CN113406227A - Novel capillary analytical column and method for realizing ultrahigh-sensitivity protein mass spectrum detection - Google Patents
Novel capillary analytical column and method for realizing ultrahigh-sensitivity protein mass spectrum detection Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Abstract
The invention belongs to the field of analytical chemistry, and particularly relates to a novel capillary analysis column and a method for realizing ultrahigh-sensitivity protein mass spectrometry detection based on the same, wherein the analysis column comprises a column tube, a capillary integrated emission tip, a filler filled in a tube cavity, and a frit for supporting the filler. Wherein, the filler is a modified silicon-based microsphere with smooth surface, uniform particle size and organic polymer-covered surface, and the particle size of the modified silicon-based microsphere is less than or equal to 3 mu m. The inner diameter of the analytical column is less than or equal to 30 mu m. When the capillary analytical column is used for carrying out mass spectrometry detection on protein, the flow velocity is less than or equal to 1nL/min through the flow dividing device, sensitive control is realized through high-precision position control, the sensitivity of mass spectrometry can be greatly improved under ultra-low flow velocity, the reduction of chromatographic resolution when the flow velocity is lower than the minimum flow velocity of Van Deemter can be compensated, the analysis and identification on biomacromolecule with lower content are effectively improved, and the capillary analytical column has wide application prospect.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a novel capillary analytical column, a chromatographic analyzer or a chromatography-mass spectrometry combined system comprising the capillary analytical column, and a method for realizing ultrahigh-sensitivity protein mass spectrometry detection.
Background
Liquid chromatography has effective separation and resolution capability on substances such as organic compounds, and mass spectrometry is an effective means for accurately identifying macromolecules. The liquid chromatogram-mass spectrum combined technology (LC-MS) formed by combining the two can directly separate a sample in a complex mixture by using the liquid chromatogram under the control of a computer, so that detected compounds in the sample enter an ion source of a mass spectrometer one by one, and can carry out electron bombardment or chemical ionization by using atmospheric pressure ionization (including electrospray ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI)), so that all compounds in the sample are ionized and converged into an ion beam with certain energy and geometric shape, and the separation from other substances in the mixture is realized by volatility, momentum charge performance and the like, and further the analysis and identification are carried out by a mass spectrometry system.
The technology can be suitable for separation and detection of substances with high molecular weight, strong polarity, low volatility and thermal instability, and is mainly completed by mass spectrometry analysis of the whole protein through LC-MS. However, since in primary or other karyotype normal cells, the phosphorylation sites of the cells are few, the generated phosphorylation signals are low and easily masked by other high signal values, and therefore, only a few specific protein phosphorylation sites of the cells can be identified by the current LC-MS analysis method. Furthermore, when enriched biological samples are tested by cell type, protein class or specific post-translational modifications, the independent absolute signal values obtained often exceed the dynamic range of current LC-MS instrumental analysis.
The capillary chromatographic column is a key part playing a role in separation in LC-MS analysis, and the preparation technology of the capillary chromatographic column is related to the performance of the column and the separation capability thereof, so that the capillary chromatographic column has an important influence on the result of subsequent mass spectrum detection. The development of new capillary columns has been a long-felt focus of attention and research in the field. Improvements in the sample introduction interface in mass spectrometry have been reported to improve the detection limit and dynamic range of the results. Improvements to the sample introduction interface in existing LC-MS proteomics include the use of small inner diameter columns and small particle packing or the use of machine controlled nozzles, which emphasize the assurance of reproducibility and stability of results under optimal chromatographic and electrospray performance conditions. The improvement of the chromatographic resolution by the small inner diameter column and the improvement of the electrospray ionization efficiency by the low flow rate have been reported in documents. However, as the flow rate continues to decrease, for example, to very low flow rates and below (equal to or below the Van Deemter minimum flow rate), electrospray ionization efficiency increases and chromatographic resolution decreases, and it is unknown whether this inherent problem affects the final mass spectral sensitivity, dynamic range, etc. However, it is difficult to construct a stable electrospray apparatus comprising a column of small internal diameter (. ltoreq.30 μm) that supports very low flow rates, and there are no reports of successful construction of this apparatus in the prior art.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a novel small-column-diameter capillary analytical column (capillary chromatographic column) and a process method for stably manufacturing the capillary analytical column. The capillary analytical column can be used as a component of a miniaturized LC-ESI for LC-MS analysis, and is helpful for overcoming the difficulty of the prior art and realizing high-sensitivity detection of a sample to be detected.
The capillary analytical column and the LC-MS detection method based on the analytical column can be suitable for detecting embryonic stem cells or other primary cells based on proteomics, can particularly provide a feasible way for realizing the analysis and identification of protein LC-MS through the detection of low-level tyrosine phosphorylation signals, and realize the purpose of quickly and efficiently analyzing and identifying complex biological samples with low content of protein components.
In order to realize the purpose of the invention, the invention provides the following technical scheme:
the present invention provides a capillary analytical column (capillary chromatography column) comprising a column tube, a capillary integrated emission tip, packing filled in the lumen, a frit for supporting the packing.
In the invention, the capillary analytical column is a packed capillary column (micro packed column), and the analytical column can be quartz, glass, metal, nylon and other pipes. Preferably, the present invention employs polyimide coated quartz capillary (polyimide coated SiO)2Capillary tube) which has the advantages of good elasticity, inert surface, long service life, etc. (since quartz capillary columns are very brittle, good elasticity is usually ensured by coating a layer of polyimide protective material on the outside).
In the present invention, the length of the capillary analytical column may be any length suitable for LC-MS analysis, and those skilled in the art can determine a suitable column length according to actual needs. Considering that the inner diameter of the analytical column of the present invention is small, the total length of the analytical column of the present invention cannot be too long in order to prevent mass transfer resistance due to the packing. Preferably, the length of the analytical column is 5-20 cm; further preferably, the length of the analytical column is 10-20 cm; further preferably, the length of the analytical column is 10-17 cm; still further preferably, the length of the analytical column may be one of 10, 11, 12, 13, 14, 15, 15.5, 16, 16.5, 17cm, etc.
In the invention, the outer diameter of the analytical column can be 200-500 μm; preferably, the outer diameter is 300-400 μm; further preferably, the outer diameter is one of 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400 μm.
In the invention, in order to realize high-efficiency and ultrahigh-sensitivity protein mass spectrum detection, the inner diameter of the analytical column is less than or equal to 30 mu m; further preferably; the inner diameter of the analytical column is less than or equal to 25 mu m; further preferably; the inner diameter of the analytical column is 5-25 μm; further preferably; the inner diameter of the analytical column is 10-25 mu m; further preferably, the analytical column has an internal diameter of one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 μm, etc.
In the invention, the diameter of the capillary integrated emission tip is less than or equal to 2 mu m; preferably, the capillary integrated emission tip diameter is less than or equal to 1.5 μm; further preferably, the capillary integrated emission tip diameter is less than or equal to 1.2 μm; still further preferably, the capillary integrated emission tip has a diameter ≦ 1 μm.
In the present invention, the end of the capillary is constructed with a frit, and the thickness of the frit (along the length of the capillary) can be reasonably determined by one skilled in the art through the total length of the capillary; preferably, when the length of the capillary tube analysis column is 5-20 cm, the thickness of the frit is 1-2.5 mm; preferably, the thickness of the frit is 1.5-2.5 mm; further preferably, the frit has a thickness of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 or 2.2 mm.
In the present invention, the skilled person can reasonably determine the distance of the frit from the capillary integrated emission tip by the total length of the capillary; preferably, the frit is located at a distance of 1-4mm, preferably 2-3 mm, from the capillary integrated emission tip when the capillary analysis column has a length of 5-20 cm (the distance is calculated from the side of the frit closest to the capillary integrated emission tip).
In the present invention, the frit is a high-strength, three-dimensional pore-plug-like polymer or solid formed by self-crosslinking of a silicate sol-gel system, which is drawn in by capillary suction, at a certain temperature. The frit has the characteristics of high strength, high permeability and the like, has the functions of blocking the stationary phase filler and preventing the loss of the filler, and simultaneously has good permeability so as to keep the solvent and a substance to be detected to pass through and prevent overhigh resistance. Wherein high strength means that the frit is capable of withstanding pressures of at least 4000psi, preferably 2500psi and above. Wherein, the pore-plug shape means that the polymer or the solid has a pore structure, the pore diameter of the pore structure should be smaller than the diameter of the filler, and can be 1 μm, 2 μm, 0.5 μm.
Wherein the silicate is selected from one or more of aluminum silicate, iron silicate, calcium silicate, magnesium silicate, potassium silicate, sodium silicate, manganese silicate and the like; preferably, the silicate is a mixture of aluminum silicate, potassium silicate and manganese silicate, wherein the molar ratio of the aluminum silicate to the potassium silicate to the manganese silicate is (9-10): (15-16): (1-1.5), preferably 9:15.5: 1.
The silicate forms a silicate sol-gel system under the condition of a solvent for promoting the formation of sol-gel, wherein the solvent for promoting the formation of sol-gel is one or more of water, glacial acetic acid, alkali, ethanol, isopropanol and the like; preferably, water and isopropanol in a volume ratio of 3:1, or glacial acetic acid and isopropanol in a volume ratio of 3: 1.
The regulation and control of parameters such as frit material, permeability, mechanical strength, thickness and the like are key links for preparing high-quality particle-filled capillary columns. The frit has the advantages of simple preparation method, convenient operation and good repeatability; the frit prepared by the method has good mechanical property, mechanical stability and permeability, and can ensure good column efficiency.
In the present invention, the filler can be any substance capable of realizing high-sensitivity protein mass spectrometry detection, including but not limited to silica gel, organic polymer, polysaccharide (sephadex, sepharose) and the like. Because the inner diameter of the capillary tube is very small, if the performance of the filler such as particle size, surface characteristics, uniformity and the like cannot be effectively controlled, the filler is difficult to fill into the cavity of the capillary tube column; therefore, a filler with suitable particle size, surface characteristics, uniformity, etc. is critical to the successful preparation of capillary analytical columns. Therefore, the filler suitable for the capillary analytical column is micron-sized particles, the surface is smooth, and the particle size of the filler is less than or equal to 3 mu m. Preferably, the particle size of the filler may be 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0 μm, or less than 1.0 μm.
In the invention, preferably, the filler of the invention specifically relates to a modified silicon-based microsphere, namely a silicon-based microsphere wrapped by a modified organic polymer, which has a smooth surface, a uniform particle size (2.0-4.0 mu m) and micropores on the surface
The modified organic polymer covers the surface of the silicon sphere, so that the friction force between the silicon-based microspheres can be reduced, the smoothness degree of the filler can be improved, and the filling of the filler in the capillary tube preparation process is facilitated.
In the invention, the preparation method of the modified silicon-based microspheres comprises the following steps: with N, N-dimethylhexadecylamine, NH4And F is used as a modifier, and in the presence of glacial acetic acid, water and ethanol, a polymer monomer is subjected to polymerization reaction to form a cross-linked polymer which covers the surface of the silicon-based microsphere, namely the modified silicon-based microsphere.
Wherein the mass of the modifier accounts for 6-9% of the total mass of the reaction system; preferably, it is 7.8%.
Wherein the mass of the polymer monomer accounts for 10-12% of the mass of the total reaction system; preferably, it is 11%.
Wherein the difference is selected according to the analysis requirementThe monomer (A) is subjected to polymer synthesis, so that the modified silicon-based microspheres capable of improving the analysis effect are obtained. Preferably, the polymer monomer comprises any one or more of trichloroethylene, divinylbenzene, styrene, vinyl pyrrolidone and vinyl pyridine. Preferably, the polymer monomer is a mixture of styrene, vinyl pyrrolidone and vinyl pyridine, the molar ratio of the styrene, the vinyl pyrrolidone and the vinyl pyridine is (4-8): (2-5): (10-15), preferably 5:3:12, the modified silicon-based microspheres obtained by the preferred method can reduce the friction force between the silicon-based microspheres and improve the surface smoothness, the particle size is less than or equal to 3 mu m, the size is uniform, and the surface micropores are surface micropores
Wherein the temperature of the polymerization reaction is 300-400 ℃; preferably, it is 380 ℃.
Wherein the time of the polymerization reaction is 5-6 h; preferably, it is 5.5 h.
The separation degree is a quantitative index of the separation degree of the protein, and the modified silicon-based microspheres prepared by the method have the particle size of less than or equal to 3 mu m, uniform size and pore diameter
The surface characteristics, the particle size, the uniformity, the pore structure and the like of the modified organic polymer coated silicon-based microspheres enable the modified organic polymer coated silicon-based microspheres to be used as a filler of a capillary column, so that a good effect can be obtained during chromatographic analysis, the aims of effectively separating protein and realizing high-sensitivity protein mass spectrometry are fulfilled, and the capillary analysis column is further determined to be suitable for separating and detecting low-content protein in a protein, especially in a complex biological sample.
In one embodiment, the step of preparing the filler comprises: dissolving polymer monomer in glacial acetic acid, water and ethanol, adding modifier N, N-dimethyl hexadecylamine and NH4F, the mass of the modifier is 6-9% of the total mass of the reaction system, andthe mass of the polymer monomer accounts for 10-12% of the total mass of the reaction system, and the polymer monomer is ultrasonically mixed for 20min to carry out polymerization reaction.
The invention also provides a method for stably and efficiently preparing the capillary analytical column, which comprises the following steps: construction of capillary end frit, pressure-variable chromatography filling of the filler, drawing of capillary integrated emission tips. The three-step preparation method is the key for successfully preparing the capillary analytical column, and the capillary analytical column can be stably and efficiently prepared by the method, namely the preparation success rate is high, and the method is simple and rapid; in addition, the performance of the capillary analytical columns prepared between different batches of the present invention can maintain the same.
Specifically, the preparation method of the capillary analytical column provided by the invention comprises the following steps:
(1) construction of a capillary end frit comprising the steps of:
(1.1) removing a part of the polyimide part from one end of the quartz capillary;
(1.2) migration of the silicate Sol-gel to the midpoint of the removal section by siphoning
Forming a sol-gel system by silicate under the condition of a solvent for promoting the formation of sol-gel, and then sucking the sol-gel system into an empty capillary tube by utilizing the siphoning effect to form a stable interface;
(1.3) forming a frit with a certain thickness by adopting a staged polymerization method;
different from the traditional method, the method of the electric soldering iron is adopted, and the sol-gel system can be locally heated in stages by using the space of about 1-2 mm at the tip of the electric soldering iron, so that the frit is obtained. The frit is a polymer with stable performance, and has good strength and permeability.
In the step (1.1), the length of polyimide removed from one end of the quartz capillary is determined by the thickness of the constructed frit; in general, when the thickness of the frit is 1 to 2.5mm for a capillary column having a length of 5 to 20cm, the length of polyimide to be removed is 2 to 3cm, and for example, it is preferably 2.5 cm. It should be noted that step (1.1) is not essential, and in other embodiments, a capillary tube with a coating on one end and an exposed capillary on the other end may be used directly.
In the step (1.2), the solvent for promoting the formation of the sol-gel is one or more of water, glacial acetic acid, alkali, methanol, ethanol, isopropanol and the like; preferably, water and isopropanol are in a volume ratio of 3:1, or glacial acetic acid and isopropanol are in a volume ratio of 3: 1. The concentration of the silicate ions in the total reaction system (including silicate and the sol-gel forming promoting solvent) is 2 to 3mol/L, preferably 2 mol/L.
In the step (1.2), the silicate is selected from one or more of aluminum silicate, iron silicate, calcium silicate, magnesium silicate, potassium silicate, sodium silicate, manganese silicate and the like; preferably a mixture of aluminum silicate, potassium silicate and manganese silicate, wherein the molar ratio of the aluminum silicate, the potassium silicate and the manganese silicate is (9-10) to (15-16) to (1-1.5), and preferably 9:10 to 1.
In the step (1.3), the polymerization refers to that a silicate sol-gel system undergoes a self-polymerization reaction at a certain temperature to obtain an inorganic polymer. Specifically, the polymerization step comprises low-temperature polymerization, cooling, high-temperature polymerization, cooling and cleaning.
Wherein the temperature of the low-temperature polymerization is 250-300 ℃; preferably 270 deg.c.
Wherein the time of the low-temperature polymerization is 60-90 s; preferably 80 s.
Wherein the cooling means cooling the reaction system to room temperature. The cooling can adopt natural cooling, air cooling, vacuum cooling and other modes; preferably, the present invention employs natural cooling.
Wherein the temperature of the high-temperature polymerization is 350-400 ℃; preferably 375 deg.c.
Wherein the time of the high-temperature polymerization is 20-30 s; preferably 25 s.
It should be noted that, while the staged polymerization method is used to form the frit in this embodiment, in other embodiments, the staged polymerization method may be used in a single-stage polymerization manner, however, compared to a single-stage polymerization, the staged polymerization method may control the thickness of the frit more precisely, and may also make the formed frit uniform and stable.
Wherein, the heating and temperature rise in the polymerization process are preferably realized by electric soldering iron. The skilled person can conveniently and accurately control the thickness of the frit formation by the position covered by the electric soldering iron.
In one embodiment, the method for building a frit comprises:
(a)SiO2one end of the capillary tube was stripped of a 2.5cm portion of polyimide;
(b) the silicate solution migrates to the midpoint of the removal portion by siphoning;
(c) the mixture was polymerized using an electric iron to form a frit of about 2 mm.
In another embodiment, the method of frit construction comprises:
sucking a quartz capillary column with the length of 5-20 cm and the inner diameter of 25 mu m into a silicate sol-gel system by using the siphoning action of a capillary, sealing the tail end with wax, and heating by an electric iron to generate a 1-2.5 mm hole plug-shaped solid frit; the silicate sol-gel system consists of A, B parts, wherein the part A is a mixed solvent of glacial acetic acid and isopropanol with the volume ratio of 3: 1; part B is a mixture of aluminum silicate, potassium silicate and manganese silicate with a molar ratio of 9:10: 1; a, B were then mixed, wherein the concentration of silicate ion was 2mol/L in the total reaction system.
(2) Pressure-variable chromatographic column packing of packing
(2.1) preparation of Filler
The filler is modified silicon-based microspheres with smooth surfaces, particle sizes of less than or equal to 3 mu m and uniform sizes.
The preparation method of the filler comprises the following steps: with N, N-dimethylhexadecylamine, NH4And F is used as a modifier, and in the presence of glacial acetic acid, water and ethanol, a polymer monomer is subjected to polymerization reaction to form a cross-linked polymer which covers the surface of the silicon-based microsphere, namely the modified silicon-based microsphere.
Wherein, the polymer monomer comprises any one or more of trichloroethylene, divinyl benzene, styrene, vinyl pyrrolidone and vinyl pyridine. Preferably, the polymer monomer is a mixture of styrene, vinyl pyrrolidone and vinyl pyridine, and the molar ratio of the styrene, the vinyl pyrrolidone and the vinyl pyridine is (4-8): (2-5): (10-15), and is preferably 5:3: 12.
Wherein the mass of the modifier accounts for 6-9% of the total mass of the reaction system.
Wherein the mass of the polymer monomer accounts for 10-12% of the mass of the total reaction system.
(2.2) pressure-variable chromatography column packing
The present invention may be used to fill the filler by any suitable method, such as high pressure homogenization, dry filling, wet filling, high viscosity filling, isopycnic filling, and the like. Preferably, the present invention employs a variable pressure chromatography column packing method.
Further, the pressure-variable chromatography column packing method comprises the following steps: the filler with the particle size less than or equal to 3 mu m prepared by the method is mixed with a certain volume of organic solvent to form a homogenate, under the condition that the initial pressure is 2500-. After the completion of the filling, the mixture was washed with water to remove the organic solvent, and then dried by introducing pressurized helium gas.
Wherein the organic solvent is selected from one or more of methanol, ethanol, isopropanol, benzene, xylene, toluene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, acetone, methyl butanone, methyl isobutyl ketone, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile, pyridine, phenol and the like; preferably, the solvent is a mixed solvent of ethylene glycol monoethyl ether and diethyl ether in a volume ratio of 10: 3.
Wherein the filling time is determined according to the length of the filler needing to be filled, and the length of a column bed filled with 12cm can be completed within 15 min.
Wherein, after the filling and cleaning are finished, the time for introducing the pressurized helium gas for drying is determined according to actual needs, generally, the time for introducing the pressurized helium gas for drying is 5min or more, and the pressure of the pressurized helium gas for drying is 2200-2800 psi.
In one embodiment, the pressure-variable chromatography column packing method of the present invention specifically comprises: under the pressure of 2500-2700psi, mixing the filler with the particle size of less than or equal to 3 microns prepared by the method with a mixed solvent of ethylene glycol monoethyl ether and diethyl ether with the volume ratio of 3:1 to form homogenate, filling the homogenate into a homogenate column, starting to fill the column, increasing the pressure by 20-30psi when filling the filler to a proper length, stopping filling, washing with water after filling, removing the organic solvent, and introducing pressurized helium gas for drying to obtain the capillary packed column.
(3) Capillary integrated emission tip drawing
In the invention, the drawing method of the capillary integrated emission tip comprises the following steps: an integrated emission tip with a diameter of 1 μm or less is formed outside the frit or at the capillary tip (when the length of the capillary analysis column is 5-20 cm, from the side of the frit closest to the integrated emission tip of the capillary at a distance of 2-4 mm) using a laser-based pipette puller, wherein the intensity of the laser is 7-9 KJ.
The preparation method of the invention also comprises a step of preprocessing the capillary column before the preparation, and the steps comprise: firstly, activating a quartz capillary column by using 0.15mol/L NaOH solution for 1.5h, and then, washing the quartz capillary column by using distilled water; and then leaching the inner wall of the quartz capillary column tube with reducing acid liquor, leaching the inner wall of the quartz capillary column tube with water (distilled water or deionized water), introducing dry inert gas (such as helium) into the quartz capillary column tube for purging, meanwhile, placing the quartz capillary column tube in a heating device, adjusting the temperature to 30-40 ℃, and baking for 10-15min to obtain the pretreated quartz capillary column tube.
The capillary analytical column with the inner diameter less than or equal to 30 mu m can be prepared by the preparation method of the invention, and other preparation methods are difficult to prepare capillary analytical columns with the inner diameter smaller than or equal to 30 mu m. It is particularly noted that the sequence of the step (1) capillary end frit construction, the step (2) packing of the pressure-variable chromatography column and the step (3) capillary integrated emission tip drawing in the preparation method of the present invention is not changeable, which is one of the keys of the present invention to distinguish other prior art methods that can successfully prepare capillary analytical columns with inner diameters of ≦ 30 μm. In addition, the conditions of raw materials, steps, parameters and the like constructed by the frit and the conditions of raw materials, steps, parameters and the like adopted by the preparation of the filler have important influence on the successful preparation of the capillary analytical column with the inner diameter less than or equal to 30 mu m. In addition, the capillary analytical column prepared by the preparation method is an integrated piece without any adapter part, so that the sensitivity and stability of the ultrahigh-sensitivity protein mass spectrometry are further guaranteed.
The present invention also provides a reliable and durable capillary analytical column made by the above-described method of preparation.
The invention also provides the application of the capillary analytical column and the capillary analytical column prepared by the preparation method in the preparation of a chromatographic analyzer or a chromatographic-mass spectrometry combined system.
The invention also provides a chromatographic analyzer or a chromatography-mass spectrometry combined system, which comprises the capillary analytical column and the capillary analytical column obtained by the preparation method.
The invention also provides the application of the capillary analytical column, the chromatographic analyzer or the chromatography-mass spectrometry combined system in the realization of ultra-high sensitivity detection of samples, in particular the application in the ultra-high sensitivity protein mass spectrometry detection.
The invention also provides an LC-MS detection method based on the capillary analytical column, namely an ultra-high sensitivity protein mass spectrum detection method, which comprises the following steps:
the method comprises the steps of construction of a capillary end frit, variable pressure filling of filler, drawing of a capillary integrated emission tip, control of shunt flow rate and high-precision position control of a chromatographic mass spectrometry interface.
Wherein the construction of the capillary end frit, the variable pressure filling of the filler, and the drawing of the capillary integrated emission tip are as described above.
Wherein, the front end of the analytical column leads the flow velocity to be less than or equal to 1nL/min through a flow dividing device.
The high-precision position control of the chromatographic mass spectrum interface realizes sensitive control through a stepping motor controlled by a high-precision computer.
In a specific embodiment, the method for realizing the ultra-high sensitivity protein mass spectrometry provided by the invention comprises the following steps:
1. construction of capillary end frits
The analytical column is made of fused SiO with an inner diameter less than or equal to 30 μm2And (4) constructing a capillary tube. From molten SiO2A 2.5cm section of polyimide was removed at about 3cm from one end of the tube and the silicate sol-gel system migrated by siphoning to the midpoint of the removed section. Next, polymerization was induced using an electric iron to carefully form a frit of about 2mm, and after excess silicate solution was drained, the frit was polymerized with an electric iron at 270 ℃ for 80 seconds and then at 400 ℃ for 25 seconds to form a capillary end frit.
2. Pressure drying filling of fillers
Filling the modified silica-based microspheres with the diameter less than or equal to 3 mu m into a column bed, increasing the pressure by 25psi when filling 1cm of filler under the initial pressure of 2500 plus 2700psi, filling the column bed with the length of 12cm in 10min, and drying for 5min by using pressurized helium.
3. Capillary integrated emission tip drawing
An integrated emitting tip with a diameter of 1 μm or less is formed 2-4mm outside the frit using a laser-based pipette puller.
4. Control of split flow rate
A passive shunt device is arranged at the front end of the analysis column, and the flow rate is controlled to be less than or equal to 1 mu L/min.
5. High precision position control of an interface
By using a computer controlled electrospray positioning device, the emission tip is automatically moved under a cleaning device, eliminating the accumulation of salt or other residue on the outer surface of the emission tip.
6. K562 cell culture and protein peptide preparation
K562 was inoculated in 10% FBSRPMI1640 complete medium plus diabody in 5% CO2The incubation was carried out in an incubator at 37 ℃ using perovanadates to inhibit tyrosine phosphatase activity and lysis using 8M urea containing 100mM ammonium bicarbonate.
The cell lysate was diluted with 100mM ammonium bicarbonate (urea reduced to 2M) and reduced using DTT (final concentration 10 mM). IAA (55mM) was added to alkylate the protein and trypsin digestion was added. Peptides were desalted using a SepPak Plus column, dried by vacuum centrifugation and diluted with 0.1% acetic acid. About 200ng of tryptic peptide was used for analysis in each experiment.
7. Automated sample analysis of peptides
The peptide fragments were injected by an autosampler and loaded onto the pre-column at a flow rate of 4. mu.L/min. And adjusting the six-way valve, introducing the mobile phase onto the pre-column, and allowing the peptide segments to flow into a waste liquid pipeline after being adsorbed on the pre-column. After a sample is loaded to the pre-column, the six-way valve is switched to close the waste liquid pipeline, the peptide fragment is eluted into a mass spectrometer by using HPLC gradient eluent, the mobile phase A is 0.2M acetic acid, the mobile phase B is 70% acetonitrile/0.2M acetic acid, and the mobile phase gradient is B: 0-100% in 50 min. Mass spectral data acquisition was performed using a hybrid linear ion trap-Orbitrap under the following conditions: MS scanning: m/z is more than or equal to 300 and less than or equal to 2000; MS/MS exclusion list, 2Da separation window and 25% relative collision energy: 8 most abundant ions (5> z > 1); ESI voltage: 1.5 kV; capillary temperature: at 150 ℃. MS/MS data were analyzed using the Mascot database.
Compared with the prior art, the invention has the advantages that,
(1) it is known in the art that a capillary chromatographic column with a small column diameter is a critical component for improving the detection sensitivity of mass spectrometry, and the thinner the chromatographic column is, the higher the detection sensitivity of mass spectrometry is, but in the prior art, it is difficult to successfully prepare a capillary chromatographic column with a small column diameter, and especially, it is difficult to stably prepare a capillary chromatographic column with a small column diameter. The invention effectively realizes the stable and high-efficiency manufacture of the capillary chromatographic column with small column diameter, realizes important breakthrough compared with the prior art, and has high success rate of preparation, simple and quick method; in addition, the performance of the capillary analytical columns prepared between different batches of the present invention can maintain the same.
(2) The capillary analytical column provided by the invention adopts N, N-dimethyl hexadecylamine and NH as fillers4F is used as a modifier to participate in the polymerization reaction of polymer monomers, so that the production cost can be effectively reduced, and the environment is protected.
The raw materials for preparing the filler are cheap and easily available, the preparation method is simple and feasible, the filler of the prepared capillary chromatographic column is modified silicon-based microspheres wrapped by a modified cross-linked high molecular compound, the particle size is less than or equal to 3 mu m, the size is uniform, the surface is smooth, the thermal stability is good, the column loss cannot be caused in a high-temperature range, and the corrosion resistance is high.
(3) The frit constructed by the invention has the advantages of cheap and easily-obtained raw materials, simple and stable column manufacturing method and good repeatability, and the frit constructed by the invention has higher strength (capable of bearing pressure of at least 2500 psi) and good permeability.
(4) The process method for preparing the capillary has natural advantages, can manufacture the capillary analytical columns (chromatographic columns) within 30min on the basis of preparing the filler preferentially, has the raw material cost of about 20 yuan per chromatographic column, and is simple and easy to master.
(5) The capillary analytical column can be used in a chromatograph, can be used together with a plurality of mass spectrometers, and has strong universality.
In the detection of the protein chromatography mass spectrum with ultrahigh sensitivity, the flow rate is less than or equal to 1nL/min through a shunting device, and the sensitive control is realized through high-precision position control. The characterization data of the invention show that the electrospray ionization efficiency determines the overall LC-MS performance and can compensate the decrease of the chromatographic performance when the flow rate is lower than the minimum flow rate of Van Deemter; that is, when the flow rate is lower than the minimum Van Deemter flow rate, the electrospray ionization efficiency is improved, and simultaneously, the decrease of the chromatographic resolution can be compensated, so that the sensitivity of the mass spectrum is greatly improved (at least up to2×10-18more than or equal to mol), can effectively separate trace protein, improve the analysis and identification capability of biomacromolecule with lower content, make up for the defect of the existing capillary chromatographic column on the aspect of analysis and detection, is suitable for the analysis, detection and identification of complex biological samples with lower content of biomacromolecule, and has wide application prospect.
(6) The capillary analytical column prepared by the method has the characteristics of satisfactory column efficiency, high stability, good separation effect, high chromatographic resolution, high mass spectrum sensitivity, short analysis time, high efficiency, good repeatability, long service life and the like.
(7) The capillary analytical column, the detection device and the detection method based on the analytical column can greatly improve the sensitivity of mass spectrum detection when used for detecting trace protein, and are suitable for discovery of disease biomarkers in clinical samples, protein interaction networks, comprehensive organelle proteomes, complete eukaryotic protein catalogues and the like. The ultra-high sensitivity protein mass spectrometry detection method based on the capillary analytical column has important significance for the application of proteomics in biomedical research.
Drawings
FIG. 1 is a diagram showing a capillary analytical column configuration.
FIG. 2 is a schematic diagram of an automatic sample injection LC/MS exhaust column.
FIG. 3 shows the number of all peptide sequences detected in the K562 lysate tryptic peptide injection.
FIG. 4 is a graph of the number of peptide sequences detected for K562 lysate tryptic peptide at different flow rates and column internal diameters.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Examples 1-8 construction of capillary end frits
EXAMPLE 1 analytical column from melt with an internal diameter of 25 μmSiO2The capillary tube is constructed as shown in figure 1. From molten SiO2A2.5 cm portion of the polyimide was removed at about 3cm from one end of the tube.
Forming a silicate sol-gel system by a mixture of aluminum silicate, potassium silicate and manganese silicate with a molar ratio of 9:15.5:1 in a solvent of glacial acetic acid and isopropanol with a volume ratio of 3:1, transferring the silicate sol-gel system to the midpoint of a removed part through siphoning, discharging excessive silicate solution, inducing polymerization by using an electric iron to carefully form a frit with the thickness of about 2mm, namely, firstly polymerizing for 80 seconds at a low temperature of 270 ℃, naturally cooling, then polymerizing for 25 seconds at a high temperature of 375 ℃, and naturally cooling to form a frit at the end of a capillary tube.
The preparation process was the same as above, and the frit was constructed with varying parameters as shown in table 1 below:
TABLE 1
Examples 9-15 preparation of fillers
With N, N-dimethylhexadecylamine, NH4F is used as a modifier, and a polymer monomer is subjected to polymerization reaction for 5.5 hours at 380 ℃ in the presence of glacial acetic acid, water and ethanol to form a crosslinked modified organic polymer which covers the surface of the silicon-based microspheres with the particle size of 4.0, so that the modified silicon-based microspheres are obtained; wherein the polymer monomers comprise styrene, vinyl pyrrolidone and vinyl pyridine in a molar ratio of 5:3: 12.
Wherein the mass of the modifier accounts for 7.8 percent of the mass of the total reaction system; the mass of the polymer monomer accounts for 11% of the mass of the total reaction system.
The preparation process was as above, and the fillers were prepared by changing the conditions of the different parameters as shown in table 2 below:
TABLE 2
Examples 16 to 24 on pressure filling
Example 16 the filler prepared in example 9 above had a smooth surface and a uniform particle size of 3 μm. The filler is added under the initial pressure of 2500-.
A frit was constructed as in example 1, and the filler (capillary inner diameter 25 μm) prepared in example 9 was packed under the conditions shown in Table 3 below, the column length was 12cm, the packing was completed, washed with water, the organic solvent was removed, and then pressurized helium gas was introduced to dry for 5min, to prepare a capillary analysis column, and the results are shown in the following Table:
TABLE 3
Note: in the column "variable pressure filling/condition," no "means that a constant pressure is maintained during the filling process.
EXAMPLE 25 capillary Integrated emission tip drawing
The capillary column (25 μm) was prepared by filling the completed capillary column of example 16 using a laser-based pipette puller with a laser intensity of 8KJ to form an integrated emitting tip with a diameter of 1 μm or less at 2-4mm outside the frit.
In the following examples, the drawing method of the capillary integrated emission tip used is the same as that of the example.
Examples 26-29 different inner diameter capillary column construction
A frit was prepared as in example 1, and the packing prepared as in example 9 was packed by the pressure-variable column packing method of example 16 (i.e., initial packing pressure 2500-.
Example 30
A frit was constructed as in example 1, and a packing prepared as in example 15 was packed by the pressure-variable column packing method of example 16 (i.e., initial packing pressure 2500-.
Example 31
A frit was constructed as in example 4, and a packing prepared by packing the method of example 9 according to the pressure-variable column packing method of example 16 (i.e., initial packing pressure 2500-.
Example 32
A frit was constructed as in example 4, and a packing prepared as in example 15 was packed by the pressure-variable column packing method of example 16 (i.e., initial packing pressure 2500-.
EXAMPLE 33 preparation of samples
Preparation of tryptic peptides
Will 105Approximately one K562 cell was lysed by adding 8M urea containing 100mM ammonium bicarbonate for 30 minutes. Then, a concentration of 600. mu.L was usedThe cell lysate was diluted with 100mM ammonium bicarbonate (urea reduced to 2M) and reduced with DTT (final concentration 10 mM). IAA (final concentration 55mM) was added to alkylate the protein, and 100mg trypsin was added for digestion. Peptides were desalted using a SepPak Plus column, dried by vacuum centrifugation and diluted with 0.1% acetic acid. About 200ng of trypsin cleavage product peptide was used for analysis in each experiment.
The process for preparing the tryptic peptides of the present invention can also be carried out by methods known in the art.
Application example 34
Analysis of the samples Using capillary analytical columns prepared in examples 25-29
First, analysis process
A passive shunt device is arranged at the front end of the analysis column. The accumulation of salt or other residues on the outer surface of the emission tip is eliminated by automatically moving the emission tip under a cleaning device using a computer controlled electrospray positioning device.
An autosampler analysis of peptides was performed using the vented column platform shown in FIG. 2, and the peptide fragment of sample A prepared in example 2 was autosampled and loaded onto the pre-column at a flow rate of 4. mu.L/min. Adjusting six-way valve (figure 2a), introducing mobile phase onto the pre-column, adsorbing peptide segment onto the pre-column, and flowing into waste liquid pipeline. After loading the sample onto the pre-column, as shown in fig. 2B, the six-way valve was switched to close the waste liquid line, and the peptide fragment was eluted into the mass spectrometer with an HPLC gradient eluent, mobile phase a was 0.2M acetic acid, mobile phase B was 70% acetonitrile/0.2M acetic acid, and mobile phase B was graded as: 0-100% in 50 min. Mass spectral data acquisition was performed using a hybrid linear ion trap-Orbitrap under the following conditions: MS scanning: m/z is more than or equal to 300 and less than or equal to 2000; MS/MS exclusion list, 2Da separation window and 25% relative collision energy: 8 most abundant ions (5> z > 1); ESI voltage: 1.5 kV; capillary temperature: at 150 ℃. MS/MS data were analyzed using the MascotDaemon database.
In the prior art, the capacity of the overall peptide identification was increased by extending the organic solvent gradient or decreasing the outflow rate. The latter (reduction of the outflow flow rate) is of interest in the present invention, since this method can improve electrospray ionization efficiency. However, when the flow rate is lower than the Van Deemter minimum flow rate, the chromatographic resolution is reduced, and when the capillary inner diameter is less than 50 μm, the column performance is less stable, so that the invention prepares a reliable and durable capillary analytical column with silicate frit at the tip, and an electrospray device based on the analytical column.
Second, analysis results
The results of the mass spectrometric analysis of sample a using the capillary analytical columns prepared in examples 25 and 29 are:
figures 3 and 4 show the chromatographic and electrospray ionisation performance characteristics of a 50 μm i.d. × 12cm (capillary analytical column prepared in example 29) and a 25 μm i.d. × 12cm (capillary analytical column prepared in example 25) capillary analytical column. As shown in FIG. 3, when the flow rate is greater than 1nL/min, the half-peak width of the 25 μm inner diameter column RIC is smaller than that of the 50 μm inner diameter column, which indicates that the chromatographic resolution of the 25 μm inner diameter column is higher, i.e., the chromatographic resolution is higher due to the smaller capillary inner diameter; when the flow rate is less than 1nL/min, Van Deemter effect appears in the RIC half-peak width of 50 μm and 25 μm inner diameter columns, and the chromatographic resolution is reduced. In the attached figure 4, when the flow rate is less than 1nL/min, the identification capability of the whole peptide of the column with the inner diameter of 25 mu m is obviously improved, which shows that the low flow rate can make up the reduction of chromatographic resolution brought by Van Deemter effect and improve the sensitivity of chromatographic mass spectrometry while improving the electrospray ionization efficiency; by the principle, the capillary tube analytical column and the chromatography-mass spectrometry combined system based on the analytical column have ultrahigh sensitivity, and can effectively improve the identification and analysis of proteins with lower content.
In conclusion, the invention provides a method for realizing ultrahigh-sensitivity protein mass spectrometry detection, and solves the problem of identification and detection of low-content protein.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.
Claims (20)
1. A capillary analytical column suitable for use in a chromatography-mass spectrometry system, the analytical column comprising a column tube, a capillary integrated emission tip, a filler material packed in the lumen, a frit for supporting the filler material; wherein the inner diameter of the analytical column is less than or equal to 30 μm; the filler is a modified silicon-based microsphere with smooth surface and uniform particle size, and the surface of the modified silicon-based microsphere is covered with an organic polymer, and the particle size of the modified silicon-based microsphere is less than or equal to 3 mu m.
2. The capillary analytical column of claim 1, wherein when the capillary analytical column is a polyimide coated quartz capillary, the analytical column comprises an exposed end of the capillary and a polyimide coated portion; and/or the length of the analytical column is 5-20 cm; and/or the outer diameter of the analytical column is 200-500 mu m; and/or the inner diameter of the analytical column is less than or equal to 25 mu m; and/or the diameter of the capillary integrated emission tip is less than or equal to 2 mu m; and/or the thickness of the frit is 1-2.5 mm; and/or the frit is at a distance of 2-4mm proximal to the capillary integrated emission tip.
3. The capillary analytical column of claim 1, wherein the frit is a high strength, three-dimensional pore plug-like polymer or solid formed by silicate sol-gel self-crosslinking that is drawn in by capillary siphoning.
4. The capillary analytical column of claim 3, wherein the silicate is selected from one or more of aluminum silicate, iron silicate, calcium silicate, magnesium silicate, potassium silicate, sodium silicate, manganese silicate; and/or forming the silicate sol-gel system by the silicate under the condition of a solvent for promoting the formation of sol-gel, wherein the solvent is one or more of water, glacial acetic acid, alkali, ethanol and isopropanol.
5. The capillary analytical column of claim 4, wherein the silicate is a mixture of aluminum silicate, potassium silicate, and manganese silicate, wherein the molar ratio of aluminum silicate, potassium silicate, and manganese silicate is (9-10): (15-16): (1-1.5).
6. The capillary analytical column of claim 1, wherein the modified silicon-based microspheres are prepared by a method comprising: with N, N-dimethylhexadecylamine, NH4F is used as a modifier, a polymer monomer is subjected to polymerization reaction in the presence of glacial acetic acid, water and ethanol to form a crosslinked modified organic polymer, and the crosslinked modified organic polymer is covered on the surface of the silicon-based microsphere to obtain the modified silicon-based microsphere; wherein, the polymer monomer comprises any one or more of trichloroethylene, divinyl benzene, styrene, vinyl pyrrolidone and vinyl pyridine.
7. The capillary analytical column of claim 6, wherein the mass of the modifier is 6 to 9% of the mass of the total reaction system; and/or the mass of the polymer monomer accounts for 10-12% of the mass of the total reaction system; and/or the temperature of the polymerization reaction is 300-400 ℃; and/or the time of the polymerization reaction is 5-6 h.
8. A method of preparing a capillary analytical column having an internal diameter of less than or equal to 30 μm, the method comprising the steps of: (1) building a frit at the end of the capillary; (2) filling a filler into the capillary under pressure; (3) drawing the capillary integral emission tip.
9. The method of claim 8, wherein the step (1) of building a frit at the capillary end comprises:
(1.2) migrating the silicate sol-gel system to the midpoint of the bare portion of the capillary by siphoning;
(1.3) heating the silicate sol-gel system to enable the silicate sol-gel system to perform self-polymerization reaction to obtain an inorganic polymer to form the frit.
10. As claimed in claimThe preparation method of the capillary analytical column is characterized in that in the step (2), the filler is modified silicon-based microspheres, and the preparation method of the filler comprises the following steps: with N, N-dimethylhexadecylamine, NH4F is used as a modifier, and in the presence of glacial acetic acid, water and ethanol, a polymer monomer is subjected to polymerization reaction to form a cross-linked polymer which covers the surface of the silicon-based microsphere, namely the modified silicon-based microsphere; wherein the polymer monomer comprises any one or more of trichloroethylene, divinyl benzene, styrene, vinyl pyrrolidone and vinyl pyridine; and/or, the step (2) of filling the capillary with the filler under pressure comprises: mixing the filler and the organic solvent into homogenate, filling the homogenate into a column bed under the condition of initial pressure of 2500-.
11. The method of claim 8, wherein the step (3) of drawing the capillary integrated emission tip comprises: an integrated emission tip with a diameter ≦ 2 μm is formed at the capillary tip using a laser-based pipette puller.
12. The preparation method according to claim 9, wherein in the step (1.2), the silicate is selected from one or more of aluminum silicate, iron silicate, calcium silicate, magnesium silicate, potassium silicate, sodium silicate and manganese silicate; and/or the solvent for promoting the formation of the sol-gel is one or more of water, glacial acetic acid, alkali, methanol, ethanol and isopropanol.
13. The method according to claim 9, wherein the step (1.3) of heating the silicate sol-gel system comprises low-temperature polymerization, cooling, high-temperature polymerization, cooling, and cleaning;
wherein the temperature of the low-temperature polymerization is 250-300 ℃; the time of the low-temperature polymerization is 60-90 s;
wherein the temperature of the high-temperature polymerization is 350-400 ℃; and/or the time of the high-temperature polymerization is 20-30 s;
the high temperature polymerization is carried out without the presence of a catalyst; the thickness of the formed frit can be controlled by the position covered by the electric soldering iron.
14. The production method according to claim 12, wherein in the step (1.2), the concentration of the silicate ion in the total reaction system is 2 to 3 mol/L; and/or the silicate is a mixture of aluminum silicate, potassium silicate and manganese silicate, wherein the molar ratio of the aluminum silicate to the potassium silicate to the manganese silicate is (9-10) to (15-16) to (1-1.5).
15. The preparation method according to claim 10, wherein in the step (2) of preparing the filler, the mass of the modifier accounts for 6-9% of the mass of the total reaction system; and/or the mass of the polymer monomer accounts for 10-12% of the mass of the total reaction system; and/or the presence of a gas in the gas,
and (2) filling a filling material into the capillary under pressure, wherein the organic solvent is selected from one or more of methanol, ethanol, isopropanol, benzene, xylene, toluene, pentane, hexane, octane, cyclohexane, cyclohexanone, toluene cyclohexanone, chlorobenzene, dichlorobenzene, dichloromethane, diethyl ether, propylene oxide, methyl acetate, ethyl acetate, propyl acetate, acetone, methyl butanone, methyl isobutyl ketone, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, acetonitrile, pyridine and phenol.
16. The preparation method according to claim 15, wherein in the step (2.2), the organic solvent is a mixed solvent of ethylene glycol monoethyl ether and diethyl ether in a volume ratio of 10: 3; and/or, in step (2.2), the pressure of the pressurized helium gas used for drying is 2200-2800 psi; and/or, in the step (3), the intensity of the laser is 7-9KJ joule.
17. A capillary analytical column produced by the production method according to any one of claims 8 to 16.
18. A chromatography-mass spectrometry system comprising the capillary analytical column according to any one of claims 1 to 7 or the capillary analytical column produced by the method of preparing the capillary analytical column according to any one of claims 8 to 16.
19. Use of the capillary analytical column according to any one of claims 1 to 7, or the capillary analytical column produced by the method according to any one of claims 8 to 16, or the chromatography-mass spectrometry system according to claim 18 for performing ultra-high sensitivity protein mass spectrometry.
20. An ultra-high sensitivity protein mass spectrometry detection method, which is implemented based on the capillary analytical column of any one of claims 1 to 7, or the capillary analytical column manufactured by the preparation method of any one of claims 8 to 16, or the chromatography-mass spectrometry system of claim 18; the method comprises the following steps:
the method comprises the steps of construction of a frit at the end of a capillary, variable-pressure chromatographic column filling of filler, drawing of an integrated emission tip of the capillary, control of shunt flow rate and high-precision position control of a chromatographic mass spectrometry interface;
wherein the construction of the capillary end frit, packing of the packing for a variable pressure chromatography column, drawing of the capillary integrated emission tip is performed by the method of any one of claims 8 to 16;
wherein, the flow velocity of the sample to be detected is less than or equal to 1nL/min through the flow dividing device at the front end of the analytical column;
the high-precision position control of the chromatographic mass spectrum interface realizes sensitive control through a stepping motor controlled by a high-precision computer.
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