase chromatographic mode to enter the sample ring to push the component to be detected into the mixer is opposite, so that the component to be detected can rapidly enter the reversed-phase chromatographic column for focusing.
Specifically, the mixer is a three-way mixer and has three ports, two of the three ports are inlets of the mixer and are respectively communicated with the outlet of the third chromatographic pump and the switching valve through pipelines, and the other one port is an outlet of the mixer and is communicated with the inlet of the reversed-phase chromatographic column through a pipeline.
Specifically, the first pump assembly comprises a first chromatographic pump and a low-pressure gradient proportional valve built in the first chromatographic pump; or equivalently, the first pump assembly is a binary high pressure gradient system comprising two chromatography pumps. The first pump assembly has the capability of delivering at least two solvents with different polarities, and can complete gradient elution of a first-dimension liquid phase.
In the system, in a first dimension, a first pump assembly can freely select the combination of 'one pump + a gradient proportional valve' or 'two pumps', and the two configurations can achieve the purposes of mixing and then feeding to realize gradient separation of the first dimension;
in the second dimension, a binary high-pressure gradient system is formed by adopting a second chromatographic pump and a third chromatographic pump, and through the specific flow path design, the purposes of sampling first and then mixing are achieved, so that the problem of solvent effect in the second dimension is solved. Specifically, in the second dimension liquid phase, the "switching valve + sample loop" constitutes an equivalent sample injection system, assuming that the volume of the sample loop is 500 μ L, the solvent is high proportion ACN (acetonitrile), which is a strong solvent for the reverse phase chromatographic column, if the ordinary "mix-first and sample-then-sample" sample injection method is adopted, the mobile phase pushes a section of 500 μ L ACN liquid section to enter the reverse phase chromatographic column, thereby causing a strong solvent effect. The solution of the invention is that the solution stored in the sample ring is taken out by the second chromatographic pump with small flow rate ACN (first sample injection), and then mixed with the water with large flow rate delivered by the third chromatographic pump at the mixer (post mixing), so that 500 mul of ACN is diluted by a large amount of water into solution with high proportion of water phase, and then enters the reversed phase chromatographic column, and the solvent effect can not be generated.
The invention also provides an analysis method of the system, which comprises the following steps:
1) loading a hydrophilic interaction chromatographic column, balancing a reversed phase chromatographic column:
the switching valve is adjusted to a state that an inlet of the detector is communicated with an outlet of the hydrophilic interaction chromatographic column, then the automatic sample injector introduces the sample into the system, the first pump assembly outputs a high proportion organic solvent to push the sample to enter the hydrophilic interaction chromatographic column, so that the sample is retained on a column head of the hydrophilic interaction chromatographic column, and the high proportion organic solvent enters the detector through the switching valve; meanwhile, the second chromatographic pump and the third chromatographic pump respectively convey the aqueous phase solvent to enter a mixer for mixing to form a high-proportion aqueous phase mixed solvent to enter the reversed-phase chromatographic column;
2) collecting weak polar substances:
the first pump assembly is used for conveying the solvent, the proportion of the water phase solvent in the solvent is increased, the weak polar substances in the sample are eluted from the hydrophilic interaction chromatographic column, the switching valve is switched to a
3) separating strong polar substances, focusing weak polar substances:
the switching valve is switched to a connection state 1, the proportion of the water phase solvent in the solvent conveyed by the first pump assembly is continuously increased, the strong polar substance retained on the hydrophilic interaction chromatographic column is subjected to gradient elution, and the strong polar substance enters the detector through the switching valve for detection; meanwhile, the second chromatographic pump outputs an organic phase solvent to push the weak polar substances in the sample ring to the mixer through the switching valve, the third chromatographic pump transmits a water phase solvent to the mixer, a high proportion water phase mixed solvent carrying the weak polar substances is formed in the mixer and enters the reversed phase chromatographic column, the weak polar substances are focused on the column head of the reversed phase chromatographic column, and the high proportion water phase mixed solvent is discharged out of the system through the switching valve;
4) balancing a hydrophilic interaction chromatographic column, and separating weak polar substances:
after the separation and detection of the strong polar substances on the hydrophilic interaction chromatographic column are finished, the switching valve is switched to a
5) and returning to the initial state:
after all the substances on the reverse phase chromatographic column are eluted to the detector, the system is restored to the state of the step 1) and waits for the next sample injection.
Specifically, in the step 2), the switching valve is switched to the
Specifically, in the step 1) and the step 4), the volume proportion of the organic phase solvent in the high proportion of the organic solvent delivered by the first pump assembly is more than 95%; in steps 1) to 3), the volume proportion of the aqueous phase solvent in the high-proportion aqueous phase mixed solvent formed in the mixer is always greater than 90%, and in step 4), the volume proportion gradient of the organic phase solvent in the mixed solvent formed in the mixer is increased to be greater than 95%.
Through the configuration, the polarity and the elution capacity of the solvent conveyed by each pump device in each step are adjusted, so that the effect of pushing the substance to be detected or gradient-eluting the substance to be detected on the chromatographic column is achieved. In addition, in step 3), a small amount of organic phase solvent is conveyed by the second chromatographic pump to take out the solution stored in the sample ring (first sample injection), and then the solution is mixed with a large amount of aqueous phase solvent conveyed by the third chromatographic pump at the mixer (post mixing), so that a high proportion of aqueous phase mixed solvent is formed and then enters the reversed phase chromatographic column, and the solvent effect is eliminated.
Specifically, the flow directions of the liquid phase in the sample ring in the step 2) and the step 3) are opposite, and by setting a proper valve cutting time, the switching valve in the step 3) starts backflushing when the substance to be detected just completely enters the sample ring, and if the sample ring is still not filled with the effluent of the hydrophilic interaction chromatographic column at the moment, the time for filling the blank tail section of the sample ring with the effluent without the sample can be saved, so that the target component can rapidly enter the reversed phase chromatographic column for focusing.
Compared with the prior art, the hydrophilic interaction chromatography-reversed phase chromatography combined two-dimensional liquid phase system and the analysis method thereof have the following advantages:
(1) the weak-polarity and strong-polarity substances are analyzed at one time, the same sample does not need to be analyzed by different methods respectively, and only one detector is needed, and spectrograms obtained by hydrophilic interaction chromatography and reversed phase chromatography are stored in one data file, so that the time is saved, and redundant data are reduced.
(2) The two analysis methods of the hydrophilic interaction chromatography and the reversed phase chromatography are connected on line and switched automatically without manual operation, so that the working efficiency is improved; the separation of the hydrophilic interaction chromatographic column is simultaneously carried out by the reversed phase chromatographic column, and vice versa, and the time for the chromatographic column to be balanced is macroscopically saved.
(3) The core connecting device of the system is only a switching valve and a sample ring, a trapping column is not needed, and the building difficulty and cost are reduced to the maximum extent.
(4) Besides being used for analyzing weak-polarity substances and strong-polarity substances, the system can be used for separating the two substances, and has the function of on-line pretreatment, namely the interference of polar substrates is removed to analyze the weak-polarity substances in the substances, or the interference of non-polar substrates is removed to analyze the strong-polarity substances in the substances.
(5) Different hydrophilic interaction chromatographic columns and reversed phase chromatographic columns can be selected according to specific analysis items, and expansibility is strong.
For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of a two-dimensional liquid system combining hydrophilic interaction chromatography and reverse phase chromatography provided by the present invention;
FIG. 2 is a flow-path-connection diagram of a two-dimensional liquid-phase system combining hydrophilic interaction chromatography and reverse-phase chromatography in example 1;
fig. 3 is a flow path connection diagram of the system when the two six-way valves of embodiment 1 are in the connected state 1;
fig. 4 is a flow path connection diagram of the system when the two six-way valves of embodiment 1 are in the connected
fig. 5 is a flow path connection diagram of the system when the two six-way valves of embodiment 1 are in the connected
FIG. 6 is a flow-path-connection diagram of a two-dimensional liquid-phase system combining hydrophilic interaction chromatography and reverse-phase chromatography in example 2;
fig. 7 is a flow path connection diagram of the system when the ten-way valve of
fig. 8 is a flow path coupling diagram of the system when the ten-way valve of
FIG. 9 is a chromatogram from analysis of 8 small molecule metabolites and 9 fatty acids using the system of example 2;
FIG. 10 is a chromatogram obtained from the analysis of 8 small molecule metabolites and 9 fatty acids using a HILIC chromatography column;
FIG. 11 is a chromatogram obtained from analysis of 8 small molecule metabolites and 9 fatty acids using a common reverse phase C18 chromatography column;
FIG. 12 is a chromatogram obtained from analysis of 8 small molecule metabolites and 9 fatty acids using a broad polarity T3 chromatographic column;
the arrows in the figure indicate the flow direction, and the corresponding relationships of the reference numbers are:
1-a first chromatography pump; 2-a second chromatography pump; 3-a third chromatographic pump; 4-autosampler; 5-a hydrophilic interaction chromatography column; 6-a first six-way valve; 7-a second six-way valve; 8-a mixer; 9-sample loop; 10-reverse phase chromatography column; 11-a detector; 12-ten way valve.
Detailed Description
Referring to fig. 1, the two-dimensional liquid system of the present invention includes a hydrophilic interaction chromatography column, a reverse phase chromatography column, a detector, an autosampler, a first pump assembly, a second chromatography pump, a third chromatography pump, a mixer, a sample ring, and a switching valve.
The first pump assembly is used for pushing a sample loading solution and carrying out gradient elution on the hydrophilic interaction chromatographic column, an outlet of the first pump assembly is communicated with an inlet of the hydrophilic interaction chromatographic column, and the automatic sample injector is arranged on a connecting pipeline between the first pump assembly and the hydrophilic interaction chromatographic column;
the second chromatographic pump and the third chromatographic pump are used for gradient elution of the reversed-phase chromatographic column, the outlet of the second chromatographic pump is communicated with the inlet of the mixer through the switching valve, the outlet of the third chromatographic pump is communicated with the inlet of the mixer, and the outlet of the mixer is communicated with the inlet of the reversed-phase chromatographic column;
the outlet of the hydrophilic interaction chromatographic column, the outlet of the reverse phase chromatographic column, the inlet of the detector and the two ports of the sample ring are respectively connected with the switching valve;
the switching valve has at least the following 2 connection states by switching its position:
connection state 1: the switching valve connects the inlet of the detector with the outlet of the hydrophilic interaction chromatography column and connects one port of the sample loop with the outlet of the second chromatography pump and the other port with the inlet of the mixer;
connection state 2: the switching valve connects the inlet of the detector with the outlet of the reverse phase chromatography column and connects one port of the sample loop with the outlet of the hydrophilic interaction chromatography column and the other port to the outside of the system.
Specifically, the switching valve is formed by connecting two six-way valves or is a ten-way valve.
The mixer is a three-way mixer and is provided with three ports, wherein two ports are inlets of the mixer and are respectively communicated with an outlet of the third chromatographic pump and the switching valve through pipelines, and the other port is an outlet of the mixer and is communicated with an inlet of the reversed-phase chromatographic column through a pipeline.
The first pump assembly comprises a first chromatographic pump and a low-pressure gradient proportional valve arranged in the first chromatographic pump; alternatively, the first pump assembly is a binary high pressure gradient system comprising two chromatography pumps.
The detector is a mass spectrum detector or an ultraviolet detector.
In a further preferred embodiment, the two ports of the sample loop are a first port and a second port, respectively; in the connection state 1, the switching valve connects a first port of the sample loop with the outlet of the second chromatographic pump and a second port of the sample loop with the inlet of the mixer; in the
The analysis method of the system comprises the following steps:
1) loading a hydrophilic interaction chromatographic column, balancing a reversed phase chromatographic column:
the switching valve is adjusted to a state of communicating the inlet of the detector with the outlet of the hydrophilic interaction chromatographic column (for example, the switching valve is switched to a connection state 1), then the automatic sample injector introduces the sample into the system, the first pump assembly outputs a high proportion of organic solvent to push the sample into the hydrophilic interaction chromatographic column, so that the sample is retained on the column head of the hydrophilic interaction chromatographic column, and the high proportion of organic solvent enters the detector through the switching valve; meanwhile, the second chromatographic pump and the third chromatographic pump respectively convey the aqueous phase solvent to enter a mixer for mixing to form a high-proportion aqueous phase mixed solvent to enter the reversed-phase chromatographic column;
2) collecting weak polar substances:
the first pump assembly is used for conveying the solvent, the proportion of the water phase solvent in the solvent is increased, the weak polar substances in the sample are eluted from the hydrophilic interaction chromatographic column, the switching valve is switched to a
3) separating strong polar substances, focusing weak polar substances:
the switching valve is switched to a connection state 1, the proportion of the water phase solvent in the solvent conveyed by the first pump assembly is continuously increased, the strong polar substance retained on the hydrophilic interaction chromatographic column is subjected to gradient elution, and the strong polar substance enters the detector through the switching valve for detection; meanwhile, the second chromatographic pump outputs an organic phase solvent to push the weak polar substances in the sample ring to the mixer through the switching valve, the third chromatographic pump transmits a water phase solvent to the mixer, a high proportion water phase mixed solvent carrying the weak polar substances is formed in the mixer and enters the reversed phase chromatographic column, the weak polar substances are focused on the column head of the reversed phase chromatographic column, and the high proportion water phase mixed solvent is discharged out of the system through the switching valve;
4) balancing a hydrophilic interaction chromatographic column, and separating weak polar substances:
after the separation and detection of the strong polar substances on the hydrophilic interaction chromatographic column are finished, the switching valve is switched to a
5) and returning to the initial state:
after all the substances on the reverse phase chromatographic column are eluted to the detector, the system is restored to the state of the step 1) and waits for the next sample injection.
Specifically, in step 2), the switching valve switches to the
Specifically, in the step 1) and the step 4), the volume proportion of the organic phase solvent in the high proportion of the organic solvent delivered by the first pump assembly is more than 95%; in steps 1) to 3), the volume ratio of the aqueous phase solvent in the high-ratio aqueous phase mixed solvent formed in the mixer is always greater than 90%, and in step 4), the volume ratio gradient of the organic phase solvent in the mixed solvent formed in the mixer is increased to greater than 95%. .
In a further preferred embodiment, step 2) is opposite to the flow direction of the liquid phase in the sample loop in step 3), corresponding to the connection of the first port and the second port of the sample loop in connection state 1 and
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