A method of manufacturing a zero dead volume connector for a compensating chromatographic microcolumn

Abstract

The application relates to a manufacturing method of a compensating type chromatographic micro-column zero-dead-volume connector and relates to the technical field of micro-fluid chromatography. A porous elastic material is used as a filler, the porous elastic filler is cut into a disc shape by a puncher, the cut disc is packaged in the connector by the puncher, and the manufacturing of the compensating type chromatographic micro-column zero-dead-volume connector is completed. The application is widely applicable, and different types of porous fillers can be packaged in different types of connectors. Meanwhile, the method is simple to operate, the compensating type chromatographic micro-column zero-dead-volume connector can effectively reduce the dead volume due to the fact that the porous elastic filler fills the possible gaps in the connector, and the peak shape is improved and the column efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chromatography technology, and in particular to a method for manufacturing a compensating chromatographic microcolumn zero dead volume connector. Background Technology

[0002] Miniaturization is a significant trend in the development of liquid chromatography separation technology, encompassing the reduction of instrument size, particle size of separation materials, and column diameter. Reducing the column diameter decreases sample and mobile phase consumption, minimizes dilution effects, and provides higher quality sensitivity, making it particularly suitable for proteomics research. Due to the low flow rate and small column volume, extra-column effects become quite significant. These effects primarily originate from voids in the tubing and defects at connections. While extra-column effects caused by voids in the tubing can be mitigated by reducing the capillary diameter, defects at connections are easily overlooked.

[0003] Misconnections at various connector types are widespread in flow path systems, and in different types of misconnections, capillary gaps can lead to severe extra-column effects. Currently, various types of commercial connectors are available, including low dead volume and zero dead volume types. However, since the ends of chromatographic microcolumns cannot be perfectly truncated, imperfections are likely to occur at the connection points. These imperfections can cause severe extra-column effects, especially when multiple capillary tubes need to be connected in series, where the extra-column effects are further amplified. Therefore, current commercial connectors have not completely solved this problem and still introduce additional dead volume. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned problems with existing connectors by providing a simple and rapid manufacturing method for a compensating chromatographic microcolumn zero-dead-volume connector that can effectively reduce dead volume, suppress peak broadening, and improve column efficiency. By selecting a porous elastic filler and cleverly encapsulating it inside the connector, a zero-dead-volume or extremely low-dead-volume connection effect is achieved, thereby significantly improving the accuracy and efficiency of chromatographic analysis. This invention can enable the manufacturing of different types of compensating chromatographic microcolumn zero-dead-volume connectors.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] A method for manufacturing a zero dead volume connector for a compensating chromatographic microcolumn includes the following steps:

[0007] 1) Select a porous elastic filler: Select a suitable porous elastic filler, including hydrogel, porous silica gel, polytetrafluoroethylene membrane or glass fiber membrane;

[0008] 2) Cutting the filling: Use a hole punch to cut the porous elastic filling into round pieces;

[0009] 3) Encapsulation filler: The cut disc is encapsulated inside the connector again using a punch, completing the manufacturing of the compensated chromatographic microcolumn zero dead volume connector.

[0010] In step 1), the type of hydrogel can be a series of substances belonging to the category of hydrogels, such as polyacrylamide hydrogel, agarose hydrogel, polyethylene glycol hydrogel, methylcellulose hydrogel, hyaluronic acid hydrogel, chitosan hydrogel, alginate hydrogel, polyvinyl alcohol hydrogel, and chitin hydrogel.

[0011] In step 1), the pore size of the hydrogel is 10 to 50 μm and the thickness can be precisely controlled, with a thickness range of 500 to 2000 μm.

[0012] In step 1), the porous elastic filler is porous and has good permeability, so it will not bring additional pressure to the system; the porous elastic filler has good elasticity and deformation ability, which can fill gaps and reduce dead volume.

[0013] In step 2), the punch has a hollow body and a punch head with a sharp edge inside. It has an ejection device that ejects the sample from the punch head, making it suitable for sampling elastic objects.

[0014] In step 2), the punch specifications can be 0.75 mm, 1.5 mm, 3.0 mm and 6 mm.

[0015] In step 2), the diameter of the porous elastic filler disc is completely consistent with the specifications of the punch, which can be 0.75 mm, 1.5 mm, 3.0 mm and 6 mm.

[0016] In step 2), the connector can be a common 1 / 16-inch connector, or a 1 / 4, 1 / 8, or 1 / 32-inch connector.

[0017] In step 3), the connector may be, but is not limited to, zero dead volume type, low dead volume type, stainless steel type, PEEK type, T-type connector, Y-type connector, two-way, three-way, four-way, etc.

[0018] In step 3), the cut-out circular piece is encapsulated inside the connector. Different types of porous elastic fillers can be encapsulated in different types of connectors to make different types of compensated chromatographic microcolumn zero dead volume connectors.

[0019] In step 3), the compensated chromatographic microcolumn zero dead volume connector has good packaging quality. The porous elastic filler can fill the gaps when it deforms, and there will be no leakage.

[0020] In step 3), the inner diameter of the chromatographic microcolumn may be, but is not limited to, 25–150 μm.

[0021] Compared with the prior art, the present invention has the following outstanding technical effects and advantages:

[0022] This invention provides a method for manufacturing a compensating chromatographic microcolumn zero-dead-volume connector. The method utilizes a punch to encapsulate the packing material inside the connector, offering a simple and rapid operation applicable to various types of connectors. Due to the material's porosity, the compensating chromatographic microcolumn zero-dead-volume connector exhibits excellent permeability. Furthermore, the material's good elasticity effectively fills gaps at the connector's connection points, reducing dead volume and thus suppressing extra-column effects and improving column efficiency. This method can easily manufacture different types of compensating chromatographic microcolumn zero-dead-volume connectors. The resulting connectors possess advantages such as high permeability, low back pressure, and effective reduction of dead volume, making them suitable for applications in analytical chemistry, bioanalysis, pharmaceutical analysis, metabolomics, and proteomics. This invention enables the manufacture of different types of compensating chromatographic microcolumn zero-dead-volume connectors, which offer advantages such as high permeability, extremely low dead volume, optimized peak shape, and improved column efficiency. Attached Figure Description

[0023] Figure 1 A schematic diagram of the manufacturing method of a compensating chromatographic microcolumn zero dead volume connector.

[0024] Figure 2 This is an image showing the effect of a zero dead volume connector for a compensating chromatography microcolumn.

[0025] Figure 3 This is a comparison chart of column efficiency for separating benzene series compounds.

[0026] The components in the diagram are labeled as follows: 1: Hole punch. 2: Porous elastic filler. 3: Small round pieces cut out using the hole punch. 4: Connector. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and several optional embodiments. It should be noted that the present invention is not limited to the following embodiments. Any technical features and implementation schemes in the following embodiments are one or more of a variety of optional technical features and implementation schemes. For the sake of simplicity, it is impossible to exhaustively list all alternative technical features and implementation schemes included in the present invention. Therefore, those skilled in the art should understand that any technical features and implementation schemes within this embodiment do not limit the scope of protection of the present invention, which includes all alternative technical features and implementation schemes adopted by those skilled in the art without creative effort. Where specific techniques and conditions are not specified in the embodiments, they are performed according to the techniques and conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0028] like Figure 1 This invention uses a porous elastic material as a filler, then cuts the porous elastic filler into circular pieces using a punch, and finally encapsulates the cut circular pieces inside a connector using another punch, thus completing the manufacturing of a compensating chromatographic microcolumn zero-dead-volume connector. This method has a wide range of applications, allowing different types of porous filler materials to be encapsulated in different types of connectors. Furthermore, this method is simple to operate. Because the porous elastic filler fills any gaps that may exist in the connector, the compensating chromatographic microcolumn zero-dead-volume connector can effectively reduce dead volume, thereby improving peak shape and column efficiency.

[0029] Example 1:

[0030] Agarose hydrogel was selected as the porous elastic packing material, with a pore size of 10 μm and a thickness of 500 μm. The hydrogel was cut into discs using a punch, and then these discs were encapsulated inside zero-dead-volume T-connectors to create a compensated chromatography microcolumn zero-dead-volume connector. The inner diameter of the chromatography microcolumn was 50 μm. A Harris Uni-Core punch from Delta Microscopies, France, was used.

[0031] Example 2:

[0032] Polyethylene glycol hydrogel was selected as the porous elastic packing material, with a pore size of 20 μm and a thickness of 1000 μm. The hydrogel was cut into discs using a punch, and then these discs were encapsulated inside a low dead volume, Y-type connector to create a compensated chromatography microcolumn zero dead volume connector. The inner diameter of the chromatography microcolumn was 50 μm.

[0033] Example 3:

[0034] Methylcellulose hydrogel was selected as the porous elastic filler, with a pore size of 15 μm and a thickness of 800 μm. The hydrogel was cut into circular pieces using a punch, and then these pieces were encapsulated inside a PEEK connector to create a zero-dead-volume connector for the chromatographic microcolumn. The inner diameter of the chromatographic microcolumn was 25 μm.

[0035] Examples 4-9:

[0036] The method is similar to that in Example 1, and the specific parameters are shown in Table 1.

[0037] Table 1

[0038]

[0039] Example 10: Fabrication and Performance Characterization of a Compensating Chromatography Microcolumn Zero Dead Volume Connector

[0040] A 30% acrylamide solution containing 29% (w / v) acrylamide and 1% (w / v) methylenebisacrylamide, 1.5M Tris-HCl (pH=8.7), and 10% ammonium persulfate (ASP) was prepared. The 30% acrylamide solution, 1.5M Tris-HCl, and ultrapure water were mixed. Under ice bath conditions, 10% ASP and tetramethylethylenediamine (TEMED) were added to prepare a reaction solution. This reaction solution was injected between two glass slides (with a 1000 μm gap) and placed in a -20°C refrigerator for 12 h to obtain a porous elastic polyacrylamide hydrogel membrane with a thickness of 1000 μm and a pore size of 50 μm. Polyacrylamide hydrogel membrane was used as a filler. The membrane was then cut into circular pieces using a 1.6 mm punch. The circular pieces were then encapsulated in stainless steel zero dead volume 2-way, 3-way (T-type and Y-type), and 4-way connectors; stainless steel low dead volume 2-way, 3-way (T-type and Y-type), and 4-way connectors; PEEK zero dead volume 2-way, 3-way (T-type and Y-type), and 4-way connectors; and PEEK low dead volume 2-way, 3-way (T-type and Y-type), and 4-way connectors, resulting in different types of compensating chromatographic microcolumn zero dead volume connectors. The inner diameter of the chromatographic microcolumn was 25 μm.

[0041] A glass fiber membrane with a pore size of 1 μm was selected as the porous elastic filler. The glass fiber membrane was then cut into circular pieces using a 1.6 mm punch. Finally, the cut circular pieces were encapsulated in stainless steel zero dead volume two-way, three-way (T-type and Y-type), and four-way connectors; stainless steel low dead volume two-way, three-way (T-type and Y-type), and four-way connectors; PEEK zero dead volume two-way, three-way (T-type and Y-type), and four-way connectors; and PEEK low dead volume two-way, three-way (T-type and Y-type), and four-way connectors, to obtain different types of compensating chromatographic microcolumn zero dead volume connectors. The inner diameter of the chromatographic microcolumn was 75 μm.

[0042] A polytetrafluoroethylene (PTFE) membrane with a pore size of 1 μm was selected as the porous elastic filler. The PTFE membrane was then cut into circular pieces using a 1.6 mm punch. Finally, the cut circular pieces were encapsulated in stainless steel zero-dead-volume two-way, three-way (T-type and Y-type), and four-way connectors; stainless steel low-dead-volume two-way, three-way (T-type and Y-type), and four-way connectors; PEEK zero-dead-volume two-way, three-way (T-type and Y-type), and four-way connectors; and PEEK low-dead-volume two-way, three-way (T-type and Y-type), and four-way connectors, to obtain different types of compensated chromatographic microcolumn zero-dead-volume connectors. The inner diameter of the chromatographic microcolumn was 100 μm.

[0043] A 25 μm inner diameter, 20 cm long empty capillary was used as a chromatographic microcolumn, eluted with 1 mg / ml thiourea as the sample, and eluted with 60% ACN mobile phase isotropically. Two more 25 μm inner diameter, 10 cm long empty capillary tubes were connected via a zero dead volume connector, eluted with 1 mg / ml thiourea as the sample, and eluted with 60% ACN mobile phase isotropically. Finally, two more 25 μm inner diameter, 10 cm long empty capillary tubes were connected via a compensated zero dead volume connector, eluted with 1 mg / ml thiourea as the sample, and eluted with 60% ACN mobile phase isotropically. The results are as follows: Figure 2 As shown, the peak broadening effect is minimal when no connector is used. When a zero-dead-volume connector is used, the peak broadening effect becomes significantly more severe due to the added dead volume. However, when the novel compensated zero-dead-volume connector of this invention is used, the peak broadening effect is significantly reduced. The results demonstrate that this novel compensated zero-dead-volume connector can reduce dead volume and suppress peak broadening.

[0044] Example 11: Compensating Chromatographic Microcolumn Zero Dead Volume Connector for the Separation and Effect Evaluation of Benzene Series Compounds

[0045] Six HPLC microcolumns with an inner diameter of 75 μm and a length of 8 cm were prepared by centrifugal packing using Ultimate XB-C18 (5 μm, 300 Å) reverse-phase chromatography packing material. Two to six microcolumns were connected in series via zero-dead-volume connectors and compensated zero-dead-volume connectors, respectively. Using 1‰ benzene series standards (toluene, ethylbenzene, propylbenzene, and butylbenzene) as samples, and eluting with 60% ACN mobile phase with isocratic elution, column efficiency was calculated using butylbenzene as the standard, and the column efficiencies were compared and evaluated. The results are as follows: Figure 3As shown, when using a zero-dead-volume connector, the presence of dead volume leads to peak broadening, severely impacting column efficiency, especially when multiple microcolumns are connected in series. However, when using the novel compensating zero-dead-volume connector of this invention, column efficiency increases almost linearly with the number of microcolumns connected in series. These results demonstrate that the manufacturing method of this novel compensating zero-dead-volume connector virtually eliminates column efficiency losses caused by defects in the original connector, minimizing dead volume.

[0046] The above embodiments are merely preferred embodiments of the present invention and should not be considered as limiting the scope of the present invention. All equivalent variations and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. A method for manufacturing a compensating chromatographic microcolumn zero dead volume connector, characterized in that... Includes the following steps: 1) Selection of porous elastic filler: Select a suitable porous elastic filler, including hydrogel, porous silica gel, polytetrafluoroethylene membrane or glass fiber membrane; the porous elastic filler is porous and has good permeability, and will not bring additional pressure to the system; the porous elastic filler has good elasticity and deformation capacity to fill gaps and reduce dead volume; 2) Cutting the filling: Use a hole punch to cut the porous elastic filling into round pieces; The punch has a hollow body and a punch head with a sharp edge inside. It is equipped with an ejection device to eject the sample from the punch head, which is suitable for sampling elastic objects. 3) Encapsulation filler: The cut disc is encapsulated inside the connector again using a punch, completing the manufacturing of the compensating chromatography microcolumn zero dead volume connector. The porous elastic filler effectively fills the gaps at the connection points in the connector, reducing the dead volume, thereby suppressing extracolumn effects and improving column efficiency. The compensated chromatography microcolumn zero dead volume connector has good packaging quality. The porous elastic filler deforms to fill the gaps, preventing leakage.

2. The method for manufacturing a compensating chromatographic microcolumn zero dead volume connector as described in claim 1, characterized in that... In step 1), the type of hydrogel is polyacrylamide hydrogel, agarose hydrogel, polyethylene glycol hydrogel, methylcellulose hydrogel, hyaluronic acid hydrogel, chitosan hydrogel, alginate hydrogel, polyvinyl alcohol hydrogel, or chitin hydrogel.

3. The method for manufacturing a compensating chromatographic microcolumn zero dead volume connector as described in claim 1, characterized in that... In step 1), the pore size of the hydrogel ranges from 10 to 50 μm, and the thickness ranges from 500 to 2000 μm.

4. The method for manufacturing a compensating chromatographic microcolumn zero dead volume connector as described in claim 1, characterized in that... In step 2), the punch has a diameter of 0.75 mm, 1.5 mm, 3.0 mm or 6 mm; the diameter of the porous elastic filler disc is the same as that of the punch, which is 0.75 mm, 1.5 mm, 3.0 mm or 6 mm.

5. The method for manufacturing a compensating chromatographic microcolumn zero dead volume connector as described in claim 1, characterized in that... In step 3), the connector is a 1 / 16-inch connector, or a 1 / 4, 1 / 8, or 1 / 32-inch connector; the connector is of zero dead volume type, low dead volume type, stainless steel type, PEEK type, T-type connector, Y-type connector, two-way, three-way, or four-way type.

6. The method for manufacturing a compensating chromatographic microcolumn zero dead volume connector as described in claim 1, characterized in that... In step 3), the process of encapsulating the cut disc inside the connector involves encapsulating different types of porous elastic fillers in different types of connectors to create different types of compensated chromatographic microcolumn zero dead volume connectors.

7. The method for manufacturing a compensating chromatographic microcolumn zero dead volume connector as described in claim 1, characterized in that... In step 3), the inner diameter of the chromatographic microcolumn is 25–150 μm.

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