A two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument

Abstract

The present invention provides a two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument, comprising: a first flow channel, a second flow channel, a third flow channel, an analytical flow channel connected to a second chromatographic column (C2), a waste liquid flow channel (L7), a first multi-channel switching valve (V1) and a second multi-channel switching valve (V2) connected to each other, and an air flow channel connected to an air filter (AIR) and an air compressor pump (P3), etc. By introducing compressed air, the present invention discharges the first mobile phase in the first chromatographic column and the pipeline connected thereto from the instrument, thereby reducing or completely removing the generation of matrix effect caused by the entry of the first mobile phase into the mass spectrometry part, and avoiding adverse effects on the response, stability and reproducibility of the mass spectrometry.

Description

Technical Field

[0001] The invention belongs to the field of liquid chromatography-mass spectrometry instruments and equipment, and in particular relates to a two-dimensional liquid chromatograph capable of reducing a liquid chromatography-mass spectrometry instrument matrix effect. Background Art

[0002] Two-dimensional liquid chromatography is a chromatographic instrument developed on the basis of ordinary liquid chromatography. It improves separation ability by increasing the number of chromatographic separation stages and the number of chromatographic columns. Since liquid chromatography cannot directly obtain the structural information of the target, it is necessary to judge the unknown by comparing it with the standard. For special substances, different detectors usually need to be replaced. For example, for compounds without UV absorption, the common UV absorption detector cannot be used for detection, and it needs to be replaced with a fluorescence detector, a differential detector or an electrochemical detector. On the other hand, due to the limitations of the detection limits of various detectors, two-dimensional liquid chromatography has obvious limitations in trace analysis. The mass spectrometer has the advantages of a wide analysis range (almost all compounds can be detected), strong separation ability, reliable qualitative analysis results, low detection limits and fast analysis time. Liquid chromatography-mass spectrometry has been widely used in many fields such as drug analysis, food analysis and environmental analysis.

[0003] Due to the technical defects of the mass spectrometer itself, the mass spectrometer needs to purify the sample to be analyzed, and it can only be analyzed after it has a certain purity. The sample is separated from the mobile phase in the mass spectrometer part, and after being ionized, the mass analyzer of the mass spectrometer separates the ion fragments according to the mass number to obtain a mass spectrum. After the sample is chromatographically separated, the co-eluting substances enter the mass spectrometer part together, which changes the ionization efficiency of the component to be measured. The resulting signal suppression or enhancement is called the matrix effect. Since the appearance of the matrix effect greatly destroys the stability of the response and the reproducibility of the results, it is regarded as a major obstacle to the liquid chromatography-mass spectrometry technology.

[0004] In order to remove endogenous impurities from biological samples in the first dimension as much as possible, existing two-dimensional liquid chromatography-mass spectrometry inevitably requires the use of ion pair reagents, buffer salts, etc. These substances are often transferred to the two-dimensional chromatography along with the target to varying degrees, and then enter the mass spectrometry part, resulting in matrix effects, affecting the response, stability and reproducibility of the mass spectrometry, and limiting the application of two-dimensional liquid chromatography-mass spectrometry in routine inspections. Summary of the invention

[0005] In order to solve the technical problems described in the background, the purpose of the present invention is to improve the two-dimensional liquid chromatograph to reduce or eliminate the matrix effect brought by the two-dimensional liquid chromatography to the mass spectrometer detector.

[0006] The present invention reduces or completely eliminates the matrix effect caused by the first mobile phase entering the mass spectrometry part by discharging the first mobile phase in the first chromatographic column and the pipeline connected thereto by introducing compressed air.

[0007] The object of the present invention is achieved by the following technical solution: A two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument, comprising: a first flow channel for transporting a first mobile phase; a second flow channel for transporting a second mobile phase;

[0008] An analysis flow channel connected to a second chromatographic column for separating and detecting the captured substances; a waste liquid flow channel for discharging waste liquid; a first multi-channel switching valve and a second multi-channel switching valve connected to each other for connecting and switching each flow channel; the first multi-channel switching valve and the second multi-channel switching valve are provided with a plurality of ports; further comprising an air flow channel connected to an air filter and an air compressor pump; a plug is provided on one of the ports of the first multi-channel switching valve, and the other two arbitrary ports are respectively connected to the first flow channel and the air flow channel; a first chromatographic column is connected between any two ports of the second multi-channel switching valve; the second multi-channel switching valve is further connected to the second flow channel, the waste liquid flow channel, and the analysis flow channel respectively on any three ports.

[0009] The first multi-channel switching valve has 4 ports, namely port 1, port 2, port 3, and port 4; the second multi-channel switching valve has 6 ports, namely port 5, port 6, port 7, port 8, port 9, and port 10; the first flow channel is connected to port 1, the air flow channel is connected to port 3, a plug is provided on port 2, port 4 and port 6 are connected, a first chromatographic column is connected to the flow channel between port 7 and port 10, port 8 is connected to the analysis flow channel, and port 9 is connected to the second flow channel.

[0010] Preferred solution: Further comprising a third flow channel for transporting a third mobile phase, and the third flow channel is connected to the first flow channel. Since only the first mobile phase in the first chromatographic column and the connected pipeline is discharged from the instrument, some inorganic salts or substances with weak ionic strength will still remain in the chromatographic column. By introducing the third mobile phase to flush the first chromatographic column and the connected pipeline, the entry of related substances into the mass spectrometry part can be further reduced, thereby reducing or eliminating the matrix effect brought by the two-dimensional liquid chromatography to the mass spectrometry detector.

[0011] A first chromatographic pump and an injector are provided on the first flow channel.

[0012] Further preferably, the third flow channel is connected to the first flow channel at the rear end of the injector.

[0013] A second chromatographic pump is provided on the second flow channel.

[0014] The analysis flow channel is connected to a detector.

[0015] The air compression pump causes the pressure in the pipeline to reach a set value of 1 Mpa - 1.2 Mpa.

[0016] The air filter can filter out particulate matter with a diameter greater than 0.2 microns in the compressed air.

[0017] The third mobile phase can be any solvent that can dissolve salts and is difficult to ionize, such as water, acid, etc.

[0018] The present invention has the following advantages and effects compared with the prior art:

[0019] 1. The two-dimensional liquid chromatograph capable of reducing the matrix effect of the liquid chromatography-mass spectrometry instrument of the present invention discharges the first mobile phase in the first chromatographic column and the pipeline connected thereto from the instrument by introducing compressed air, thereby reducing or completely removing the generation of matrix effect caused by the first mobile phase entering the mass spectrometry part.

[0020] 2. The two-dimensional liquid chromatograph capable of reducing the matrix effect of the liquid chromatography-mass spectrometry instrument of the present invention filters out particulate matter with a diameter greater than 0.2 microns in the compressed air through the AIR air filter, improves the purity of the compressed air, reduces the influence of impurities on the equipment, and extends the service life of the equipment.

[0021] 3. The two-dimensional liquid chromatograph capable of reducing the matrix effect of the liquid chromatography-mass spectrometry instrument of the present invention flushes the first chromatographic column and the connected pipeline by introducing the third mobile phase, which can further reduce the entry of some inorganic salts or substances with weak ionic strength into the mass spectrometry part, thereby reducing or eliminating the generation of the two-matrix effect and avoiding adverse effects on the response, stability, and reproducibility of the mass spectrometry.

[0022] 4. The two-dimensional liquid chromatograph capable of reducing the matrix effect of the liquid chromatography-mass spectrometry instrument of the present invention increases the injection volume by adding the third mobile phase during injection. When not added, the maximum injection volume is usually 50 microliters. After adding the third mobile phase, the injection volume reaches 1 milliliter, obtaining an ideal dot injection effect, reducing injection error, and improving injection accuracy and repeatability. Brief Description of the Drawings

[0023] Figure 1 It is a schematic structural diagram of the liquid chromatograph of Example 1;

[0024] Figure 2 It is a working state diagram of the two-dimensional liquid chromatograph of Example 1;

[0025] Figure 3 It is a working state diagram of the two-dimensional liquid chromatograph of Example 1;

[0026] Figure 4 It is a working state diagram of the two-dimensional liquid chromatograph of Example 1;

[0027] Figure 5 It is a schematic structural diagram of the liquid chromatograph in Embodiment 2;

[0028] Figure 6 It is a working state diagram of a two-dimensional liquid chromatograph in Embodiment 2;

[0029] Figure 7 It is a working state diagram of a two-dimensional liquid chromatograph in Embodiment 2;

[0030] Figure 8 It is a working state diagram of a two-dimensional liquid chromatograph in Embodiment 2;

[0031] Figure 9 It is a working state diagram of a two-dimensional liquid chromatograph in Embodiment 2;

[0032] Figure 10 It is a working state diagram of a two-dimensional liquid chromatograph in Embodiment 2;

[0033] Among them, S1 is the first mobile phase, S2 is the second mobile phase, and S3 is the third mobile phase; P1 is the first chromatographic pump, P2 is the second chromatographic pump, P3 is the air compression pump, and P4 is the third chromatographic pump; L1 is the first section of the first flow channel, L2 is the second section of the first flow channel, L3 is the third section of the first flow channel, L4 is the first connecting pipeline, L5 is the second connecting pipeline, L6 is the third connecting pipeline, L7 is the waste liquid flow channel, L8 is the first section of the second flow channel, L9 is the second section of the second flow channel, L10 is the first section of the analysis flow channel, L11 is the second section of the analysis flow channel, L12 is the first section of the air pipeline, L13 is the second section of the air pipeline, L14 is the first section of the third flow channel, and L15 is the second section of the third flow channel; V1 is the first multi-channel switching valve; V2 is the second multi-channel switching valve; T1 is the plug; C1 is the first chromatographic column; C2 is the second chromatographic column; W1 is the waste liquid port; D1 is the detector; SIL is the automatic sampler; AIR is the air filter; J1 is the tee; The thick solid lines in the figure represent the mobile phase flow routes, and the dashed lines represent the non-flowing routes. Specific Embodiments

[0034] The present invention will be further explained and described below with reference to the accompanying drawings and embodiments. Embodiment 1

[0035] A two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument, comprising: a first flow channel for conveying a first mobile phase S1; a second flow channel for conveying a second mobile phase S2; an analysis flow channel connected to a second chromatographic column C2 for separating and detecting the captured substances; a waste liquid flow channel L7 for discharging waste liquid; a first multi-channel switching valve V1 and a second multi-channel switching valve V2 connected to each other for connecting and switching each flow channel; the first multi-channel switching valve V1 and the second multi-channel switching valve V2 are provided with a plurality of ports; further comprising an air flow channel connected to an air filter AIR and an air compressor pump P3; a plug T1 is provided on one of the ports of the first multi-channel switching valve V1, and the other two arbitrary ports are respectively connected to the first flow channel and the air flow channel; a first chromatographic column C1 is connected between any two ports of the second multi-channel switching valve V2; the second multi-channel switching valve V2 is further respectively connected to the second flow channel, the waste liquid flow channel L7 and the analysis flow channel on any three ports.

[0036] As Figure 1 shown, the first multi-channel switching valve has 4 ports, namely port 1, port 2, port 3 and port 4; the second multi-channel switching valve has 6 ports, namely port 5, port 6, port 7, port 8, port 9 and port 10; the first flow channel is connected to port 1, the air flow channel is connected to port 3, a plug T1 is provided on port 2, port 4 and port 6 are connected, a first chromatographic column C1 is connected to the flow channel between port 7 and port 10, port 8 is connected to the analysis flow channel, and port 9 is connected to the second flow channel.

[0037] As Figure 2 [Status 1] shown, at this time, the position state of the first multi-channel switching valve V1 is that 1 is connected to 4 and 2 is connected to 3, and the position state of the second multi-channel switching valve V2 is that 5 is connected to 10, 6 is connected to 7, and 8 is connected to 9.

[0038] The air compressor pump P3 is started and conveys the air filtered by the air filter AIR to the second section L13 of the air pipeline. Since the passage of port 2 of the first multi-channel switching valve V1 is blocked by the plug T1, the air compressor pump P3 suspends working after the pressure in the pipeline reaches the set value (1 Mpa). The compressed air input into the device by the P3 air pump needs to be filtered by the AIR air filter to remove the particulate matter with a diameter greater than 0.2 microns in the air to improve the service life of the device.

[0039] The first mobile phase S1 is transported by the first chromatographic pump P1 through the first section L1 of the first flow channel and the second section L2 of the first flow channel. The sample is carried by the autosampler SIL and passes through the third section L3 of the first flow channel, the first connecting pipeline L4, and the second connecting pipeline L5, and then leads to the first chromatographic column C1 for primary separation of the sample. The impurities with a retention strength less than that of the target on the first chromatographic column C1 first pass through the third connecting pipeline L6 and the waste liquid flow channel L7, and then are discharged from the instrument through the waste liquid port W1.

[0040] As Figure 3 As shown in [Status 2], before the target and the impurities with a similar retention strength to the target on the first chromatographic column C1 are eluted from the first chromatographic column C1, the first multi-channel switching valve V1 switches its position to 1 to 2, 3 to 4. The position of the second multi-channel switching valve V2 remains unchanged.

[0041] At the same time, the first chromatographic pump P1 pauses operation and stops the transportation of the first mobile phase S1. Compressed air passes through the second section L13 of the air pipeline, the first connecting pipeline L4, the second connecting pipeline L5, the third connecting pipeline L6, and the waste liquid flow channel L7, and discharges the first mobile phase S1 in the pipelines passed through and the first chromatographic column C1 from the instrument through the waste liquid port W1. When the pressure of the compressed air in the pipeline is lower than the set value of 1 Mpa, the air compressor pump P3 will automatically start to maintain the pressure in the pipeline.

[0042] As Figure 4 As shown in [Status 3], when all the first mobile phase S1 in the second connecting pipeline L5, the third connecting pipeline L6, and the first chromatographic column C1 is discharged from the instrument through compressed air, the position state of the first multi-channel switching valve V1 is 1 to 4, 2 to 3, and the position state of the second multi-channel switching valve V2 is 5 to 6, 7 to 8, 9 to 10.

[0043] At this time, the first chromatographic pump P1 transports the first mobile phase S1 through the first section L1 of the first flow channel, the second section L2 of the first flow channel, the third section L3 of the first flow channel, the first connecting pipeline L4, the waste liquid flow channel L7, and discharges it from the instrument through the waste liquid port W1, or the first chromatographic pump P1 pauses operation and stops transporting the first mobile phase S1.

[0044] At the same time, the second chromatographic pump P2 transports the second mobile phase S2 through the first section L8 of the second flow channel, the second section L9 of the second flow channel, and the second connecting pipeline L5, and leads to the first chromatographic column C1 to elute the target from the first chromatographic column C1. Then it passes through the third connecting pipeline L6 and the first section L10 of the analysis flow channel and leads to the second chromatographic column C2 to complete the separation, and then enters the detector D through the second section L11 of the analysis flow channel for detection. Example 2

[0045] As Figure 5As shown in the figure, a two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument, on the basis of Example 1, further includes a third flow channel for delivering a third mobile phase S3; the third flow channel is connected to the first flow channel and is located on the flow channel behind the injector. Since only the first mobile phase S1 in the first chromatographic column C1 and the connected pipelines is discharged from the instrument, some inorganic salts or substances with weak ionic strength will still remain in the chromatographic column. By introducing the third mobile phase S3 to flush the first chromatographic column C1 and the connected pipelines, the entry of related substances into the mass spectrometry part can be further reduced, thereby reducing or eliminating the matrix effect brought by the two-dimensional liquid chromatography to the mass spectrometry detector.

[0046] As Figure 6 As shown in [Optimization State 1] of the injection state, at this time, the position state of the first multi-channel switching valve V1 is that port 1 is connected to port 4, and port 2 is connected to port 3. The position state of the second multi-channel switching valve V2 is that port 5 is connected to port 10, port 6 is connected to port 7, and port 8 is connected to port 9.

[0047] The air compressor pump P3 starts and delivers filtered air through the air filter AIR to the second section L13 of the air pipeline. Since the passage of port 2 of the first multi-channel switching valve V1 is blocked by the plug T1, the air compressor pump P3 pauses working after the pressure in the pipeline reaches the set value (1 Mpa).

[0048] The first mobile phase S1 is delivered by the first chromatographic pump P1 through the first section L1 and the second section L2 of the first flow channel, and the sample is carried by the autosampler SIL. The third chromatographic pump P4 delivers the third mobile phase S3, which is mixed with the first mobile phase S1 through the tee joint J1, and then passes through the third section L3 of the first flow channel, the first connecting pipeline L4, and the second connecting pipeline L5 to reach the first chromatographic column C1. Adding the third mobile phase S3 during injection can increase the injection volume. Without adding it, the maximum injection volume is usually 50 μL. After adding the third mobile phase S3, when the injection volume reaches 1 mL, an ideal dot-like injection effect can still be obtained, and the injection error can be reduced.

[0049] As Figure 7 As shown in [Optimization State 2], after the injection is completed, the third chromatographic pump P4 pauses working and stops delivering the third mobile phase S3. The first mobile phase S1 delivered by the first chromatographic pump P1 performs primary separation on the sample through the first chromatographic column C1; the impurities with a retention strength less than that of the target on the first chromatographic column C1 first pass through the third connecting pipeline L6 and the waste liquid flow channel L7 and are discharged from the instrument through the waste liquid port W1.

[0050] As Figure 8 As shown in [Optimization State 3], before the target and the impurities with a similar retention strength to the target on the first chromatographic column C1 are eluted from the first chromatographic column C1, at this time, the first multi-channel switching valve V1 switches its position to port 1 connected to port 2, and port 3 connected to port 4. The position of the second multi-channel switching valve V2 remains unchanged.

[0051] Meanwhile, the first chromatographic pump P1 and the third chromatographic pump P4 are suspended, halting the delivery of the first mobile phase S1 and the third mobile phase S3. Compressed air passes through the second section L13 of the air pipeline, the first connecting pipeline L4, the second connecting pipeline L5, the third connecting pipeline L6, and the waste liquid flow channel L7, discharging the first mobile phase S1 in the pipelines and the first chromatographic column C1 through the waste liquid port W1 out of the instrument. When the pressure of the compressed air in the pipeline is lower than the set value of 1 Mpa, the air compressor pump P3 will automatically start to maintain the pressure in the pipeline.

[0052] As Figure 9 [Optimization state 4] shows that after all of the first mobile phase S1 in the second connecting pipeline L5, the third connecting pipeline L6, and the first chromatographic column C1 is discharged from the instrument through compressed air, the position state of the first multi-channel switching valve V1 is 1 to 4, 2 to 3, and the position state of the second multi-channel switching valve V2 remains unchanged.

[0053] At this time, the first chromatographic pump P1 remains suspended and does not deliver the first mobile phase S1. Meanwhile, the third chromatographic pump P4 starts, delivering the third mobile phase S3 through the first section L14 of the third flow channel, the second section L15 of the third flow channel, the third section L3 of the first flow channel, and the first connecting pipeline L4, eluting the substances such as inorganic salts and other difficult-to-ionize substances in the remaining first mobile phase S1 in the second connecting pipeline L5, the third connecting pipeline L6, and the first chromatographic column C1 and discharging them through the third connecting pipeline L6 and the waste liquid flow channel L7 through the waste liquid port W1 out of the instrument.

[0054] After the substances such as inorganic salts and other difficult-to-ionize substances in the remaining first mobile phase S1 in the second connecting pipeline L5, the third connecting pipeline L6, and the first chromatographic column C1 are eluted, the state of discharging the first mobile phase S1 with compressed air is repeated to discharge the third mobile phase S3 in the second connecting pipeline L5, the third connecting pipeline L6, and the first chromatographic column C1 from the instrument.

[0055] As Figure 10 [Optimization state 5] shows that after all of the third mobile phase S3 in the second connecting pipeline L5, the third connecting pipeline L6, and the first chromatographic column C1 is discharged from the instrument through compressed air, at this time, the position state of the first multi-channel switching valve V1 is 1 to 4, 2 to 3, and the position state of the second multi-channel switching valve V2 is 5 to 6, 7 to 8, 9 to 10.

[0056] At this time, the first chromatographic pump P1 delivers the first mobile phase S1 through the first section L1 of the first flow channel, the second section L2 of the first flow channel, the third section L3 of the first flow channel, the first connecting pipeline L4, and the waste liquid flow channel L7 and discharges it through the waste liquid port W1 out of the instrument or the first chromatographic pump P1 is suspended and stops delivering the first mobile phase S1. The third chromatographic pump P4 is suspended and stops delivering the third mobile phase S3.

[0057] Meanwhile, the second chromatographic pump P2 delivers the second mobile phase S2 through the first section L8 of the second flow channel, the second section L9 of the second flow channel, and the second connecting pipeline L5 to the first chromatographic column C1, eluting the target substance from the first chromatographic column C1, passing through the third connecting pipeline L6 and the first section L10 of the analysis flow channel, leading to the second chromatographic column C2 for separation, and entering the D detector through the second section L11 of the analysis flow channel to complete the detection.

Claims

1. A two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument, comprising: A first flow channel for transporting a first mobile phase (S1); A second flow channel for transporting a second mobile phase (S2); An analysis flow channel connected to a second chromatographic column (C2) for separating and detecting the captured substances; A waste liquid flow channel (L7) for discharging waste liquid; A first multi-channel switching valve (V1) and a second multi-channel switching valve (V2) connected to each other for connecting and switching each flow channel; The first multi-channel switching valve (V1) and the second multi-channel switching valve (V2) are provided with a plurality of ports; Characterized in that it further comprises an air flow channel connected to an air filter (AIR) and an air compressor pump (P3); a plug (T1) is provided on one port of the first multi-channel switching valve (V1), and the other any two ports are respectively connected to the first flow channel and the air flow channel; a first chromatographic column (C1) is connected between any two ports of the second multi-channel switching valve (V2); a second flow channel, a waste liquid flow channel (L7), and an analysis flow channel are respectively connected to any three ports of the second multi-channel switching valve (V2); The first multi-channel switching valve (V1) has 4 ports, namely port 1, port 2, port 3, and port 4; the second multi-channel switching valve (V2) has 6 ports, namely port 5, port 6, port 7, port 8, port 9, and port 10; the first flow channel is connected to port 1, the air flow channel is connected to port 3, a plug (T1) is provided on port 2, port 4 and port 5 are connected, a first chromatographic column (C1) is connected to the flow channel between port 7 and port 10, port 8 is connected to the analysis flow channel, and port 9 is connected to the second flow channel; It further comprises a third flow channel for transporting a third mobile phase (S3), and the third flow channel is connected to the first flow channel.

2. The two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument according to claim 1, characterized in that A first chromatographic pump (P1) and an injector (SIL) are provided on the first flow channel.

3. The two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument according to claim 2, characterized in that The third flow channel is connected to the first flow channel behind the injector.

4. The two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument according to claim 1, characterized in that A second chromatographic pump (P2) is provided on the second flow channel.

5. The two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument according to claim 1, characterized in that The analysis flow channel is connected to a detector (D1).

6. The two-dimensional liquid chromatograph capable of reducing the matrix effect of a liquid chromatography-mass spectrometry instrument according to claim 1, characterized in that The air filter (AIR) is a filter capable of filtering out particulate matter with a diameter greater than 0.2 microns in compressed air.

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