CN114994208A - Nanoliter flow rate double-column capillary chromatography device and its application and use method - Google Patents

Abstract

The invention discloses a nanoliter flow rate double-column capillary chromatographic device and its application and use method. The device comprises an automatic sampler, a first chromatographic pump, a second chromatographic pump, a switching valve, a first enrichment column, and a second chromatographic pump. Two enrichment columns, the first capillary chromatographic column, the second capillary chromatographic column, the first stop valve, the second stop valve, the first waste liquid bottle, the second waste liquid bottle, and the mass spectrometer. The beneficial effects of the present invention are as follows: the device of the present invention adopts a double capillary chromatographic column to connect the components separated by the capillary chromatographic column with the ESI ionization source on-line through a switching valve (10-port-2-position), so that the mass spectrometer is always in the Collecting components from capillary column separation to achieve high-throughput and low sample cross-contamination, high-throughput analysis of tryptic digests of proteins in complex biological samples (plasma samples from cancer patients) Finding biomarkers closely related to disease from biological samples provides an efficient analytical technique.

Description

Nanoliter flow rate double-column capillary chromatography device and its application and using method

technical field

The invention belongs to the technical field of analysis and identification instruments for biological samples, and in particular relates to a nanoliter flow rate double-column capillary chromatography device and an application and use method thereof.

Background technique

Finding biomarkers closely related to diseases (such as cancer) from biological samples (such as blood and urine) for disease diagnosis and prognosis evaluation is an urgent need in clinical medicine, which has an extremely important role in improving disease diagnosis and treatment. Significance. Since these biomarkers usually come from diseased tissues and organs, and the concentrations after secretion into biological fluids are low (<0.5ng/mL), it is crucial to establish an analytical technique with high sensitivity and no cross-contamination between samples. Nanoliter flow rate capillary high-performance liquid chromatography-mass spectrometry (CapLC-MS) online analytical methods are widely used for the analysis and detection of various biomarkers in complex biological samples, including proteins, nucleic acids, metabolites and lipid molecules.

The existing nanoliter flow rate capillary high performance liquid chromatography-mass spectrometry analysis method adopts a single capillary chromatographic column and a mass spectrometer electrospray (ESI) ionization source directly combined, and the components separated from the capillary chromatographic column are subjected to ESI ionization. The method enters the mass spectrometer in the state of gaseous ions for analysis and detection. Since the method adopts a nanoliter/minute (nL/min) flow rate, it can provide a detection sensitivity dozens of times higher than that of the conventional flow rate of microliter/minute to milliliter/minute (uL/min to mL/min). However, the existing nanoliter flow rate capillary high performance liquid chromatography-mass spectrometry technology has the following main shortcomings:

① The analysis throughput is low, and only one sample can be analyzed at a time; when the chromatographic column is cleaned, the mass spectrometer is in a standby or invalid data acquisition state.

②The residual sample in the chromatographic column leads to cross-contamination between samples, which affects the accuracy of sample analysis.

③ The waste liquid from cleaning the chromatographic column enters the mass spectrometer directly through the ESI ionization source, causing unnecessary pollution to the mass spectrometer and increasing the number and time of downtime caused by cleaning the instrument.

④The residual and accumulation of samples in the chromatographic column reduces the separation efficiency of the chromatographic column and shortens the service life of the chromatographic column.

⑤ The residual and accumulation of samples in the chromatographic column increases the probability of clogging and scrapping of the chromatographic column.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a nanoliter flow rate dual-column capillary chromatography device for analyzing complex biological samples and its application and use method.

To achieve the above object, the present invention provides the following technical solutions:

A nanoliter flow rate dual-column capillary chromatographic device, comprising an automatic sampler, a first chromatographic pump, a second chromatographic pump, a switching valve, a first enrichment column, a second enrichment column, a first capillary chromatographic column, a second Capillary chromatographic column, first stop valve, second stop valve, first waste liquid bottle, second waste liquid bottle, mass spectrometer;

The mobile phase flows through the automatic sampler under the action of the first chromatographic pump, the solution flowing out from the automatic sampler enters the switching valve, and the switching valve passes through the first enrichment column to the liquid that flows through the second stop valve and the first capillary chromatographic column. Equilibrate, and the liquid flowing through the first capillary chromatographic column enters the mass spectrometer through the switching valve;

The mobile phase flows through the switching valve under the action of the second chromatographic pump, the switching valve balances the liquid flowing through the first stop valve and the second capillary chromatographic column through the second enrichment column, and the liquid flowing through the second capillary chromatographic column is switched The valve flows into the first waste liquid bottle;

The outlets of the first shut-off valve and the second shut-off valve are both communicated with the second waste liquid bottle.

The mass spectrometer employed in this application can be any commercial electrospray ionization mass spectrometer, including quadrupole-time-of-flight mass spectrometry, quadrupole-orbitrap mass spectrometry, ion trap mass spectrometry, and the like.

The above-mentioned nanoliter flow rate dual-column capillary chromatography device, as a preferred embodiment, the automatic sampler includes a sampling pump, a sampling valve, a sampling needle and a sample tray, and the sampling valve is 6- port-2-position injection valve; the sample tray contains at least 2 samples to be tested, and the two ends of the sample loop are respectively connected to the inlet 2 of the injection valve and the inlet 5 of the injection valve; preferably, the sample ring is 20ul PEEK tube.

The injection pump is communicated with the inlet 3 of the injection valve, one end of the injection needle is communicated with the inlet 4 of the injection valve, and the other end of the injection needle is connected with the sample tray Pass.

In the above-mentioned nanoliter flow rate dual-column capillary chromatography device, as a preferred embodiment, the switching valve is a 10-port-2-position switching valve, and the dead volume between the internal channels of the switching valve is less than 7nL;

Both the first shut-off valve and the second shut-off valve are 6-port-2-position shut-off valves.

The above-mentioned nanoliter flow rate dual-column capillary chromatographic device, as a preferred embodiment, the first chromatographic pump is communicated with the inlet 1 of the injection valve, and the outlet 6 of the injection valve is connected to the switch The valve inlet 10 is connected;

The outlet 1 of the switching valve is communicated with the first enrichment column, and the outlet of the first enrichment column is communicated with the inlet 4 of the second stop valve and the first capillary chromatographic column through a tee, respectively. The capillary chromatographic column is communicated with the inlet 4 of the switching valve, the outlet 5 of the switching valve is communicated with the mass spectrometer, and the outlet 3 of the switching valve is communicated with the first waste liquid bottle; A plug is provided on the outlet 3 of the second shut-off valve.

The above-mentioned one nanoliter flow rate dual-column capillary chromatographic device, as a preferred embodiment, the second chromatographic pump is communicated with the inlet 8 of the switching valve, and the outlet 9 of the switching valve is connected with the second enrichment column The outlet of the second enrichment column is communicated with the inlet 2 of the first shut-off valve and the second capillary chromatographic column respectively through the tee, and the second capillary chromatographic column is communicated with the inlet 6 of the switching valve. ; A plug is provided on the outlet 1 of the first shut-off valve.

Above-mentioned a kind of nanoliter flow rate double column capillary chromatographic device, as a kind of preferred embodiment, the outlet 3 of described first stop valve and the outlet 5 of described second stop valve are all communicated with described second waste liquid bottle .

In the above-mentioned dual-column capillary chromatography device with nanoliter flow rate, as a preferred embodiment, the first enrichment column and the second enrichment column are both enrichment columns of 75-200 μm, ID x 2 cmL; The first capillary chromatographic column and the second capillary chromatographic column are both capillary chromatographic columns of 50-100 μm, ID x 10-50 cmL.

A second aspect of the present application provides an application of the above nanoliter flow rate dual-column capillary chromatography device in biological sample analysis.

A third aspect of the present application provides a method for analyzing complex biological samples by the above-mentioned nanoliter flow rate dual-column capillary chromatography device, comprising the following steps:

(1) Loading of the first sample: Adjust the injection valve so that the inlet 3, inlet 2, inlet 5, and inlet 4 of the injection valve are connected in sequence, and the inlet 1 of the injection valve is connected to the outlet 6 of the injection valve. Pass;

Adjust the switching valve: Connect the outlet 3, inlet 2, inlet 7, and inlet 6 of the switching valve in sequence, connect the inlet 4 and outlet 5 of the switching valve, and connect the outlet 1 and inlet 10 of the switching valve, so that the switching valve is connected. The inlet 8 and outlet 9 are connected;

Adjust the first shut-off valve: make the inlet 5 and the inlet 6 of the first shut-off valve communicate with each other, make the inlet 2 and the outlet 1 of the first shut-off valve communicate with each other, and communicate with the inlet 4 and the outlet 3 of the first shut-off valve;

Adjust the second globe valve: connect the inlet 1 and the inlet 2 of the second globe valve, connect the inlet 6 and the outlet 5 of the second globe valve, and connect the inlet 4 and the outlet 3 of the second globe valve;

The syringe draws the first sample 5uL;

The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 300nL/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min;

(2) Enrichment of the first sample: adjust the injection valve so that the inlet 3 and the inlet 4 of the injection valve are connected, and the inlet 1, the inlet 2, the inlet 5 and the outlet 6 of the injection valve are connected in sequence;

Adjust the switching valve so that the inlet 6, inlet 7, inlet 2 and outlet 3 of the switching valve are connected in turn, so that the inlet 4 and outlet 5 of the switching valve are connected, and the inlet 10 and outlet 1 of the switching valve are connected, so that the switching valve is connected. The inlet 8 and outlet 9 are connected;

Adjust the second globe valve: connect the inlet 1 and the inlet 6 of the second globe valve, connect the inlet 2 and the outlet 3 of the second globe valve, and connect the inlet 4 and the outlet 5 of the second globe valve;

The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 5ul/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min;

(3) Gradient elution and mass spectrometry detection:

Adjust the inlet and outlet communication modes of the injection valve, the switching valve, the first shut-off valve and the second shut-off valve in the same manner as in step (1);

The first chromatography pump performs separation gradient elution: the elution mode is:

0min<elution time≤0.1min, linear change from 95% mobile phase A and 5% mobile phase B to 90% mobile phase A and 10% mobile phase B, the eluent flow rate is 300nl/min;

0.1min＜elution time≤96min, linear change from 90% mobile phase A and 5% mobile phase B to 67% mobile phase A and 33% mobile phase B, the eluent flow rate is 300nl/min;

96min<elution time≤106min, linear change from 67% mobile phase A and 33% mobile phase B to 50% mobile phase A and 50% mobile phase B, the eluent flow rate is 300nl/min;

106min<elution time≤111min, use 50% mobile phase A and 50% mobile phase B, linearly change from 50% mobile phase A and 50% mobile phase B to 0% mobile phase A and 100% mobile phase B, elution The liquid flow rate is 300nl/min;

111min＜elution time≤120min, use 0% mobile phase A and 100% mobile phase B, and the eluent flow rate is 1000nl/min;

The mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B, and the flow rate of the mobile liquid is 1.0ul/min;

(4) Loading of the second sample: Adjust the inlet and outlet communication modes of the injection valve, the first shut-off valve and the second shut-off valve are the same as in step (1);

The outlet 1, inlet 2, inlet 7 and inlet 8 of the adjustment switch valve are connected in sequence, the inlet 10 and outlet 9 of the adjustment switch valve are connected, the inlet 4 and outlet 3 of the adjustment switch valve are connected, and the inlet 6 and the outlet 3 of the adjustment switch valve are connected. Exit 5 is connected;

The syringe draws the second sample 5uL;

The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 300nL/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min;

(5) Enrichment of the second sample: adjust the injection valve so that the inlet 3 and the inlet 4 of the injection valve are connected, and the inlet 1, the inlet 2, the inlet 5 and the outlet 6 of the injection valve are connected in sequence;

Adjust the switching valve so that the outlet 1, inlet 2, inlet 7, and inlet 8 of the switching valve are connected in sequence, so that the inlet 10 and outlet 9 of the switching valve are connected, and the inlet 6 and outlet 5 of the switching valve are connected, so that the switching valve is connected. The inlet 4 and outlet 3 are connected;

Adjust the first stop valve so that the outlet 1 and the inlet 6 of the first stop valve are connected, the inlet 4 and the inlet 5 of the first stop valve are connected, and the inlet 2 and the outlet 3 of the first stop valve are connected;

Adjust the second shut-off valve so that the inlet 1 and the inlet 2 of the second shut-off valve are communicated, the inlet 4 and the outlet 3 of the second shut-off valve are communicated, and the inlet 6 and the outlet 5 of the second shut-off valve are communicated;

The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 5ul/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min;

(6) Gradient elution and mass spectrometry detection:

Adjusting the inlet and outlet communication modes of the injection valve, the switching valve and the second shut-off valve are the same as in step (4);

Adjust the inlet 4 and the inlet 5 of the first stop valve to be connected, adjust the inlet 2 and the outlet 3 of the first stop valve to be connected, and adjust the inlet 6 of the first stop valve to be connected to the outlet 1;

The first chromatographic pump performs cleaning gradient elution: the elution mode is:

0min<elution time≤28min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

28min<elution time≤30min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

30min<elution time≤58min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

58min<elution time≤60min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul min;

60min<elution time≤88min, linear change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

88min<elution time≤90min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

90min＜elution time≤118min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

118min＜elution time≤120min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

The mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B, and the flow rate of the mobile liquid is 1.0ul/min;

(7) Repeat steps (1) to (6) to analyze the remaining samples in the sample tray by dual-column capillary chromatography with a nanoliter flow rate.

As a preferred embodiment of the above-mentioned nanoliter flow rate dual-column capillary chromatography device for analyzing complex biological samples, the mobile phase A is an aqueous solution of 0.1% formic acid, and the mobile phase B is an acetonitrile solution of 0.1% formic acid.

The beneficial effects of the invention are as follows: the nanoliter flow rate dual-column capillary chromatographic device of the invention adopts the dual-capillary chromatographic column to ionize the components separated by the capillary chromatographic column and ESI through a switching valve (10-port-2-position). The source is connected online, so that the mass spectrometer is always collecting the components separated from the capillary chromatographic column, so as to achieve the effect of high throughput and low sample cross-contamination, and can be used for the trypsin digestion of proteins in complex biological samples (plasma samples of cancer patients). The product was subjected to high-throughput analysis.

The nanoliter flow rate dual-column capillary chromatographic device of the invention avoids the low analytical throughput, cross-contamination between samples, short capillary chromatographic column life, and capillary chromatographic column cleaning waste liquid pollution of the existing single-capillary column chromatographic separation technology. Mass spectrometry analysis It can be used for nanoscale flow rate capillary chromatography-mass spectrometry analysis of metabolites, nucleic acids, proteins and protein enzymolysis products in complex biological samples such as blood, urine and tissue, and is also suitable for high-sensitivity analysis of environmental pollutants and drug residues. analyze.

The nanoliter flow rate dual-column capillary chromatographic device of the present invention provides a high-throughput, no cross-contamination between samples, prolongs the life of the chromatographic column and reduces the contamination of the mass spectrometer for finding and discovering biomarkers closely related to diseases from biological samples. efficient analytical techniques.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other implementation drawings can also be obtained according to the extension of the drawings provided without creative efforts.

The structures, proportions, sizes, etc. shown in this specification are only used to cooperate with the contents disclosed in the specification, so as to be understood and read by those who are familiar with the technology, and are not used to limit the conditions for the implementation of the present invention, so there is no technical The substantive meaning above, any modification of the structure, the change of the proportional relationship or the adjustment of the size should still fall within the technical content disclosed in the present invention without affecting the effect and the purpose that the present invention can produce. within the range that can be covered.

Figure-1A is a schematic structural diagram of the first sample loading of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-1B is a schematic structural diagram of the first sample enrichment of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-1C is a schematic structural diagram of the first sample gradient elution and mass spectrometry detection of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-1D is a schematic structural diagram of the second sample loading of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-1E is a schematic structural diagram of the second sample enrichment of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-1F is a schematic structural diagram of the second sample gradient elution and mass spectrometry detection of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-2A is the separation and cleaning conditions of the first sample of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Figure-2B is the separation and cleaning conditions of the second sample of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

Fig. 3 is a comparison diagram of cleaning effect of the nanoliter flow rate dual-column capillary chromatographic device online combined analysis and single-column capillary chromatography-mass spectrometry online combined analysis of the present invention;

4 is a comparison of the analysis results between the nanoliter flow rate dual-column capillary chromatography device of the present invention and the single-column capillary chromatography-mass spectrometry;

Fig. 5 is the reproducibility of the analysis of the nanoliter flow rate dual-column capillary chromatography device according to the present invention;

In the figure: 1. Injection pump; 2. Injection valve; 3. Injection needle; 4. Sample tray; 5. First chromatography pump; 6. Second chromatography pump; 7. Switching valve; 8. First rich Collecting column; 9. Second enrichment column; 10. First capillary chromatographic column; 11. Second capillary chromatographic column; 12. First shut-off valve; 13. Second shut-off valve; 14. First waste liquid bottle; 15 , the second waste liquid bottle; 16, mass spectrometer.

Detailed ways

The embodiments of the present invention are described below by specific specific embodiments. Those who are familiar with the technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", The orientation or positional relationship indicated by "vertical", "horizontal", "horizontal", "longitudinal", etc. is based on the orientation or positional relationship shown in the drawings. These terms are primarily used to better describe the present application and its embodiments, and are not intended to limit the fact that the indicated device, element, or component must have a particular orientation, or be constructed and operated in a particular orientation.

In addition, some of the above-mentioned terms may be used to express other meanings besides orientation or positional relationship. For example, the term "on" may also be used to express a certain attachment or connection relationship in some cases. For those of ordinary skill in the art, the specific meanings of these terms in the present application can be understood according to specific situations.

The invention provides a nanoliter flow rate double-column capillary chromatography device, comprising an automatic sampler, a first chromatography pump, a second chromatography pump, a switching valve, a first enrichment column, a second enrichment column, and a first capillary chromatography column, second capillary chromatographic column, first stop valve, second stop valve, first waste liquid bottle, second waste liquid bottle, mass spectrometer;

The mobile phase flows through the automatic sampler under the action of the first chromatographic pump, the solution flowing out from the automatic sampler enters the switching valve, and the switching valve passes through the first enrichment column to the liquid that flows through the second stop valve and the first capillary chromatographic column. Equilibrate, and the liquid flowing through the first capillary chromatographic column enters the mass spectrometer through the switching valve;

The mobile phase flows through the switching valve under the action of the second chromatographic pump, the switching valve balances the liquid flowing through the first stop valve and the second capillary chromatographic column through the second enrichment column, and the liquid flowing through the second capillary chromatographic column is switched The valve flows into the first waste liquid bottle;

The outlets of the first shut-off valve and the second shut-off valve are both communicated with the second waste liquid bottle.

The injection valve used in this application is a 6-port-2-position injection valve, and the switching valve used is a 10-port-2-position switching valve. The dead volume between the internal channels of the switching valve is less than 7nL, so Both the first shut-off valve and the second shut-off valve used are 6-port-2-position shut-off valves.

The automatic sampler described in this application includes a sampling pump, a sampling valve, a sampling needle and a sample tray; and the sample tray contains at least two samples to be tested, and the two ends of the sample loop are respectively connected to the inlet of the sampling valve. 2 is communicated with the inlet 5 of the injection valve.

The injection pump is communicated with the inlet 3 of the injection valve, one end of the injection needle is communicated with the inlet 4 of the injection valve, and the other end of the injection needle is communicated with the sample tray.

The first chromatographic pump is communicated with the inlet 1 of the injection valve, and the outlet 6 of the injection valve is communicated with the inlet 10 of the switching valve;

The outlet 1 of the switching valve is communicated with the first enrichment column, and the outlet of the first enrichment column is communicated with the inlet 4 of the second stop valve and the first capillary chromatographic column through a tee, respectively. The capillary chromatographic column is communicated with the inlet 4 of the switching valve, the outlet 5 of the switching valve is communicated with the mass spectrometer, and the outlet 3 of the switching valve is communicated with the first waste liquid bottle; A plug is provided on the outlet 3 of the second shut-off valve.

The second chromatographic pump is communicated with the inlet 8 of the switching valve, the outlet 9 of the switching valve is communicated with the second enrichment column, and the outlet of the second enrichment column is respectively connected to the first stop through a tee. The inlet 2 of the valve is communicated with the second capillary chromatographic column, and the second capillary chromatographic column communicates with the inlet 6 of the switching valve; the outlet 1 of the first shut-off valve is provided with a plug.

The outlet 3 of the first shut-off valve and the outlet 5 of the second shut-off valve are both communicated with the second waste liquid bottle.

In order to achieve large-volume sample loading, the first enrichment column and the second enrichment column both use a 75-200 μm, ID x 2 cmL enrichment column; the first capillary chromatographic column and the second capillary chromatographic column All used capillary chromatographic columns of 50-100 μm, ID x 10-50 cmL.

Example 1

A method for analyzing complex biological samples by a nanoliter flow rate dual-column capillary chromatography device, comprising the following steps:

(1) Loading of the first sample: Adjust the injection valve so that the inlet 3, inlet 2, inlet 5, and inlet 4 of the injection valve are connected in sequence, and the inlet 1 of the injection valve is connected to the outlet 6 of the injection valve. Pass;

Adjust the switching valve: Connect the outlet 3, inlet 2, inlet 7, and inlet 6 of the switching valve in sequence, connect the inlet 4 and outlet 5 of the switching valve, and connect the outlet 1 and inlet 10 of the switching valve, so that the switching valve is connected. The inlet 8 and outlet 9 are connected;

Adjust the first shut-off valve: make the inlet 5 and the inlet 6 of the first shut-off valve communicate with each other, make the inlet 2 and the outlet 1 of the first shut-off valve communicate with each other, and communicate with the inlet 4 and the outlet 3 of the first shut-off valve;

Adjust the second globe valve: connect the inlet 1 and the inlet 2 of the second globe valve, connect the inlet 6 and the outlet 5 of the second globe valve, and connect the inlet 4 and the outlet 3 of the second globe valve;

The first chromatographic pump draws a certain volume of sample (5uL) from the first sample bottle in the sample tray through the injection needle into the sample loop of the injection valve. The first chromatographic pump flows 95% mobile phase-A and 5% mobile phase-B through the injection valve at a flow rate of 300nL/min, the liquid flowing out of the injection valve flows into the switching valve, and the switching valve flows through the first enrichment column. The second shut-off valve is equilibrated with the liquid in the first capillary column. The solution flowing out from the first enrichment column enters the inlet 4 of the second stop valve and the first capillary chromatographic column respectively after passing through the three-way. Because the outlet 3 of the second stop valve is connected with a plug, all the solution flows through the first capillary chromatographic column. The solution flowing out from the first capillary chromatographic column enters the inlet 4 of the switching valve and flows out from the outlet 5 to enter the electrospray ionization source of the mass spectrometer, and the ionization voltage is "0" kV at this time;

The second chromatographic pump flows through the switching valve at a flow rate of 1.0 uL/min (the mobile liquid is 95% mobile phase A and 5% mobile phase B), and the switching valve flows through the first stop valve and the second enrichment column through the second enrichment column. The liquid in the capillary column is equilibrated. The solution flowing out from the second enrichment column enters the inlet 2 of the first stop valve and the second capillary chromatographic column respectively after passing through the three-way. Because the outlet 1 of the first stop valve is connected with a plug, all the solution flows through the second capillary In the chromatographic column (elution and cleaning of the second capillary chromatographic column), the solution flowing out from the second capillary chromatographic column enters the inlet 6 of the switching valve and then flows out from the outlet 3 and enters the first waste liquid bottle.

(2) Enrichment of the first sample: adjust the injection valve so that the inlet 3 and the inlet 4 of the injection valve are connected, and the inlet 1, the inlet 2, the inlet 5 and the outlet 6 of the injection valve are connected in sequence;

Adjust the switching valve so that the inlet 6, inlet 7, inlet 2 and outlet 3 of the switching valve are connected in turn, so that the inlet 4 and outlet 5 of the switching valve are connected, and the inlet 10 and outlet 1 of the switching valve are connected, so that the switching valve is connected. The inlet 8 and outlet 9 are connected;

Adjust the second globe valve: connect the inlet 1 and the inlet 6 of the second globe valve, connect the inlet 2 and the outlet 3 of the second globe valve, and connect the inlet 4 and the outlet 5 of the second globe valve;

After the first chromatographic pump enters 95% mobile phase A and 5% mobile phase B into the inlet 1 of the injection valve at a flow rate of 5uL/min, the sample in the sample loop of the injection valve is pushed out from the outlet 6 of the injection valve and enters the first through the switching valve. An enrichment column. The sample is enriched in the first enrichment column, and the solution flowing out from the first enrichment column passes through the tee and then enters the inlet 4 of the second stop valve and the first capillary chromatographic column, because the outlet 5 of the second stop valve is connected To the second waste liquid bottle, the resistance (back pressure) of the stationary phase in the first capillary column causes all the solution to flow into the second waste liquid bottle.

The second chromatographic pump flows through the switching valve at a flow rate of 1.0 uL/min (the mobile liquid is 95% mobile phase A and 5% mobile phase B), and the switching valve flows through the first stop valve and the second enrichment column through the second enrichment column. The liquid in the capillary column is equilibrated. The solution flowing out from the second enrichment column enters the inlet 2 of the first shut-off valve and the second capillary chromatographic column respectively after passing through the tee. Because the outlet 1 of the first shut-off valve is connected with a plug, all the solution flows through the second capillary chromatographic column. column. The solution flowing out from the second capillary chromatographic column enters the inlet 6 of the switching valve and then flows out from the outlet 3 to enter the first waste liquid bottle.

(3) Gradient elution and mass spectrometry detection:

Adjust the inlet and outlet communication modes of the injection valve, the switching valve, the first shut-off valve and the second shut-off valve in the same manner as in step (1);

The first chromatographic pump flows the mobile phase A and the mobile phase B through the injection valve and the switching valve according to the gradient elution procedure to perform gradient elution on the first enrichment column and the first capillary chromatographic column. The sample component solution eluted from the first enrichment column enters the inlet 4 of the second stop valve and the first capillary chromatographic column respectively after passing through the tee. Because the outlet 3 of the second stop valve is connected to a plug, the sample group The fractional solution all flows through the first capillary chromatographic column. The sample components separated from the first capillary chromatographic column enter the inlet 4 of the switching valve and flow out from the outlet 5 to enter the electrospray ionization source (ionization voltage of 2.0kV) of the mass spectrometer. The ionized sample components enter the mass spectrometer. instrument and be tested;

The first chromatography pump performs separation gradient elution: the elution mode is:

0min<elution time≤0.1min, linear change from 95% mobile phase A and 5% mobile phase B to 90% mobile phase A and 10% mobile phase B, the eluent flow rate is 300nl/min;

0.1min＜elution time≤96min, linear change from 90% mobile phase A and 10% mobile phase B to 67% mobile phase A and 33% mobile phase B, the eluent flow rate is 300nl/min;

96min<elution time≤106min, linear change from 67% mobile phase A and 33% mobile phase B to 50% mobile phase A and 50% mobile phase B, the eluent flow rate is 300nl/min;

106min<elution time≤111min, use 50% mobile phase A and 50% mobile phase B, linearly change from 50% mobile phase A and 50% mobile phase B to 0% mobile phase A and 100% mobile phase B, elution The liquid flow rate is 300nl/min;

111min＜elution time≤120min, use 0% mobile phase A and 100% mobile phase B, and the eluent flow rate is 1000nl/min;

The mobile liquid used in the second chromatography pump was 95% mobile phase A and 5% mobile phase B, and the flow rate of the mobile liquid was 1.0 ul/min.

The second chromatographic pump flows through the switching valve and the first shut-off valve at a flow rate of 1.0 uL/min (95% mobile phase A and 5% mobile phase B) to the second enrichment column and the second capillary column. Gradient wash. The solution flowing out from the second enrichment column enters the inlet 2 of the first stop valve and the second capillary chromatographic column respectively after passing through the three-way. Because the outlet 1 of the first stop valve is connected with a plug, all the solution flows through the second capillary chromatographic column. The solution flowing out from the second capillary chromatographic column enters the inlet 6 of the switching valve and then flows out from the outlet 3 to enter the first waste liquid bottle.

(4) Loading of the second sample: Adjust the inlet and outlet communication modes of the injection valve, the first shut-off valve and the second shut-off valve are the same as in step (1);

The outlet 1, inlet 2, inlet 7 and inlet 8 of the adjustment switch valve are connected in sequence, the inlet 10 and outlet 9 of the adjustment switch valve are connected, the inlet 4 and outlet 3 of the adjustment switch valve are connected, and the inlet 6 and the outlet 3 of the adjustment switch valve are connected. Exit 5 is connected;

The first chromatographic pump draws a certain volume of sample (5uL) from the second sample bottle in the sample tray through the injection needle into the sample loop of the injection valve. The first chromatographic pump flows 95% mobile phase A and 5% mobile phase B through the injection valve at a flow rate of 300nL/min, the liquid flowing out of the injection valve flows into the switching valve, and the switching valve flows through the first enrichment column through the second enrichment column. The shut-off valve and the liquid in the second capillary column are equilibrated. The solution flowing out from the second enrichment column enters the inlet 2 of the first shut-off valve and the second capillary chromatographic column respectively after passing through the tee. Because the outlet 1 of the first shut-off valve is connected with a plug, all the solution flows through the second capillary chromatographic column. column. The solution flowing out from the second capillary chromatographic column enters the inlet 6 of the switching valve and then flows out from the outlet 5 to enter the electrospray ionization source of the mass spectrometer. At this time, the ionization voltage is "0" kV; the second chromatographic pump runs at 1.0uL/min (The mobile liquid is 95% mobile phase A and 5% mobile phase B) The flow rate flows through the switching valve, which equilibrates the liquid flowing through the second shut-off valve and the first capillary chromatography column through the first enrichment column. The solution flowing out from the first enrichment column enters the inlet 4 of the second stop valve and the first capillary chromatographic column respectively after passing through the three-way. Because the outlet 3 of the second stop valve is connected to a plug, all the solution flows through the first capillary chromatograph column. The solution flowing out from the first capillary chromatographic column enters the inlet 4 of the switching valve and then flows out from the outlet 3 to enter the first waste liquid bottle.

(5) Enrichment of the second sample: adjust the injection valve so that the inlet 3 and the inlet 4 of the injection valve are connected, and the inlet 1, the inlet 2, the inlet 5 and the outlet 6 of the injection valve are connected in sequence;

Adjust the switching valve so that the outlet 1, inlet 2, inlet 7, and inlet 8 of the switching valve are connected in sequence, so that the inlet 10 and outlet 9 of the switching valve are connected, and the inlet 6 and outlet 5 of the switching valve are connected, so that the switching valve is connected. The inlet 4 and outlet 3 are connected;

Adjust the first stop valve so that the outlet 1 and the inlet 6 of the first stop valve are connected, the inlet 4 and the inlet 5 of the first stop valve are connected, and the inlet 2 and the outlet 3 of the first stop valve are connected;

Adjust the second shut-off valve so that the inlet 1 and the inlet 2 of the second shut-off valve are communicated, the inlet 4 and the outlet 3 of the second shut-off valve are communicated, and the inlet 6 and the outlet 5 of the second shut-off valve are communicated;

The first chromatographic pump pushes 95% mobile phase A and 5% mobile phase B into the inlet 1 of the injection valve at a flow rate of 5uL/min, and pushes the sample in the sample loop of the injection valve from the outlet 6 of the injection valve into the inlet of the switching valve. 10, and then flow out from the switching valve outlet 9 and enter the second enrichment column. The sample is enriched in the second enrichment column, and the solution flowing out from the second enrichment column passes through the tee and then enters the inlet 2 of the first stop valve and the second capillary chromatographic column, because the outlet 3 of the first stop valve is connected The resistance (back pressure) to the second waste bottle and the stationary phase of the second capillary column causes all the solution to flow into the second waste bottle.

The second chromatographic pump flows through the switching valve and the first shut-off valve at 1.0ul/min (the mobile liquid is 95% mobile phase A and 5% mobile phase B) to perform a gradient on the first enrichment column and the first capillary column Elution wash. The solution flowing out from the first enrichment column enters the inlet 4 of the second stop valve and the first capillary chromatographic column respectively after passing through the three-way. Because the outlet 3 of the second stop valve is connected to a plug, all the solution flows through the first capillary chromatograph column. The solution flowing out from the first capillary chromatographic column enters the inlet 4 of the switching valve and then flows out from the outlet 3 to enter the first waste liquid bottle.

(6) Gradient elution and mass spectrometry detection:

Adjusting the inlet and outlet communication modes of the injection valve, the switching valve and the second shut-off valve are the same as in step (4);

Adjust the inlet 4 and the inlet 5 of the first stop valve to be connected, adjust the inlet 2 and the outlet 3 of the first stop valve to be connected, and adjust the inlet 6 of the first stop valve to be connected to the outlet 1;

The first chromatography pump flows the mobile phase A and the mobile phase B through the injection valve and the switching valve according to the gradient elution procedure at a flow rate of 350nL/min to perform gradient elution on the second enrichment column and the second capillary chromatography column. The sample component solution eluted from the second enrichment column enters the inlet 2 of the first stop valve and the second capillary chromatographic column respectively after passing through the three-way. Because the outlet 3 of the first stop valve is connected to a plug, all the solution flows through the second capillary column. The sample components separated from the second capillary chromatographic column enter the inlet 6 of the switching valve and flow out from the outlet 5 to enter the electrospray ionization source (ionization voltage of 2.0kV) of the mass spectrometer. The ionized sample components enter the mass spectrometer. instrument and get tested.

The first chromatographic pump performs cleaning gradient elution: the elution mode is:

0min<elution time≤28min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

28min<elution time≤30min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

30min<elution time≤58min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

58min＜elution time≤60min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the flow rate of the eluent is 1.0ul/min;

60min<elution time≤88min, linear change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and % mobile phase B, the eluent flow rate is 1.0ul/min;

88min<elution time≤90min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

90min＜elution time≤118min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

118min＜elution time≤120min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

The second chromatographic pump flows through the switching valve and the second shut-off valve at 1.0 uL/min (95% mobile phase A and 5% mobile phase B in the mobile liquid) to gradient the first enrichment column and the first capillary column Elution wash. The solution flowing out from the first enrichment column enters the inlet 4 of the second stop valve and the first capillary chromatographic column respectively after passing through the three-way. Because the outlet 3 of the second stop valve is connected to a plug, all the solution flows through the first capillary chromatograph column. The solution flowing out from the first capillary chromatographic column enters the inlet 4 of the switching valve and then flows out from the outlet 3 to enter the first waste liquid bottle.

(7) Repeat steps (1) to (6) to analyze the remaining samples in the sample tray by dual-column capillary chromatography with a nanoliter flow rate.

The application of the nanoliter flow rate dual-column capillary chromatography device of the present invention in the analysis of complex biological samples:

For the nanoliter flow rate dual-column capillary chromatographic device described in the present application, the inventors have designed reasonable chromatographic separation and analysis conditions and chromatographic column cleaning conditions. (As shown in Figure-2):

Figure-2A is a gradient elution curve with a flow rate of 300nL/min and an analysis time of 120 minutes. At this time, one set of enrichment columns and capillary chromatographic columns in the system (the first enrichment column and the first enrichment column in Figure-1C) Capillary chromatographic column) and mass spectrometer are used for online analysis to achieve high-sensitivity analysis and detection. The gradient separation and elution procedures are as follows:

0min<elution time≤0.1min, linear change from 95% mobile phase A and 5% mobile phase B to 90% mobile phase A and 10% mobile phase B, the eluent flow rate is 300nl/min;

0.1min＜elution time≤96min, linear change from 90% mobile phase A and 10% mobile phase B to 67% mobile phase A and 33% mobile phase B, the eluent flow rate is 300nl/min;

96min<elution time≤106min, linear change from 67% mobile phase A and 33% mobile phase B to 50% mobile phase A and 50% mobile phase B, the eluent flow rate is 300nl/min;

106min＜elution time≤111min, linear change from 50% mobile phase A and 50% mobile phase B to 0% mobile phase A and 100% mobile phase B, the eluent flow rate is 300nl/min;

111min<elution time≤120min, use 0% mobile phase A and 100% mobile phase B, and the eluent flow rate is 1000nl/min.

Figure-2B shows another set of enrichment column and capillary chromatographic column in the high-flow gradient cleaning system with a flow rate of 1.0uL/min (the second enrichment column and the second capillary chromatographic column in Figure-1C). Instead of entering the mass spectrometer, it directly enters the waste liquid collection bottle, which avoids the pollution of the instrument due to the waste liquid from cleaning the column entering the mass spectrometer. The gradient cleaning and elution procedure is as follows:

0min<elution time≤28min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

28min<elution time≤30min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

30min<elution time≤58min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

58min＜elution time≤60min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the flow rate of the eluent is 1.0ul/min;

60min<elution time≤88min, linear change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

88min<elution time≤90min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min;

90min＜elution time≤118min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min;

118min<elution time≤120min, use 5% mobile phase A and 95% mobile phase B, and the eluent flow rate is 1.0ul/min.

300nL/min is used for the separation of the sample in the enrichment column and the capillary column (such as the first enrichment column and the first capillary chromatographic column) in one of the nanoliter flow rate dual-column capillary chromatography devices to ensure the sensitivity of mass spectrometry detection. The first enrichment column and the first capillary chromatographic column are connected with the mass spectrometer online, as shown in Figure-1C, and the gradient elution separation time is 120 minutes; 1.0uL/min is used for the other one in the nanoliter flow rate dual-column capillary chromatographic device. High-flow gradient cleaning of enrichment and capillary columns (e.g., second enrichment and second capillary columns) to maximize the removal of residual sample on second enrichment and second capillary columns, each high flow rate The gradient elution wash time was 30 min. At this time, the second enrichment column and the second capillary chromatographic column are connected offline with the mass spectrometer, as shown in Figure-1C, the cleaning waste liquid does not enter the mass spectrometer, but directly enters the waste liquid collection bottle, which avoids the waste liquid from cleaning the chromatographic column entering the mass spectrometer. contamination of the instrument. When the switching valve is switched to another position, the system will separate and elute another sample through the second enrichment column and the second capillary chromatographic column at a flow rate of 300nL/min and perform online mass spectrometry analysis and detection, that is, the second enrichment column and the second capillary chromatographic column is connected to the mass spectrometer online (see Figure-1F); at the same time, the last sample remaining in the first enrichment column and the first capillary chromatographic column is washed with 1.0uL/min high flow gradient elution, The cleaning waste liquid does not enter the mass spectrometer, but directly enters the waste liquid collection bottle, which avoids the pollution of the instrument due to the waste liquid from cleaning the chromatographic column entering the mass spectrometer (see Figure-1F).

1. Use the nanoliter flow rate dual-column capillary chromatography device of the present invention to analyze the trypsin hydrolysis products of plasma proteins from breast cancer patients, and use the conventional single-column capillary chromatography-mass spectrometry online for comparative analysis.

Detect the signal intensities of peptide molecules with different mass-to-charge ratios (m/z) detected at different retention times for two-dimensional map display (as shown in Figure-3). The gray and black levels in the figure represent the detected peptides The signal strength of the segment molecule.

The lower right part of Figure 3 is: using the commonly used single-column capillary chromatography-mass spectrometry online analysis, after the sample analysis is completed, the capillary chromatographic column is cleaned three times, and the mass spectrometry analysis results after each cleaning are obtained;

The lower left part of FIG. 3 is: the same sample is analyzed online by using the nanoliter flow rate dual-column capillary chromatographic device described in the present application, and the capillary chromatographic column is cleaned once to obtain the mass spectrometry analysis result after cleaning.

As can be seen from Figure 3:

By comparing the results after one cleaning of the capillary column: it can be seen from Figure 3 that the residue of the separated sample in the single capillary column is significantly higher than that of the separated sample in the double capillary column described in this application (Figure-3 The first picture in the lower right part and the lower left part of Figure-3).

Even if the conventional single capillary chromatographic column is cleaned three times, the results of the third cleaning show that the residue of the separated sample of the capillary chromatographic column is higher than that of the separated sample of the double capillary chromatographic column described in this application after one cleaning ( The third image in the lower right part of Figure-3 and the lower left part of Figure-3).

Five peptide molecules with different mass-to-charge ratios (m/z) (m/z: 515.627; 651.563; 667.718; 789.906; 979.275) were randomly selected, and they were analyzed and compared in the online dual-column capillary chromatography-mass spectrometry described in this application. The hyphenated analysis results and the conventional single-column capillary chromatography-mass spectrometry online hyphenated analysis results further verify that the dual-column capillary chromatography-mass spectrometry online hyphenation technique described in this application is between reducing the residue of the sample in the capillary chromatographic column and reducing the sample. The cross-contamination is far superior to the conventional single-column capillary chromatography-mass spectrometry online method. The verification results are shown in Figure 4.

2. Test of reproducibility and reliability of the nanoliter flow rate dual-column capillary chromatography device described in this application when analyzing complex biological samples

Three replicate analyses of trypsin digests from the same plasma sample were performed on the nanoliter flow rate dual-column capillary chromatography-mass spectrometry system described in this application to evaluate the reproducibility of the present technique in the analysis of actual biological samples (see Figure- 5).

It can be seen from Figure 5 that the results show that the baseline peak intensities of the three replicate analyses are 1.35E7, 1.33E7, and 1.32E7, respectively. The standard deviation is 0.015, and the coefficient of variation is 1.15%, that is, the nanoliter flow rate dual-column capillary chromatography device of the present invention has good reproducibility and reliability when analyzing complex biological samples.

Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (10)

1. a nanoliter flow rate dual-column capillary chromatographic device, is characterized in that, comprises autosampler, the first chromatographic pump, the second chromatographic pump, switching valve, the first enrichment column, the second enrichment column, the first Capillary chromatographic column, second capillary chromatographic column, first stop valve, second stop valve, first waste liquid bottle, second waste liquid bottle, mass spectrometer; The mobile phase flows through the automatic sampler under the action of the first chromatographic pump, the solution flowing out from the automatic sampler enters the switching valve, and the switching valve passes through the first enrichment column to the liquid that flows through the second stop valve and the first capillary chromatographic column. Equilibrate, and the liquid flowing through the first capillary chromatographic column enters the mass spectrometer through the switching valve; The mobile phase flows through the switching valve under the action of the second chromatographic pump, the switching valve balances the liquid flowing through the first stop valve and the second capillary chromatographic column through the second enrichment column, and the liquid flowing through the second capillary chromatographic column is switched The valve flows into the first waste liquid bottle; The outlets of the first shut-off valve and the second shut-off valve are both communicated with the second waste liquid bottle. 2. The nanoliter flow rate dual-column capillary chromatography device according to claim 1, wherein the automatic sampler comprises a sampling pump, a sampling valve, a sampling needle and a sample tray, and the sampling valve is 6 -port-2-position injection valve; the sample tray contains at least 2 samples to be tested, and the two ends of the sample loop are respectively connected to the inlet 2 of the injection valve and the inlet 5 of the injection valve; The injection pump is communicated with the inlet 3 of the injection valve, one end of the injection needle is communicated with the inlet 4 of the injection valve, and the other end of the injection needle is connected with the sample tray Pass. 3. The nanoliter flow rate dual-column capillary chromatography device according to claim 1, wherein the switching valve is a 10-port-2-position switching valve, and the dead volume between the internal passages of the switching valve is less than 7nL; Both the first shut-off valve and the second shut-off valve are 6-port-2-position shut-off valves. 4. The nanoliter flow rate dual-column capillary chromatographic device according to claim 3, wherein the first chromatographic pump is communicated with the inlet 1 of the injection valve, and the outlet 6 of the injection valve is connected to the inlet 1 of the injection valve. The switching valve inlet 10 is connected; The outlet 1 of the switching valve is communicated with the first enrichment column, and the outlet of the first enrichment column is communicated with the inlet 4 of the second stop valve and the first capillary chromatographic column through a tee, respectively. The capillary chromatographic column is communicated with the inlet 4 of the switching valve, the outlet 5 of the switching valve is communicated with the mass spectrometer, and the outlet 3 of the switching valve is communicated with the first waste liquid bottle; A plug is provided on the outlet 3 of the second shut-off valve. 5. The nanoliter flow rate dual-column capillary chromatographic device according to claim 3, wherein the second chromatographic pump is communicated with the inlet 8 of the switching valve, and the outlet 9 of the switching valve is connected with the second enrichment The columns are connected, and the outlet of the second enrichment column is respectively connected with the inlet 2 of the first stop valve and the second capillary chromatographic column through the tee, and the second capillary chromatographic column is connected with the inlet 6 of the switching valve. A plug is provided on the outlet 1 of the first shut-off valve. 6. The nanoliter flow rate dual-column capillary chromatographic device according to claim 3, wherein the outlet 3 of the first shut-off valve and the outlet 5 of the second shut-off valve are all connected with the second waste liquid bottle Pass. 7. The nanoliter flow rate dual-column capillary chromatography device according to claim 1, wherein the first enrichment column and the second enrichment column are both enrichment columns of 75-200 μm, ID x 2 cmL; Both the first capillary chromatographic column and the second capillary chromatographic column are capillary chromatographic columns of 50-100 μm, ID x 10-50 cmL. 8. The application of the nanoliter flow rate dual-column capillary chromatography device of any one of claims 1-7 in the analysis of biological samples. 9. the method for the analysis of complex biological samples by the nanoliter flow rate dual-column capillary chromatographic device described in any one of claims 1-7, is characterized in that, comprises the following steps: (1) Loading of the first sample: Adjust the injection valve so that the inlet 3, inlet 2, inlet 5, and inlet 4 of the injection valve are connected in sequence, and the inlet 1 of the injection valve is connected to the outlet 6 of the injection valve. Pass; Adjust the switching valve: Connect the outlet 3, inlet 2, inlet 7, and inlet 6 of the switching valve in sequence, connect the inlet 4 and outlet 5 of the switching valve, and connect the outlet 1 and inlet 10 of the switching valve, so that the switching valve is connected. The inlet 8 and outlet 9 are connected; Adjust the first shut-off valve: make the inlet 5 and the inlet 6 of the first shut-off valve communicate with each other, make the inlet 2 and the outlet 1 of the first shut-off valve communicate with each other, and communicate with the inlet 4 and the outlet 3 of the first shut-off valve; Adjust the second globe valve: connect the inlet 1 and the inlet 2 of the second globe valve, connect the inlet 6 and the outlet 5 of the second globe valve, and connect the inlet 4 and the outlet 3 of the second globe valve; The syringe draws the first sample 5uL; The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 300nL/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min; (2) Enrichment of the first sample: adjust the injection valve so that the inlet 3 and the inlet 4 of the injection valve are connected, and the inlet 1, the inlet 2, the inlet 5 and the outlet 6 of the injection valve are connected in sequence; Adjust the switching valve so that the inlet 6, inlet 7, inlet 2 and outlet 3 of the switching valve are connected in turn, so that the inlet 4 and outlet 5 of the switching valve are connected, and the inlet 10 and outlet 1 of the switching valve are connected, so that the switching valve is connected. The inlet 8 and outlet 9 are connected; Adjust the second globe valve: connect the inlet 1 and the inlet 6 of the second globe valve, connect the inlet 2 and the outlet 3 of the second globe valve, and connect the inlet 4 and the outlet 5 of the second globe valve; The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 5ul/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min; (3) Gradient elution and mass spectrometry detection: Adjust the inlet and outlet communication modes of the injection valve, the switching valve, the first shut-off valve and the second shut-off valve in the same manner as in step (1); The first chromatography pump performs separation gradient elution: the elution mode is: 0min<elution time≤0.1min, linear change from 95% mobile phase A and 5% mobile phase B to 90% mobile phase A and 10% mobile phase B, the eluent flow rate is 300nl/min; 0.1min＜elution time≤96min, linear change from 90% mobile phase A and 10% mobile phase B to 67% mobile phase A and 33% mobile phase B, the eluent flow rate is 300nl/min; 96min<elution time≤106min, linear change from 67% mobile phase A and 33% mobile phase B to 50% mobile phase A and 50% mobile phase B, the eluent flow rate is 300nl/min; 106min＜elution time≤111min, linear change from 50% mobile phase A and 50% mobile phase B to 0% mobile phase A and 100% mobile phase B, the eluent flow rate is 300nl/min; 111min＜elution time≤120min, use 0% mobile phase A and 100% mobile phase B, and the eluent flow rate is 1000nl/min; The mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B, and the flow rate of the mobile liquid is 1.0ul/min; (4) Loading of the second sample: Adjust the inlet and outlet communication modes of the injection valve, the first shut-off valve and the second shut-off valve are the same as in step (1); The outlet 1, inlet 2, inlet 7 and inlet 8 of the adjustment switch valve are connected in sequence, the inlet 10 and outlet 9 of the adjustment switch valve are connected, the inlet 4 and outlet 3 of the adjustment switch valve are connected, and the inlet 6 and the outlet 3 of the adjustment switch valve are connected. Exit 5 is connected; The syringe draws the second sample 5uL; The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 300nL/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min; (5) Enrichment of the second sample: adjust the injection valve so that the inlet 3 and the inlet 4 of the injection valve are connected, and the inlet 1, the inlet 2, the inlet 5 and the outlet 6 of the injection valve are connected in sequence; Adjust the switching valve so that the outlet 1, inlet 2, inlet 7, and inlet 8 of the switching valve are connected in sequence, so that the inlet 10 and outlet 9 of the switching valve are connected, and the inlet 6 and outlet 5 of the switching valve are connected, so that the switching valve is connected. The inlet 4 and outlet 3 are connected; Adjust the first stop valve so that the outlet 1 and the inlet 6 of the first stop valve are connected, the inlet 4 and the inlet 5 of the first stop valve are connected, and the inlet 2 and the outlet 3 of the first stop valve are connected; Adjust the second shut-off valve so that the inlet 1 and the inlet 2 of the second shut-off valve are communicated, the inlet 4 and the outlet 3 of the second shut-off valve are communicated, and the inlet 6 and the outlet 5 of the second shut-off valve are communicated; The mobile liquid used by the first chromatography pump is 95% mobile phase A and 5% mobile phase B, the flow rate of the mobile liquid is 5ul/min, and the mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B The mobile phase B, the flow rate of the mobile liquid is 1.0ul/min; (6) Gradient elution and mass spectrometry detection: Adjusting the inlet and outlet communication modes of the injection valve, the switching valve and the second shut-off valve are the same as in step (4); Adjust the inlet 4 and the inlet 5 of the first stop valve to be connected, adjust the inlet 2 and the outlet 3 of the first stop valve to be connected, and adjust the inlet 6 of the first stop valve to be connected to the outlet 1; The first chromatographic pump performs cleaning gradient elution: the elution mode is: 0min<elution time≤28min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min; 28min<elution time≤30min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min; 30min<elution time≤58min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min; 58min＜elution time≤60min, linear change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the flow rate of the eluent is 1.0ul/min; 60min<elution time≤88min, linear change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min; 88min<elution time≤90min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min; 90min＜elution time≤118min, linearly change from 95% mobile phase A and 5% mobile phase B to 5% mobile phase A and 95% mobile phase B, the eluent flow rate is 1.0ul/min; 118min＜elution time≤120min, linearly change from 5% mobile phase A and 95% mobile phase B to 95% mobile phase A and 5% mobile phase B, the eluent flow rate is 1.0ul/min; The mobile liquid used by the second chromatography pump is 95% mobile phase A and 5% mobile phase B, and the flow rate of the mobile liquid is 1.0ul/min; (7) Repeat steps (1) to (6) to analyze the remaining samples in the sample tray by dual-column capillary chromatography with a nanoliter flow rate. 10 . The method for analyzing complex biological samples with a nanoliter flow rate dual-column capillary chromatography device according to claim 10 , wherein the mobile phase A is an aqueous solution of 0.1% formic acid, and the mobile phase B is an acetonitrile solution of 0.1% formic acid. 11 .

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