Compensation type chromatographic microcolumn zero dead volume connector manufacturing method

Abstract

The invention discloses a manufacturing method of a compensation type chromatographic micro-column zero dead volume connector, and relates to the technical field of microfluidic chromatography. A porous elastic material is used as a filler, then a puncher is used for cutting the porous elastic filler into a circular sheet shape, finally, the puncher is used for packaging the cut circular sheet in the connector, and manufacturing of the compensation type chromatographic microcolumn zero dead volume connector is completed. And the application range is very wide, and different types of porous filling materials can be packaged in different types of connectors. Meanwhile, the method is simple to operate, and the gap possibly existing in the connector is filled with the porous elastic filler, so that the compensation type chromatographic microcolumn zero-dead-volume connector can effectively reduce the dead volume, thereby improving the peak shape and improving the column efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of microfluidic chromatography, and in particular to a method for manufacturing a compensation type chromatography microcolumn zero dead volume connector. Background Art

[0002] Miniaturization is one of the important trends in the development of liquid chromatography separation technology, including the miniaturization of instrument dimensions, the miniaturization of separation material particle size, and the miniaturization of chromatographic column inner diameter. The reduction of the inner diameter of the chromatographic column can reduce the consumption of samples and mobile phases, while reducing the dilution effect, with higher mass sensitivity, and is particularly suitable for proteomics research. Due to the low flow rate and the small volume of the chromatographic column, the extra-column effect becomes very significant. The extra-column effect mainly comes from the gaps in the pipeline and the defects at the joints. The extra-column effect caused by the gaps in the pipeline can be solved by reducing the inner diameter of the capillary, but the defects at the joints are easily overlooked.

[0003] Among various types of connectors, misconnections caused by the connection are widely present in the flow path system, and in different types of misconnections, the capillary gap will cause serious extra-column effects. At present, there are different types of commercial connectors, including low dead volume type, zero dead volume type, etc. However, since the port of the chromatographic microcolumn cannot be perfectly truncated, it is very likely that there will be defects at the connection, and the defects will cause serious extra-column effects, especially when multiple capillaries need to be connected in series, the extra-column effect will be further amplified, so the current commercial connectors have not completely solved this problem and will still bring additional dead volume. Summary of the invention

[0004] The purpose of the present invention is to address the above-mentioned problems existing in existing connectors and to provide a method for manufacturing a compensating chromatographic microcolumn zero dead volume connector that is simple and quick to operate, can effectively reduce dead volume, inhibit peak broadening, and improve column efficiency. By selecting porous elastic fillers and cleverly encapsulating them 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, and the manufacture of different types of compensating chromatographic microcolumn zero dead volume connectors can be realized.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector comprises the following steps:

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

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

[0009] 3) Encapsulating filler: Use the puncher again to encapsulate the cut disc inside the connector to complete the manufacture of the compensation type chromatographic microcolumn zero dead volume connector.

[0010] In step 1), the type of the hydrogel can be a series of substances belonging to the hydrogel category, 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 10-50 μm and the thickness can be precisely controlled, ranging from 500 to 2000 μm.

[0012] In step 1), 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 ability, can fill the gap and reduce the dead volume.

[0013] In step 2), the puncher has a hollow body, a punch head with a sharp edge, and an ejection device for ejecting the sample from the punch head, which is suitable for sampling elastic objects.

[0014] In step 2), the punch specifications may 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 may be a common 1 / 16-inch connector, or may be a 1 / 4-inch, 1 / 8-inch, or 1 / 32-inch connector.

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

[0018] In step 3), the cut disc is encapsulated inside the connector. Different types of porous elastic fillers can be encapsulated in different types of connectors to produce different types of compensation type chromatographic microcolumn zero dead volume connectors.

[0019] In step 3), the compensating chromatographic microcolumn zero dead volume connector has good packaging quality, and the porous elastic filler can fill the gaps when deformed, and no leakage occurs.

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

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

[0022] The present invention provides a method for manufacturing a compensating chromatographic microcolumn zero dead volume connector, which uses a puncher to encapsulate a filler inside the connector, is simple and fast to operate, and can be applied to different types of connectors. Because the material is porous, the compensating chromatographic microcolumn zero dead volume connector has good permeability; because the material has good elasticity, it can effectively fill the gap existing in the connection of the connector, reduce the dead volume, thereby inhibiting the extra-column effect and improving the column efficiency. This method can simply manufacture different types of compensating chromatographic microcolumn zero dead volume connectors, and the obtained compensating chromatographic microcolumn zero dead volume connector has a series of advantages such as good permeability, low back pressure, and can effectively reduce the dead volume, and can be applied to analytical chemistry, biological analysis, drug analysis, metabolomics, proteomics and other fields. The present invention can realize the manufacture of different types of compensating chromatographic microcolumn zero dead volume connectors, and the obtained compensating chromatographic microcolumn zero dead volume connector has the advantages of high permeability, extremely low dead volume, optimized peak shape, and improved column efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the manufacturing method of the compensation type chromatography microcolumn zero dead volume connector.

[0024] Figure 2 This is the effect diagram of the zero dead volume connector of the compensation type chromatography microcolumn.

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

[0026] The parts in the figure are numbered as follows: 1: puncher. 2: porous elastic filler. 3: small disc cut by the puncher. 4: connector. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and several optional embodiments. It should be noted that the present invention is not limited to the following examples. Any technical features and implementation schemes in the following examples are one or more of a variety of optional technical features and a variety of optional implementation schemes. In order to describe the need for simplicity, it is impossible to exhaustively enumerate all the alternative technical features and implementation schemes included in the present invention, so those skilled in the art should know that any technical features and implementation schemes in this embodiment do not limit the scope of protection of the present invention, and the scope of protection includes any alternative technical features and implementation schemes taken by all those skilled in the art without paying creative labor. If the specific technology and conditions are not indicated in the examples, they are carried out according to the technology and conditions described in the literature in this field or according to the product instructions. If the reagents or instruments used do not indicate the manufacturer, they are all conventional products that can be purchased commercially.

[0028] like Figure 1 The present invention uses a porous elastic material as a filler, then uses a puncher to cut the porous elastic filler into discs, and finally uses a puncher to encapsulate the cut discs inside a connector to complete the manufacture of a compensating chromatographic microcolumn zero dead volume connector. The scope of application of this method is very wide, and different types of porous filling materials can be encapsulated in different types of connectors. At the same time, this method is simple to operate. Since the porous elastic filler fills the gaps that may exist in the connector, the compensating chromatographic microcolumn zero dead volume connector can effectively reduce the dead volume, thereby improving the peak shape and improving the column efficiency.

[0029] Embodiment 1:

[0030] Agarose hydrogel is selected as a porous elastic filler with a pore size of 10 μm and a thickness of 500 μm. The hydrogel is cut into discs using a puncher, and then the cut discs are encapsulated inside a zero dead volume type, T-type connector to make a compensation type chromatographic microcolumn zero dead volume connector. The inner diameter of the chromatographic microcolumn is 50 μm. The puncher can be a Harris Uni-Core puncher produced by DeltaMicroscopies in France.

[0031] Embodiment 2:

[0032] Polyethylene glycol hydrogel was selected as the porous elastic filler with a pore size of 20 μm and a thickness of 1000 μm. The hydrogel was cut into discs using a puncher, and then the cut discs were encapsulated inside a low dead volume, Y-shaped connector to make a compensation type chromatographic microcolumn zero dead volume connector. The inner diameter of the chromatographic microcolumn is 50 μm.

[0033] Embodiment 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 discs using a puncher, and then the cut discs were encapsulated inside a PEEK connector to make a compensation type chromatographic microcolumn zero dead volume connector. The inner diameter of the chromatographic microcolumn is 25 μm.

[0035] Embodiment 4~9:

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

[0037] Table 1

[0038]

[0039] Example 10: Fabrication and effect characterization of zero dead volume connector for compensatory chromatographic microcolumn

[0040] A 30% acrylamide solution containing 29% (w / v) acrylamide and 1% methylenebisacrylamide (w / v), 1.5M Tris-HCl (PH=8.7), and 10% ammonium persulfate (ASP) were prepared. First, 30% acrylamide solution, 1.5M Tris-HCl, and ultrapure water were mixed, and 10% ASP and tetramethylethylenediamine (TEMED) were added under ice bath conditions to prepare a reaction solution, which was injected between two glass slides (the gap between the glass slides was 1000 μm) 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. The polyacrylamide hydrogel membrane was used as a filler, and then the polyacrylamide hydrogel membrane was cut into discs using a puncher with a specification of 1.6 mm. Finally, the cut discs were encapsulated in stainless steel zero dead volume two-way, three-way (T-type and Y-type), four-way, stainless steel low dead volume two-way, three-way (T-type and Y-type), four-way; PEEK zero dead volume two-way, three-way (T-type and Y-type), four-way, PEEK low dead volume two-way, three-way (T-type and Y-type), four-way, to obtain different types of compensation type chromatographic microcolumn zero dead volume connectors, and the inner diameter of the chromatographic microcolumn was 25μm.

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

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

[0043] An empty capillary with an inner diameter of 25 μm and a length of 20 cm was used as a chromatographic microcolumn, and 1 mg / ml thiourea was used as a sample and 60% ACN mobile phase was used for isocratic elution; then two empty capillaries with an inner diameter of 25 μm and a length of 10 cm were connected through a zero dead volume connector, and 1 mg / ml thiourea was used as a sample and 60% ACN mobile phase was used for isocratic elution; finally, two empty capillaries with an inner diameter of 25 μm and a length of 10 cm were connected through a compensation type zero dead volume connector, and 1 mg / ml thiourea was used as a sample and 60% ACN mobile phase was used for isocratic elution. The results are shown in the figure. Figure 2 As shown in the figure, when no connector is used for connection, the peak broadening effect is minimal. When a zero dead volume type connection is used, the peak broadening effect becomes significantly more serious due to the additional dead volume. However, when the novel compensating zero dead volume connector of the present invention is used for connection, the peak broadening effect is significantly reduced. The results show that this novel compensating zero dead volume connector can reduce dead volume and inhibit peak broadening.

[0044] Example 11: Compensation-type chromatographic microcolumn zero dead volume connector for the separation of benzene series and effect evaluation

[0045] The reversed phase chromatographic filler Ultimate XB-C18 (5 μm, 300 Å) was used to prepare 6 liquid chromatography microcolumns with an inner diameter of 75 μm and a length of 8 cm by centrifugal column filling. 2-6 chromatographic microcolumns were connected in series through zero dead volume connectors and compensation type chromatographic microcolumn zero dead volume connectors. 1‰ benzene series standards (toluene, ethylbenzene, propylbenzene, butylbenzene) were used as samples, 60% ACN mobile phase was used for isocratic elution, and butylbenzene was used as the standard for calculating the column efficiency, and the column efficiency was compared and evaluated. The results are shown in the figure. Figure 3As shown, when a zero dead volume connector is used, the presence of dead volume causes peak broadening, which seriously affects the growth of column efficiency, especially when multiple chromatographic microcolumns are connected in series. However, when the novel compensatory chromatographic microcolumn zero dead volume connector of the present invention is used, the column efficiency increases almost linearly with the number of series connections. The above results show that through the manufacturing method of this novel compensatory chromatographic microcolumn zero dead volume connector, the column efficiency loss caused by the defects of the original connector can be almost eliminated, and the dead volume can be reduced to a minimum.

[0046] The above embodiments are only preferred embodiments of the present invention and cannot be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector, characterized in that The following steps are involved: 1) Select porous elastic filler: Select appropriate porous elastic filler, including hydrogel, porous silica gel, polytetrafluoroethylene membrane or glass fiber membrane; 2) Cutting the filling material: Use a hole punch to cut the porous elastic filling material into discs; 3) Packaging filler: Use the puncher again to pack the cut disc into the connector to complete the manufacture of the compensation type chromatographic microcolumn zero dead volume connector. The porous elastic filler effectively fills the gap in the connection of the connector, reduces the dead volume, thereby inhibiting the extra-column effect and improving the column efficiency.

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

3. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 1), the pore size of the hydrogel is in the range of 10 to 50 μm, and the thickness is in the range of 500 to 2000 μm.

4. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 1), 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 ability to fill the gap and reduce the dead volume.

5. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 2), the puncher has a hollow body, a punch head with a sharp edge, and an ejection device for ejecting the sample from the punch head, which is suitable for sampling elastic objects.

6. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 2), the punch has a specification of 0.75 mm, 1.5 mm, 3.0 mm or 6 mm; the diameter of the disc of the porous elastic filler is consistent with the punch specification, which is 0.75 mm, 1.5 mm, 3.0 mm or 6 mm.

7. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 3), the connector may be a 1 / 16-inch connector, or a 1 / 4-, 1 / 8-, or 1 / 32-inch connector; the connector may be a zero dead volume type, a low dead volume type, a stainless steel type, a PEEK type, a T-shaped connector, a Y-shaped connector, a two-way connector, a three-way connector, or a four-way connector.

8. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 3), the cut disc is encapsulated inside the connector, which is to encapsulate different types of porous elastic fillers in different types of connectors to make different types of compensation type chromatographic microcolumn zero dead volume connectors.

9. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 3), the compensating chromatographic microcolumn zero dead volume connector has good packaging quality, the porous elastic filler is deformed to fill the gaps, and no leakage occurs.

10. A method for manufacturing a compensation type chromatographic microcolumn zero dead volume connector as claimed in claim 1, characterized in that In step 3), the inner diameter of the chromatographic microcolumn is 25-150 μm.

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