CN115541797A - Integrated chromatographic analysis probe assembly, device and chromatographic analysis method - Google Patents
Integrated chromatographic analysis probe assembly, device and chromatographic analysis method Download PDFInfo
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Abstract
The invention discloses an integrated chromatographic analysis probe component, which comprises a probe body with a mobile phase channel and a chromatographic column channel, wherein one end of the chromatographic column channel is provided with a sampling port; the mobile phase capillary tube is arranged on the probe body and communicated with the mobile phase channel; the chromatographic column is detachably fixed in the chromatographic column channel, and the inlet of the chromatographic column corresponds to the sampling port; a mobile phase liquid inlet gap is reserved between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is communicated with the outlet of the mobile phase channel and the sampling port at the same time. The invention also discloses an analysis device and method. The device has the advantages of high integration level, high strength and hardness, simple structure, small volume, suitability for batch processing and simple and convenient operation, and mainly aims at micro samples with small volume, complex composition and low concentration, so that the loss of the micro samples in transfer and pipeline transportation is reduced to the maximum extent.
Description
Technical Field
The invention belongs to the field of chromatographic analysis, and particularly relates to an integrated chromatographic analysis probe assembly, an integrated chromatographic analysis probe device and a chromatographic analysis method.
Background
The chromatographic analysis technology has been widely concerned by researchers for a long time due to the strong separation and analysis capability of the chromatographic analysis technology on complex samples, and has wide development potential and application prospect. At present, high performance liquid chromatography and gas chromatography have become one of the most important analytical techniques in the fields of chemistry, biomedicine, agriculture, environmentology, dietetics, and the like.
The traditional high performance liquid chromatography sample injection method usually depends on an automatic sample injector or a sample injection valve, and sample injection is realized through the matching of a quantitative ring, valve switching and a liquid pump. In recent years, with the increasing demand of various research fields for trace sample analysis, more and more work puts forward a trace sample introduction requirement on the sample introduction link of the liquid chromatography method. Different from the traditional liquid chromatography sample injection method, the target of the micro-chromatography sample injection method is usually a micro sample with extremely small volume, extremely low concentration and complex components. At present, the main application fields of the micro-chromatography sample introduction technology comprise single cell proteomics analysis, metabonomics analysis, mass spectrometry imaging, space multiomics analysis, micro high-throughput screening and the like.
At present, in the field of micro-chromatography sampling, the literature usually adopts a droplet microextraction method, a micro-fluidic chip method, a probe sampling method and an in-situ reaction method to transfer a micro sample into a chromatographic column. However, these approaches have limitations, including: the sample cannot be completely transferred, and the sample has residual loss; the addition of a sample reagent is difficult, and the compatibility with complex pretreatment operation is difficult; when the probe sampling method is adopted, the device based on the capillary processing probe body cannot bear high pressure due to structural strength, and practical application of the device is limited due to insufficient working durability and reliability of the device.
Disclosure of Invention
The invention provides an integrated chromatographic analysis probe assembly and a chromatographic analysis method, which integrate chromatographic sampling, sample introduction and separation functions into a probe and can realize chromatographic trace sample introduction and separation analysis with extremely low loss.
An integrated chromatography probe assembly comprising:
the probe body is provided with a mobile phase channel and a chromatographic column channel, and one end of the chromatographic column channel is provided with a sampling port;
the mobile phase capillary tube is arranged on the probe body and communicated with the mobile phase channel;
the chromatographic column is detachably fixed in the chromatographic column channel, and the inlet of the chromatographic column corresponds to the sampling port;
a mobile phase liquid inlet gap is reserved between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is communicated with the outlet of the mobile phase channel and the sampling port at the same time.
The sampling port is used for covering a sample area and forming a sealed space with the sample chip;
the conduction of the outlet of the mobile phase channel, the sampling port and the inlet of the chromatographic column is realized through the liquid inlet gap of the mobile phase, and the entering of the mobile phase and the sample introduction are realized.
The integrated chromatographic analysis probe component adopts a modular structure. Preferably, the integrated chromatography probe assembly can use a split structure with different integration degrees until a one-piece structure that all parts are integrally processed on the probe body is adopted.
Furthermore, a mobile phase capillary fixing interface, a mobile phase channel, a sampling port, a chromatographic column channel and a chromatographic column fixing interface are processed on the body of the integrated chromatographic analysis probe, and the mobile phase capillary and the chromatographic column are fixed on the body of the integrated chromatographic analysis probe by utilizing a capillary fixing component.
More preferably, the integrated chromatography probe is configured to have a higher degree of integration, and the mobile phase capillary fixing port, the mobile phase channel, the sampling port, the column channel, and the column are integrally formed in the probe body.
Preferably, the mobile phase channel and the chromatographic column channel are respectively provided with a fixed interface at one end and a fixing component for fixing the mobile phase capillary tube and the chromatographic column at the corresponding fixed interfaces
The fixed interface of the mobile phase channel is matched with the capillary tube fixing component and used for connecting the mobile phase capillary tube with the mobile phase channel on the probe body and keeping sealing, and the other end of the mobile phase capillary tube is connected with the fluid driving device and used for introducing the mobile phase into the integrated chromatographic analysis probe.
The fixed interface of the chromatographic column channel is matched with the chromatographic column fixing component to connect and keep the chromatographic column with the chromatographic column channel of the probe sealed, so that the mobile phase is prevented from leaking from the chromatographic column.
More preferably, the fixing means includes a column fixing means and a mobile phase capillary fixing means, and both may have the same structure, or may be adaptively adjusted according to the difference in structure between the column and the mobile phase capillary. As an aspect, the fixing assembly includes: a positioning sleeve for realizing the positioning of the chromatographic column or the mobile phase capillary and a locking piece for fixing the sleeve and the probe body with each other. The outer wall of the positioning sleeve is provided with a positioning bulge. The locking member may be a threaded locking member; for example, the locking piece is a hollow piece, one end of the locking piece is provided with an inner folded edge for clamping the positioning bulge, and the other end of the locking piece is provided with an internal thread; at this time, an external thread matched with the locking piece can be arranged at the corresponding channel interface of the probe body.
Preferably, the device further comprises a sample chip for carrying the sample and a moving device for controlling the displacement of the sample chip. The position of the sample chip can be adjusted by using the mobile device.
Preferably, all components included in the integrated chromatography probe, including the probe body, the mobile phase capillary, the chromatographic column and the sample chip, are made of solvent-inert inorganic materials or polymer materials, such as metal, glass, quartz, polyetheretherketone, polytetrafluoroethylene, or a composite material of two or more of the above, respectively, so as to resist adverse effects such as swelling caused by long-term liquid contact.
Furthermore, the integrated chromatography probe body is made of hard metal materials, high molecular polymer materials or other materials.
Preferably, the sample chip material is different from the material of the integrated chromatography probe body in order to improve the sealing property, stability, life and pressure resistance of the integrated chromatography probe.
Preferably, the hardness of the probe body material corresponding to the sampling port is greater than or equal to the hardness of the sample chip material.
Preferably, the sample chip is made of a material having a hardness lower than that of the probe body material corresponding to the sampling port and having a relatively elasticity.
Further, the processing of integration chromatography probe body and subassembly adopt monoblock material to subtract material processing mode and process, with pressure resistance, structural strength and the life of guaranteeing the probe.
Furthermore, the integrated chromatographic analysis probe is made of a stainless steel material or a titanium alloy material and is processed by adopting a program-controlled automatic machine tool (CNC) technology, so that the pressure resistance of a channel in the probe reaches more than 30 MPa, the strength and the service life of the integrated chromatographic analysis probe are improved, and the integrated chromatographic analysis probe is suitable for batch production.
The channel configuration in the integrated chromatographic analysis probe is a T-shaped structure or a Y-shaped structure, or a structure similar to the T-shaped structure or the structure similar to the Y-shaped structure. And the bottom of the T-shaped structure or the Y-shaped structure is provided with a sampling port. The mobile phase channel is communicated with the chromatographic column channel and is connected with the inlet of the chromatographic column, and the joint of the mobile phase channel and the inlet of the chromatographic column is a sampling port. Further, a sampling port may be provided at the inlet of the chromatography column.
According to the invention, the inner diameter (diameter) or inner side length of the mobile phase channel, the chromatographic column and the sampling port on the integrated chromatographic analysis probe ranges from 0.05 micrometer to 5 millimeters. The diameter of the chromatographic column channel is larger than the outer diameter of the chromatographic column. Preferably, the difference between the diameter of the chromatography column channel and the outer diameter of the chromatography column is reduced, reducing the dead volume in the probe channel.
And external threads, or internal threads, or buckles, or the combination of the above types are processed at the mobile phase capillary tube fixing interface and the chromatographic column fixing interface, so that the mobile phase capillary tube fixing interface and the chromatographic column fixing interface are used for sealing the capillary tube fixing component.
Preferably, the outer surface of the sample port, and the portion of the sample chip that contacts the outer surface of the sample port, are polished or otherwise planarized.
Preferably, the mobile phase capillary tube fixing interface and the chromatographic column fixing interface are provided with external threads, and the scheme is combined with a split type capillary tube fixing component provided with internal threads, so that a good sealing effect can be provided, and the probe has higher capacity of resisting hydraulic pressure in a channel.
In the invention, the material of the chromatographic column tube and the mobile phase capillary is glass, quartz, metal, or high molecular polymer, or other materials. The material has a tolerance to the liquid and mobile phase flowing through the probe passage.
Preferably, the mobile phase capillary is a quartz capillary with a coating coated on the surface, and has good pressure resistance, corrosion resistance and toughness.
In the invention, the chromatographic column is a packed column, an open tubular column, a chemical synthesis monolithic column, a micro-processing monolithic column or a composite configuration of the above configurations.
Preferably, an electrospray spray tip is processed at the outlet of the chromatographic column and is used for connecting with an electrospray mass spectrometry detection system, and a sample flows out through the spray tip at the outlet of the chromatographic column to form electrospray.
According to the invention, the sample chip is made of a material with hardness smaller than that of the probe body material. The material is inorganic material, organic material or high molecular polymer material.
According to the invention, the hardness and elasticity matching between the integrated chromatography probe material and the sample chip material needs to be screened and optimized to obtain satisfactory sealing effect and service life.
Preferably, the sample chip is made of a material having relative elasticity, which can significantly improve the sealing capability between the probe sampling port and the sample chip.
Preferably, a sample chip is made of a Polyetheretherketone (PEEK) material or a material with similar hardness and elasticity, and the sample chip is matched with an integrated chromatographic analysis probe processed by stainless steel or titanium alloy, so that the sealing effect meeting the analysis requirement of a high performance liquid chromatography device (the pressure resistance exceeds 30 MPa) is obtained, and the use frequency and the service life of the sample chip are obviously improved.
According to the present invention, the shape of the sample chip is a circular piece having a thickness, a square piece having a thickness, a polygonal piece having a thickness, a cylinder, a cube, a cylinder having steps, a cube having steps, or other three-dimensional shapes.
Preferably, the sample chip is manufactured by CNC one-time processing, and is in a cylindrical shape with steps, so that the sample chip is convenient to fix and position in the pits.
According to the invention, one or more pits for bearing micro samples are processed on the sample chip, and the volume range of the pits is 1 attoliter to 1 milliliter; the diameter range of the pits is 1 micrometer to 10 centimeters; the depth of the pits ranges from 1 micron to 1 centimeter.
Further, the sample-bearing pits on the sample chip are cylindrical, or cross-shaped, or hemispherical, or microstructures with other shapes, and play a role in fixing the position of the sample on the surface of the sample chip.
According to the invention, the initial state of the sample carried by the sample chip is a liquid sample, a solid sample or a solid-liquid mixed sample, and the final state of the sample can be the liquid sample or the solid-liquid mixed sample or the solid sample formed after the liquid sample is dried.
According to the invention, the sample chip is driven by the moving device for controlling the displacement of the sample chip to contact with the outer surface of the sampling port of the integrated chromatographic analysis probe and form a pressing force, and the pressing force is utilized to realize the sealing between the sample chip and the outer surface of the sampling port of the integrated chromatographic analysis probe.
Further, the pressing force applied to the sample chip and the probe is in the range of 0.01 kg to 100 kg. Preferably, the extrusion force output by the pressure driving device is adjusted to the highest hydraulic pressure of the system using the fluid driving device under the condition of no liquid leakage through theoretical calculation according to actual experimental requirements.
Preferably, the outer surface of the sampling port of the integrated chromatography probe and the portion of the sample chip that contacts the outer surface of the sampling port of the integrated chromatography probe are polished or otherwise planarized to ensure the hermeticity between the sample chip and the integrated chromatography probe.
According to the invention, the sample chip is contacted with the outer surface of the sampling port of the integrated chromatographic analysis probe to form the operation of extrusion sealing, and the integrated chromatographic analysis probe can be driven to be contacted with the sample chip by adopting a moving device, or the integrated chromatographic analysis probe and the sample chip are driven to move in the same direction by adopting the moving device respectively, so that the contact and extrusion sealing of the integrated chromatographic analysis probe and the sample chip are realized.
Preferably, a precise groove is processed on a driving device of the integrated chromatographic analysis probe and the sample chip and used for fixing the sample chip; or the driving device is provided with a mechanical structure, an adhesive structure or an integrated sample chip for accurately positioning the sample bearing area.
According to the invention, when the sample chip contacts with the outer surface of the sampling port of the integrated chromatographic analysis probe and forms a compression seal, the inlet of the chromatographic column in the probe is higher than, or flush with or lower than the sealing surface of the sample chip.
Preferably, after the sample chip is contacted with the outer surface of the sampling port of the integrated chromatographic analysis probe and forms extrusion sealing, the inlet of the chromatographic column in the probe is lower than the sealing surface of the chip, namely the inlet end of the chromatographic column is inserted into the pit of the sample chip, so that the dead volume is reduced, the elution speed of the sample in the pit is accelerated, and the analysis speed is improved.
Preferably, the sample chip is processed with one or more pits for carrying micro samples.
A chromatography apparatus comprising an integrated chromatography probe assembly as described in any preceding claim, a fluid actuating means and a detection means.
The mobile phase channel is connected with a fluid driving device for providing a chromatographic mobile phase through a mobile phase capillary tube so as to control the mobile phase to flow into the integrated chromatographic analysis probe, and the outlet of the chromatographic column is connected with a detection device;
preferably, when the outlet of the chromatographic column is connected with the electrospray mass spectrometry detection device, the mass spectrometry electrospray spray tip is integrated and processed at the outlet of the chromatographic column, so that four functions of chromatographic sampling, sample introduction, separation and mass spectrometry electrospray are integrally completed on the probe.
A method of performing chromatography using an integrated chromatography probe assembly according to any of the preceding claims, comprising:
(1) Moving a sample chip carrying a sample to a target position, aligning a sampling port of the integrated chromatographic analysis probe assembly to the sample position, and enabling the outer surface of the sampling port to be in contact with the sample chip and form extrusion to realize sealing;
(2) A fluid driving device is used for leading a mobile phase into a mobile phase liquid inlet gap of a chromatographic column channel through a mobile phase capillary and a mobile phase channel, and then the mobile phase enters a chromatographic column by carrying a sample on a sample chip to a sampling port;
(3) Driven by the subsequent mobile phase, the sample component entering the chromatographic column is separated in the chromatographic column and flows out of the chromatographic column outlet to a detector for detection.
The invention is suitable for the fields of single cell analysis, multidimensional chromatographic analysis, ultra-micro sample analysis, high-throughput screening and the like which need to carry out rapid and high-throughput chromatographic analysis on trace samples. And is also suitable for field analysis requiring small volume or portable chromatographic analysis devices.
A method of using an integrated chromatography probe assembly, comprising:
(a) Adding a sample into a pit of a sample chip, and directly carrying out subsequent sample introduction operation on the sample or carrying out in-situ pretreatment on the sample in the pit;
(b) Fixing a sample chip on a moving device for controlling the displacement of the sample chip;
(c) Driving the sample chip by using a moving device for controlling the displacement of the sample chip, accurately aligning the sampling port of the integrated chromatographic analysis probe to a pit bearing a target sample on the sample chip, and then contacting the sample chip on the outer surface of the sampling port of the integrated chromatographic analysis probe to form extrusion for realizing sealing;
(d) A fluid driving device is utilized to introduce a mobile phase into the integrated chromatographic analysis probe through a mobile phase capillary and a mobile phase channel inlet, the mobile phase flows through a probe sampling port to carry a liquid sample in a pit of a sample chip or dissolve and elute a solid sample in the pit and enters a chromatographic column inlet;
(e) Driven by a subsequent mobile phase, the sample component entering the chromatographic column is separated in the chromatographic column and flows out of the chromatographic column outlet to enter a detector for detection;
(f) And (e) repeating the operation steps from (a) to (e) to finish sampling, sample introduction and analysis of different samples on the sample chip, or samples on different sample chips, or different spatial positions of the same sample.
Preferably, the pressing force acting on the sample chip and the integrated chromatography probe is in the range of 0.01 kg to 100 kg. Preferably, the gas pressure generated by the sample chip moving device (for example, the gas pressure generated by an air pump arranged in the sample chip moving device) is used for driving the sealing operation of the sample chip and the sampling port of the integrated chromatographic analysis probe; or the sample chip is fixed on a support with certain elasticity on a mobile device for controlling the displacement of the sample chip so as to realize the closed operation of the sample chip and the sampling port of the integrated chromatographic analysis probe in a mode of compensating the deformation of the sample chip in real time. The two methods can compensate the pressure change generated by the deformation of the sample chip in real time, and provide continuous thrust to ensure the long-time sealing of the sample chip and the sampling port of the integrated chromatographic analysis probe.
Preferably, the inlet of the chromatographic column in the integrated chromatography probe is higher, or flush, or lower than the sealing surface of the sample chip after the sample chip is brought into contact with the outer surface of the sampling port of the integrated chromatography probe and forms a compression seal. Preferably, the inlet of the column in the probe is lower than the sealing surface of the chip, i.e. the inlet end of the column is inserted into the pit of the sample chip to reduce the dead volume and increase the analysis speed.
According to the invention, when a sample is analyzed, after a liquid sample or a solid sample is added into the pit of the sample chip, the sample chip is installed in the positioning device of the mobile device for controlling the displacement of the sample chip, and the aim of accurately positioning the pit of the sample chip relative to the sampling port of the integrated chromatographic analysis probe is achieved.
Further, after adding a liquid sample or a solid sample to the well of the sample chip, a multi-step and complicated pretreatment operation including a reagent addition operation and a reaction step of the pretreatment is performed in the well, and then the sample chip is mounted to a positioning device of a moving device for controlling the displacement of the sample chip.
Preferably, the method of adding the original sample to the pit, performing in-situ sample pretreatment in the pit and performing subsequent in-situ sample injection is adopted for ultramicro biological samples with complex compositions, such as single cells, subcellular organelles, biological tissue samples or other trace samples needing pretreatment operation, so that the loss of the samples caused by transfer in the pretreatment and sample injection processes can be greatly reduced, even the complete lossless sample injection is realized, and the sample utilization rate and the detection sensitivity are improved.
Preferably, in order to reduce the dead volume inside the integrated chromatography probe device, when the chromatographic column is manufactured, the stationary phase is filled in the chromatographic column and the chromatographic column channel connected with the chromatographic column channel, even the probe sampling port, so that the zonal broadening and the dilution effect generated after the sample enters the sampling port and the interior of the chromatographic column can be reduced.
According to the invention, the integrated chromatographic analysis probe sampling port is contacted with the sample chip to realize the sealing operation, and the effect of tight fit can be realized by utilizing the thrust provided by a pressure driving device, or the thrust propelled by a motor, or a screw-thread fit screwing mode, or a buckle locking mode, or other modes.
Preferably, the gas pressure generated by the gas pump arranged in the sample chip moving device is used for driving the sealing operation of the sample chip and the integrated chromatographic analysis probe sampling port, and the continuous thrust can be provided to ensure the long-time sealing of the sample chip and the integrated chromatographic analysis probe sampling port. Because the sample chip has certain deformability, the sample chip is easy to deform in a long-time extrusion process, so that the pressure between the sample chip and the sample chip is reduced, the airtightness is poor, and the phenomenon that fluid flows out or leaks from the airtight interface is caused. When methods such as motor-propelled thrust are adopted, pressure change caused by deformation of the sample chip is difficult to compensate in real time, and interface leakage is easy to generate.
Preferably, the sample chip is fixed on a support having a certain elasticity on a moving device for controlling the displacement of the sample chip, and the pressure change caused by the deformation can be compensated in real time by using the elasticity of the support.
Preferably, the contact area between the sampling port and the sample chip is smaller, so that the extrusion force can be obviously reduced, and the loss of the material of the sample chip and the material of the integrated chromatographic analysis probe body is reduced.
According to the invention, the sealing effect of the integrated chromatographic analysis probe sampling port and the sealing realized by the sample chip is positively correlated with the ultimate pressure resistance of the system, so that the ultimate pressure resistance is improved, the device can be suitable for chromatographic columns with thinner inner diameter, thinner filler granularity, longer filler granularity and higher back pressure, and the separation efficiency, the separation degree, the detection sensitivity and the like of the sample can be improved.
According to the invention, in the sampling and sample feeding processes, the fluid driving device controls the mobile phase to inject into the integrated chromatographic analysis probe, when the mobile phase flows through the pit carrying the sample, the mobile phase is used for carrying all or most of the sample at one time to enter the chromatographic column for separation and analysis, or the mobile phase gradually elutes the sample carried on the pit to enter the chromatographic column for separation and analysis, or the mobile phase with different compositions and concentrations gradually elutes components with different properties carried on the pit to enter the chromatographic column for separation and analysis.
According to the invention, the composition, concentration and flow rate of the mobile phase carrying or eluting the sample into the chromatographic column can be the same as or different from the conditions of the mobile phase used in the subsequent separation and analysis. The main consideration is how to obtain better sampling effect. Alternatively, the sampling and sample injection operations may be performed using a gas mobile phase, and the separation operations may be performed using a liquid mobile phase.
According to the present invention, preferably, the sample chip can be reused. After sampling, sample introduction and separation analysis of one sample are completed, the surface bearing the sample is thoroughly cleaned by using a cleaning solution before analysis of the next or next batch of samples is carried out, so that cross contamination among different samples is reduced.
According to the invention, the detection method adopted by the detection device of the chromatographic system is an optical analysis method, an electrochemical analysis method, a mass spectrometry method or a combination of two or more detection methods. The optical analysis method comprises ultraviolet-visible spectrophotometry, fluorescence spectroscopy, infrared spectroscopy, raman spectroscopy, nuclear magnetic resonance spectroscopy, optical imaging analysis, optical sensing analysis, atomic spectroscopy, etc. Electrochemical analysis methods include potentiometry, electrolysis and coulometry, voltammetry, conductimetry, electrochemical sensing analysis, and the like. The mass spectrometry comprises electron bombardment mass spectrometry, field desorption mass spectrometry, fast atom bombardment mass spectrometry, matrix-assisted laser desorption mass spectrometry, electrospray mass spectrometry, in-situ ionization mass spectrometry, inductively coupled plasma mass spectrometry and the like.
According to the invention, an array sample introduction system consisting of a plurality of integrated chromatographic analysis probes is adopted to carry out sample analysis in parallel, which is beneficial to improving the analysis flux of the system;
according to the invention, the integrated chromatographic analysis probe is suitable for being applied to liquid chromatographic analysis, gas chromatographic analysis or supercritical fluid chromatographic analysis of trace samples.
It should be noted that the application field of the integrated chromatography probe is not limited to the chromatography field, but can be applied to other flow analysis fields, such as direct injection ionization mass spectrometry, microfluidic analysis, flow injection analysis, continuous flow analysis, capillary electrophoresis analysis, bubble space flow analysis, or analysis using a combination of the above analysis systems.
The main advantages of the invention include: the integrated chromatographic analysis probe integrates three main steps of sampling, sample introduction and separation in chromatographic analysis, combines the advantages of a micro-fluidic chip and a probe sample introduction method, directly covers and seals a sample area on the sample chip by a sampling port of the integrated chromatographic analysis probe aiming at micro samples with small volume, complex composition and low concentration, and avoids the loss of the micro samples caused by transfer and transportation. The integrated chromatographic analysis probe device has the advantages of small volume, short pipeline, small dead volume and high sampling speed, and can obviously improve the analysis flux. The integrated probe is designed in a detachable mode, parts are easy and convenient to replace, the service life of the integrated chromatographic analysis probe body is prolonged, and the cost is reduced. By utilizing the device, the sample pretreatment is completed in situ on the sample chip, and the combination of the in-situ pretreatment and the in-situ analysis can be realized.
The main advantages of the invention are:
(1) The device has high integration level, high strength and hardness, simple structure, small volume, suitability for batch processing and simple and convenient operation, integrates the functions of sampling, sample introduction and separation in the chromatographic analysis process into the chromatographic analysis probe, shortens the pipeline length of a sample introduction module and the distance between a sample and a chromatographic column inlet, obviously shortens the sampling and sample introduction time, and improves the analysis speed and the analysis flux;
(2) The device adopts the strategy of normal-pressure in-situ reactor pretreatment-high-pressure integrated chromatography probe sealing sample injection analysis, realizes the combination of sample pretreatment and online separation analysis, simultaneously realizes the zero transfer of all the processes from pretreatment to sample injection of samples, solves the problems of sample transfer and sample injection of trace and ultra-small volume samples which are difficult to solve by an automatic sample injector in the conventional chromatographic system, reduces the loss of trace samples in transfer and pipeline transportation to the maximum extent, and improves the analysis sensitivity;
(3) The integrated chromatographic analysis probe has small size and is suitable for automation, and a scheme for miniaturizing a conventional liquid chromatographic system is provided; by further reducing the size of the sampling port, the method can be applied to the high-spatial-resolution in-situ imaging analysis of biological, environmental and cultural relic samples;
(4) The ultrahigh strength and hardness of the integrated probe and the ultrahigh pressure resistance of the device successfully adapt to common ultrahigh pressure liquid chromatography pumps on the market, and favorable conditions are provided for popularization and adaptation of the device and the method.
(5) The system disclosed by the invention is expected to be applied to the fields of multidimensional chromatographic analysis of trace complex samples, high-throughput drug screening analysis, unicellular omics analysis, biological tissue imaging analysis, space multiomics analysis, field analysis and the like.
Drawings
FIG. 1 is a schematic diagram showing the structure of an integrated chromatography probe module of example 1, which is made of polyetheretherketone.
FIG. 2a is a schematic diagram showing the structure of an integrated chromatography probe made of stainless steel in example 2.
Fig. 2b is a schematic diagram of various sampling ports and sample carrying structures on sample chips used in embodiments 2 and 4.
FIG. 3 shows the results of liquid chromatography-electrospray mass spectrometry analysis of a mixed solution sample containing four standard peptide fragments in a gradient elution mode using the analysis apparatus of example 2.
FIG. 4 is a schematic diagram of the structure of an integrated chromatography probe processed in Y-configuration of example 4.
In the figure, 1-an integrated chromatographic analysis probe body, 2-a mobile phase channel, 3-a sampling port, 4-a chromatographic column, 5-a chromatographic column channel, 6-a mobile phase capillary tube fixing interface, 7-a chromatographic column fixing interface, 8-a capillary tube fixing component, 9-a chromatographic column outlet, 10-a sample chip, 11-a pressure moving device for controlling the displacement of the sample chip, 12-a sample, 13-a detection device, 14-a fluid driving device, 15-a mobile phase capillary tube and 16-a sample chip pit.
Detailed Description
The technical solutions of the present invention are further described below with reference to specific examples, but the scope of the present invention is not limited thereto.
Preferred embodiments according to the present invention will be described in detail below with reference to the accompanying drawings.
Example 1
FIG. 1 is a schematic diagram of the structure of an integrated chromatography probe assembly constructed in accordance with the present invention. The probe comprises a probe body 1 with a mobile phase channel 2 and a chromatographic column channel 5, wherein one end of the chromatographic column channel is a sampling port 3; a mobile phase capillary 15 provided on the probe body and communicating with the mobile phase channel; a chromatographic column 4 detachably fixed in the chromatographic column channel, wherein the inlet of the chromatographic column corresponds to the sampling port; a mobile phase liquid inlet gap is reserved between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is simultaneously communicated with the outlet of the mobile phase channel, the inlet of the chromatographic column and the sampling port. The mobile phase channel and the chromatographic column channel are respectively provided with a fixed interface at one end, namely a mobile phase capillary tube fixed interface 6 and a chromatographic column fixed interface 7, the mobile phase capillary tube fixed interface 6 is provided with a capillary tube fixed component 8 for fixing the mobile phase capillary tube 15, and the chromatographic column fixed interface 7 is provided with a chromatographic column fixed component 8a for fixing the chromatographic column 4.
The capillary holding unit 8 and the column holding unit 8a are similar in structure and each include a positioning sleeve for positioning the column or the mobile phase capillary and a locking member for fixing the sleeve and the probe body to each other. The outer wall of the positioning sleeve is provided with a positioning bulge. The locking member may be a threaded locking member; in the embodiment, the locking piece is a hollow piece, namely a sleeve structure, one end of the locking piece is provided with an inner folded edge clamped with the positioning bulge and used for axially limiting the positioning sleeve, and the other end of the hollow piece is provided with an internal thread; at this time, an external thread matched with the locking piece can be arranged on the corresponding fixing interface of the probe body. In fig. 1, the locking element of the capillary tube retaining assembly 8 is in a locked state, the locking element of the column retaining assembly 8a is in an open state, and the column 4 is also in an uninstalled state.
The probe body 1 is made of a bulk material of polyetheretherketone, and in practical use, the probe body can be matched with a sample chip 10 and a pressure moving device 11 (see fig. 2 a) for fixing the sample chip 10, and can also be matched with a liquid chromatography pump fluid driving device 14 and an electrospray mass spectrometer as a detection device 13. FIG. 2a shows a schematic flow direction of a chromatographic mobile phase by arrows.
A self-made chromatographic column with an integrated spray tip and an inner diameter of 75 micrometers and an outer diameter of 360 micrometers is fixed in a chromatographic column channel of the probe body 1, a quartz mobile phase capillary tube 15 with an inner diameter of 75 micrometers, which is in sealing butt joint with a mobile phase channel, is simultaneously fixed on the probe body 1, and the fixing of the quartz mobile phase capillary tube 15 and the mobile phase capillary tube is realized through a fixed capillary tube fixing component 8 and a chromatographic column fixing component 8a respectively.
In this embodiment, the pressure moving device 11 may be a three-dimensional moving platform, or may be a moving platform with an air pump (which may be a common one-dimensional moving platform, two-dimensional moving platform, or three-dimensional moving platform), and the like. The fluid driving means may employ a chromatographic pump.
When the integrated chromatographic analysis probe body is processed, polyether-ether-ketone is used as a probe manufacturing material, the probe is processed by using a CNC (computerized numerical control) technology, so that a chromatographic column channel and a mobile phase channel in the probe are in a T-shaped structure, and a capillary tube fixing interface 6 for fixing a mobile phase capillary tube and a chromatographic column fixing interface 7 for fixing a chromatographic column 4 are processed; the outlet of the fixed interface of the tube chromatographic column is provided with a cylindrical chromatographic column channel 5 which is used for installing the chromatographic column 4 and has the diameter of 400 microns and the length of 9 millimeters and is communicated with a sampling port. The outlet of the chromatographic column channel is provided with a circular sampling port, and the center of the circular sampling port is provided with a conical cavity. When the integrated chromatographic analysis probe is used, the circular ring surface of the sampling port is a contact surface and is positioned at a position where a mobile phase cannot flow through; the cone cavity is used to cover the sample in a position where the mobile phase flows through, facilitating cleaning to avoid cross contamination.
The cylindrical sample chip in the two-end type is made of a polyether-ether-ketone material, a single-hole chip scheme is adopted, a pit for bearing a sample is processed at the top, the diameter of the pit is 700 micrometers, and the depth of the pit is 300 micrometers.
The specific method of use of the apparatus of example 1 is as follows: (1) Fixing the sample chip 10 in the pressure moving device 11 having the groove; (2) Starting an air pump, wherein the pressure moving device carries the sample chip to move towards a sampling port 3 of the integrated chromatographic analysis probe, so that the sampling port is attached to and sealed with a sample bearing surface of the sample chip; (3) The sample connection that keeps integrating the chromatography analysis probe and the sealing of sample chip, start fluid drive arrangement 10 liquid chromatography pump promptly, with the chromatogram mobile phase along the mobile phase passageway 2 continuous injection that integrates the chromatography analysis probe subassembly integrate the internal mobile phase feed liquor clearance of chromatography analysis probe to reach the sample connection, get into the chromatographic column.
After the mobile phase with the pressure of 30 MPa is injected into the system and lasts for 8 hours, no obvious mobile phase leakage phenomenon is observed near the sampling port 3, the mobile phase channel fixing interface (the mobile phase capillary fixing interface 6) and the chromatographic column fixing interface 7, and the device of the embodiment is proved to keep good sealing stability in a long-term high-pressure mobile phase experiment process.
Example 2
FIG. 2a is a schematic view showing the structure of an integrated chromatography probe manufactured in stainless steel for improving the strength and pressure resistance of the system in example 2; the chromatographic column and the flow channel are made of thinner quartz capillary with the inner diameter of 50 microns instead. The sample chip 10 is formed with a depression 16.
Example 2 the specific method of use of the device is as follows: (1) Fixing the sample chip 10 in the pressure moving device 11 having the groove; (2) Starting the air pump, and moving the sample chip carried by the pressure moving device to the sampling port 3 of the integrated chromatographic analysis probe to ensure that the sampling port is attached to and sealed with the sample bearing surface of the sample chip; (3) Keeping the sealing of the integrated chromatographic analysis probe and the sampling port of the integrated chromatographic analysis probe, starting a liquid chromatographic pump of a fluid driving device 14, continuously injecting a chromatographic mobile phase into the integrated chromatographic analysis probe along a mobile phase channel of the integrated chromatographic analysis probe, enabling the chromatographic mobile phase to flow through a mobile phase liquid inlet gap in the channel of the chromatographic column to reach the sampling port, and carrying a sample 12 into the chromatographic column; (3) Adjusting the ratio of the mobile phase delivered by the liquid chromatography pump of the fluid driving device, wherein the mobile phase flows through the sample to be separated on the chromatographic column; (4) Starting an electrospray mass spectrometer detection device, ionizing a sample which is separated on a chromatographic column and reaches an outlet 9 of the chromatographic column under the action of an electric field, and detecting the sample in the mass spectrometer detection device; (5) And (4) repeating the steps (1) to (4) to finish sampling, sample introduction and analysis of samples in different sample chips.
By using the chromatographic analysis device and the method of using the same of embodiment 2, the pressure of the mobile phase of the injection device is gradually increased to 120 mpa in one minute, and then the pressure of the mobile phase of the device is gradually decreased to 0 mpa in one minute, and no obvious leakage phenomenon of the mobile phase is observed near the sampling port, the mobile phase channel fixing interface and the chromatographic column fixing interface, so that the device is proved to maintain good sealing stability in the short-time ultrahigh-pressure mobile phase experiment process, and the system has the ultimate pressure-resistant capacity of at least 120 mpa of the pressure of the mobile phase.
Fig. 2b is a schematic diagram of various sample ports and sample-on-chip sample-carrying structures that can be used in embodiments 2 and 4. Wherein, the configuration of b1 in fig. 2b is that the whole probe is a T-shaped structure, the sampling port 3 is an inverted cone, the pit 16 on the sample chip 10 is a cylinder, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;
the configuration of b2 in fig. 2b is that the whole probe is in a T-shaped structure, the probe sampling port 3 is in a circular tube shape identical to the chromatographic column channel 5, the pit 16 on the sample chip 10 is in a cylindrical shape, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;
the configuration of b3 in fig. 2b is that the whole probe is a T-shaped structure, the probe sampling port 3 has a configuration with a minimum dead volume, the chromatographic column channel 5 is a tapered cone, the pit 16 on the sample chip 10 is a hemisphere, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;
in the configuration b4 in fig. 2b, the whole probe is a T-shaped structure, the sampling port 3 of the probe is an inverted cone, the pit 16 on the sample chip 10 is a cylinder, and the inlet end of the capillary chromatographic column 4 is lower than the upper surface of the sample chip 10, i.e. the capillary chromatographic column 4 is inserted into the pit 16 on the sample chip 10 to reduce the dead volume of sample injection;
in the configuration of b5 in fig. 2b, the whole probe is in a T-shaped structure, the sampling port 3 of the probe is in an inverted cone shape, the pit 16 is not processed on the sample chip 10, the surface of the sample chip 10 is directly utilized to bear a sample, and the inlet end of the capillary chromatographic column 4 is slightly higher than the upper surface of the sample chip 10;
the configuration of b6 in fig. 2b is that the whole probe is in a T-shaped structure, the sampling port 3 of the probe is in an inverted cone shape, the pit 16 on the sample chip 10 is in a cone shape, and the inlet end of the capillary chromatographic column 4 is flush with the upper surface of the sample chip 10;
the configuration of b7 in fig. 2b is that the probe is a V-shaped structure as a whole, the probe sampling port 3 is an inverted cone, the pit 16 on the sample chip 10 is a cylinder, the capillary chromatographic column 4 is inclined, and the inlet end of the capillary chromatographic column is basically flush with the upper surface of the sample chip 10;
in b1, b2, b3, b4, b5, b6, b7 of FIG. 2b, the diameter or side length of the sampling port 3 of the probe is larger than that of the pit 16 of the sample chip 10.
In the configuration of b8 in fig. 2b, the whole probe is in a V-shaped structure, the probe sampling port 3 is in a V-shaped channel configuration, the pit 16 on the sample chip 10 is in a cylindrical shape, the side length of the probe sampling port 3 is equal to the diameter of the pit 16 on the sample chip 10, the capillary chromatographic column 4 is obliquely arranged, and the inlet end of the capillary chromatographic column is far away from the upper surface of the sample chip 10;
the configuration of b9 in fig. 2b is that the whole probe is a V-shaped structure, the probe sampling port 3 is a V-shaped channel configuration, the pit 16 on the sample chip 10 is cylindrical, the diameter of the pit 16 is larger than the side length of the sampling port 3, the capillary chromatographic column 4 is inclined, and the inlet end of the capillary chromatographic column is far away from the upper surface of the sample chip 10.
Fig. 3 is a liquid chromatography-electrospray mass spectrometry result of a mixed solution sample containing four standard peptide fragments in a gradient elution mode, which is realized by using the chromatography apparatus and the using method thereof in example 2.
Specifically, the mixed solution sample of the four standard peptide fragments contains 10 ng/muL of each of the four peptide fragments, the volume of a sample droplet is 200 nanoliters, six sampling times are carried out in parallel, and used chromatographic mobile phases are respectively as follows: (1) mobile phase A: an aqueous solution containing 0.01% formic acid (phase a); (2) mobile phase B: acetonitrile solution containing 0.01% formic acid (phase B). The sample was taken at a mobile phase ratio of 100% mobile phase a for 4.5 minutes and combined in an elution gradient. The overall chromatographic mobile phase gradient was: 0-4.5 minutes, 0% mobile phase B (the remaining 100% is mobile phase a); from 4.5 to 9 minutes, from 0 to 50% of mobile phase B (the remaining 100 to 50% being mobile phase A); from 9 to 10 minutes, from 50 to 65% of mobile phase B (the remaining 50 to 35% being mobile phase A); 10-14 minutes, 65% mobile phase B (the remaining 35% mobile phase a); 14 th to 14.1 th minutes, 65 to 0% of mobile phase B (the remaining 35 to 100% of mobile phase A); from 14.1 to 15 minutes, 0% of mobile phase B (the remaining 100% being mobile phase a); the electrospray flow rate in the mass spectrometer detection device was 1.9 microliters per minute and the spray voltage was 2.2 kilovolts.
The six times of separation analysis of the mixed sample by the method in the embodiment shows that the relative standard deviations of the retention times of the four peptide fragments are respectively 0.5%, 0.6%, 0.8% and 0.3%, and the relative standard deviations of the peak areas of the four peptide fragments are respectively 4.9%, 9.7%, 6.0% and 8.7%, which proves that the device has good stability and reproducibility when analyzing the simple mixed sample.
Liquid chromatography-electrospray mass spectrometry of trace HeLa protein digests was achieved using the chromatography apparatus of example 2 and its method of use.
Specifically, the concentration of the used HeLa protein digest solution sample is 1 ng/mul, the sample with the volume of 200 nanoliters is accurately added into a pit on the sample chip, five parallel sample injection analyses are continuously carried out, and the control group is pure water. The analysis time was 40 minutes, and the chromatographic mobile phases used were: mobile phase A: an aqueous solution containing 0.01% formic acid (phase a); mobile phase B: 20% water-80% acetonitrile solution containing 0.01% formic acid (phase B). The electrospray flow rate in the mass spectrometer detection device was 100 nanoliters per minute and the spray voltage was 1.75 kv.
Five times of analysis of the HeLa protein digest samples by using the method in the embodiment averagely identifies 3909 peptide fragments and 866 protein groups, wherein 620 proteins are detected in the five continuous groups of analysis and account for 50.4 percent of the total identification number; by way of comparison, an average of 3481 peptides and 842 proteomes were identified by a commercial autosampler in five replicates. The results show that the proteome identified by the method of this example is slightly higher than the auto-sampler, and the time consumed for analysis is only half of that of the commercial auto-sampler, demonstrating that the device is suitable for analysis of a small amount of complex mixed sample and has advantages in throughput.
Example 3
The integrated chromatographic analysis probe and the use method in the preferred embodiment 2 are utilized to realize the combination of the sample chip in-situ 293 single cell pretreatment and in-situ liquid chromatography-electrospray mass spectrometry separation analysis.
Specifically, six single 293 cells are sequentially sucked and added into a pit of a sample chip array, and a cell lysis reagent RapidGest, a protein reduction reagent TCEP, a protein alkylation reagent IAA, a proteolysis reagent Trypsin and Lys-C are sequentially added into the pit to perform pretreatment steps such as cell disruption, proteolysis and the like. The sample chip is then removed and transferred to a pressure driven device for analysis. The analysis time was 40 minutes.
Six single-cell analyses performed by the method in this example identified 1508 peptide fragments at most, 256 proteomes, 3140 peptide fragments in total, and 418 proteomes. The group of proteins identified together in all groups accounted for 14.4% of the total. The proving device can analyze the biological samples at single cell level, and has the potential of single cell proteomics research.
Example 4
FIG. 4 is a schematic representation of the integrated chromatography probe processed in the Y-configuration of example 4. The system consists of an integrated chromatographic analysis probe body 1 made of hard materials and provided with a Y-shaped configuration, a pressure moving device 11 used for fixing a sample chip 10 and providing high extrusion force, a fluid driving device 14 (a liquid chromatographic pump) and a detection device 13 (an electrospray mass spectrometer). The arrows in FIG. 4 show the direction of flow of the chromatographic mobile phase. The rest of the numbering names are the same as those in FIGS. 1 and 2, see example 1 and example 2.
When the Y-shaped integrated chromatography analysis probe body is processed, the CNC technology is utilized to process, and a chromatographic column channel and a mobile phase channel in the Y-shaped integrated chromatography analysis probe body are in a Y-shaped structure; the outlet of the capillary fixing interface for fixing the chromatographic column is provided with a cylindrical chromatographic column channel 5 which is used for installing the chromatographic column 4 and has the diameter of 400 microns and the length of 5 millimeters and is communicated with the sampling port. The adoption of a shorter chromatographic column channel is beneficial to reducing the dead volume of the device and reducing the zone broadening and the dilution effect generated after a sample enters a sampling port and enters the interior of the chromatographic column. When the integrated chromatographic analysis probe is used, the assembled Y-type integrated chromatographic analysis probe is inclined, the probe is fixed after the sampling port is vertically downward, and the axis of the sampling port is aligned with the axis of the sample chip.
Example 4 the specific method of use of the device is as follows: (1) Fixing the sample chip in a pressure driving device with a groove; (2) Starting the air pump, wherein the pressure driving device carries the sample chip to move towards the sampling port of the integrated chromatographic analysis probe, so that the sampling port is attached to and sealed with the sample bearing surface of the sample chip; (3) Keeping the sealing between the sampling port of the integrated chromatographic analysis probe and the sample chip of the integrated chromatographic analysis probe, starting a liquid chromatographic pump of a fluid driving device, continuously injecting chromatographic mobile phase into the integrated chromatographic analysis probe along the mobile phase channel of the integrated chromatographic analysis probe, flowing the chromatographic mobile phase through the chromatographic column channel to reach the sampling port, and carrying a sample into the chromatographic column; (3) Adjusting the ratio of the mobile phase delivered by the liquid chromatography pump of the fluid driving device, wherein the mobile phase flows through the sample to be separated on the chromatographic column; (4) Starting an electrospray mass spectrometer detection device, ionizing a sample which is separated on a chromatographic column and reaches an outlet of the chromatographic column under the action of an electric field, and detecting the sample in the mass spectrometer detection device; (5) And (4) repeating the steps (1) to (4) to finish sampling, sample introduction and analysis of samples in different sample chips.
Claims (11)
1. An integrated chromatography probe assembly, comprising:
the probe body is provided with a mobile phase channel and a chromatographic column channel, and one end of the chromatographic column channel is provided with a sampling port;
the mobile phase capillary tube is arranged on the probe body and communicated with the mobile phase channel;
the chromatographic column is detachably fixed in the chromatographic column channel, and the inlet of the chromatographic column corresponds to the sampling port;
a mobile phase liquid inlet gap is reserved between the chromatographic column channel and the outer wall of the chromatographic column, and the gap is communicated with the outlet of the mobile phase channel and the sampling port at the same time.
2. The integrated chromatography probe assembly according to claim 1, wherein the mobile phase channel and the chromatographic column channel are provided with fixing interfaces at one end, and fixing assemblies respectively used for fixing the mobile phase capillary tube and the chromatographic column to the corresponding fixing interfaces.
3. The integrated chromatography probe assembly of claim 1, further comprising a sample chip for carrying a sample and a movement device for controlling the displacement of the sample chip.
4. The integrated chromatography probe assembly of claim 3, wherein the hardness of the probe body material corresponding to the sampling port is greater than or equal to the hardness of the sample chip material.
5. The integrated chromatography probe assembly of claim 4, wherein the sample chip is made of a material having a hardness less than that of the probe body material corresponding to the sampling port and a relative elasticity.
6. The integrated chromatography probe assembly of claim 3, wherein the outer surface of the sample port, and the portion of the sample chip that contacts the outer surface of the sample port, is polished or otherwise planarized.
7. The integrated chromatography probe assembly of claim 3, wherein the sample chip is formed with one or more micro-sample-bearing wells.
8. A chromatography apparatus comprising the integrated chromatography probe unit according to any one of claims 1 to 7, a fluid driving device, and a detection device.
9. A method for performing chromatography using the chromatography apparatus according to claim 8, comprising:
(1) Moving a sample chip carrying a sample to a target position, aligning a sampling port of the integrated chromatographic analysis probe assembly to the sample position, and enabling the outer surface of the sampling port to be in contact with the sample chip and form extrusion to realize sealing;
(2) A fluid driving device is used for leading a mobile phase into a mobile phase liquid inlet gap of a chromatographic column channel through a mobile phase capillary and a mobile phase channel, and then the mobile phase enters a chromatographic column by carrying a sample on a sample chip to a sampling port;
(3) Driven by the subsequent mobile phase, the sample component entering the chromatographic column is separated in the chromatographic column and flows out of the chromatographic column outlet to enter a detection device for detection.
10. The method for performing chromatography with the integrated chromatography probe assembly of claim 9, wherein in the step (1), the inlet of the chromatography column is lower than the sealing surface of the sample chip, i.e., the inlet end of the chromatography column is inserted into the pit of the sample chip.
11. The method for chromatography by the integrated chromatography probe assembly according to claim 9, wherein the gas pressure generated by the sample chip moving device is used to drive the sample chip and the sampling port of the integrated chromatography probe assembly to be sealed, or further comprising an elastic member for sealing the sample chip and the sampling port of the integrated chromatography probe assembly.
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| CN117517696A (en) * | 2023-12-29 | 2024-02-06 | 杭州谱聚医疗科技有限公司 | System and sampling method for obtaining samples in situ on line |
| CN117517696B (en) * | 2023-12-29 | 2024-03-29 | 杭州谱聚医疗科技有限公司 | System and sampling method for obtaining samples in situ on line |
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