Manufacturing method of narrow-inner-diameter integrated spray needle capillary chromatographic column

Abstract

The invention discloses a manufacturing method of a narrow-inner-diameter integrated spray needle capillary chromatographic column, and relates to the field of capillary chromatographic column preparation. The inlet end is machined into the bevel opening, the angle of the bevel opening ranges from 10 degrees to 40 degrees, the cross section of the inlet end is remarkably increased, the acute angle is formed between the direction in which air pressure or hydraulic pressure acts on the cross section and an inner channel of the capillary tube, and therefore filler is difficult to accumulate at the inlet of the capillary tube to block the inlet. The problem that the chromatographic filler is interrupted due to the fact that the inlet end of the chromatographic filler is polished into an inclined opening through abrasive paper and the narrow-inner-diameter chromatographic column with the inner diameter of 5-50 microns can be prevented from being blocked in the filling process is solved.

Description

Technical Field

[0001] The present invention relates to the field of capillary column preparation, and more particularly to a method for manufacturing a narrow-inner-diameter integrated needle capillary column. Background Art

[0002] Proteins are the direct executors of cell functions. Qualitative and quantitative analysis of trace proteome samples such as single cells, trace body fluids, or micro-region tissues can more accurately evaluate the detailed physiological state of an organism, help doctors perform targeted personalized precision treatment, improve the success rate of disease treatment, help researchers further understand the mechanism of action of the immune system and the occurrence mechanism of tumors, and reveal the detailed differentiation mechanism at different stages during embryonic development. However, due to the extremely low protein content in the above-mentioned trace samples and the inability to amplify them, powerful analytical methods are required.

[0003] In recent years, with the continuous improvement of the sensitivity of mass spectrometry technology and the pressure resistance of liquid chromatographs, the bottom-up "shotgun method" based on liquid chromatography-mass spectrometry coupling technology has become one of the most mainstream analytical technologies for studying trace sample proteomics. Since the components of proteome samples are extremely complex and the dynamic range is extremely wide, extremely high requirements are imposed on the core component of liquid chromatography separation analysis - the liquid chromatography column. Currently, since most of the commercial chromatographic columns on the market have an inner diameter of hundreds of micrometers, they are prone to adsorbing samples and have a relatively large dead volume in the corresponding flow path, which is not suitable for separating trace proteome samples. Therefore, most researchers in this field currently use self-made capillary packed columns to achieve the separation function. The integrated needle capillary column is widely used because it does not require the production of a sieve plate and can effectively avoid the post-column dead volume caused by an external mass spectrometry needle.

[0004] Theoretically, the narrower the inner diameter of the chromatographic column, the higher the separation efficiency. However, the inner diameter of the integrated needle capillary column commonly used in the current chromatographic field is basically 50 - 100 μm. The main reasons why chromatographic columns with a narrower inner diameter have not been popularized are mainly two: one is that the integrated needle of the capillary chromatographic column is usually drawn by a laser microelectrode puller, and it is often difficult to draw a needle with an ideal inner and outer diameter suitable for generating mass spectrometry electrospray when the inner diameter of the capillary is less than 50 μm; the other is that the narrower the inner diameter of the chromatographic column, the easier it is for the chromatographic packing to block the inlet of the capillary chromatographic column during filling, thus blocking the filling of the capillary chromatographic column.

[0005] In the prior art, an integrated injection needle of a capillary is processed by a laser microelectrode puller or manual pulling, or hydrofluoric acid etching, or a combination of the above techniques. By means of a magnetic stir bar or ultrasonic technology, or a combination of the above techniques, the packing is evenly distributed and the settlement of the packing is prevented, so that the packing can be evenly and continuously filled into the capillary under air pressure or hydraulic pressure. The packing inlet end of the capillary is 90° vertical. However, the success rate of the prior art is not high when filling a narrow-inner-diameter chromatographic column with an inner diameter of 5 - 50 μm. The reason is that the packing does not exist in the form of single-particle dispersion in the solvent, and there will also be multi-particle aggregation phenomena. Therefore, when filling a narrow-inner-diameter capillary with an inner diameter of 5 - 50 μm, the packing in the form of multi-particle aggregation is likely to form a blockage at the inlet end. Moreover, the smaller the inner diameter of the capillary, the shorter the time for the blockage at the capillary inlet end to occur, resulting in a significant decrease in the filling success rate of the capillary chromatographic column or even inability to fill.

[0006] Therefore, there is an urgent need to develop a method for fabricating a narrow-inner-diameter integrated injection needle capillary chromatographic column, so that the narrow-inner-diameter integrated injection needle capillary chromatographic column can be conveniently and stably prepared. Summary of the Invention

[0007] Based on the deficiencies in the prior art, the present invention provides a method for fabricating a narrow-inner-diameter integrated injection needle capillary chromatographic column. The tail end of the capillary with an integrated injection needle is formed into an inclined mouth by techniques such as grinding or cutting, greatly increasing the success rate of chromatographic column preparation.

[0008] The technical solution of the present invention is specifically as follows:

[0009] A method for fabricating a narrow-inner-diameter integrated injection needle capillary chromatographic column includes the following steps:

[0010] (1) Provide a quartz capillary with an integrated tip formed during processing;

[0011] (2) Immerse the integrated tip of the quartz capillary in step (1) into hydrofluoric acid for etching to expand the inner diameter at the integrated tip;

[0012] (3) Process the other end of the quartz capillary etched in step (2) relative to the integrated tip to form an inclined mouth, and the angle of the inclined mouth is 10° - 40°;

[0013] (4) Disperse the chromatographic packing in a solvent to obtain a suspension, insert one end with the inclined mouth of the quartz capillary polished in step (3) into the suspension, so that the suspension enters the quartz capillary from the inclined mouth end, and the chromatographic packing accumulates in the inner cavity of the quartz capillary to obtain a chromatographic column.

[0014] Wherein, in step (1), the processing can be pulling with a laser microelectrode puller, pulling after softening the capillary by flame heating, or grinding with sandpaper.

[0015] In step (2), the mass percentage concentration of the hydrofluoric acid is 20% - 50%, and the volume is 1 μL - 100 mL;

[0016] When the integrated tip of the quartz capillary is immersed in hydrofluoric acid for etching, the immersion depth is 1 μm - 1 cm. After the inner diameter of the tip is etched to the required size, the quartz capillary is taken out of the hydrofluoric acid, and the etching is completed. The required size is 3 - 15 μm and does not exceed the inner diameter of the main body part of the quartz capillary.

[0017] Specifically, in step (2), before the integrated tip of the quartz capillary is immersed in hydrofluoric acid, the other end of the quartz capillary relative to the integrated tip is connected to an injection pump, and water is continuously pumped into the quartz capillary through the injection pump, wherein the flow rate of the pumped water is 10 - 1000 nL / min.

[0018] In the present invention, the inner diameter of the main body part of the quartz capillary is 5 - 50 μm;

[0019] In step (2), the inner diameter of the etched integrated tip is 3 - 15 μm and does not exceed the inner diameter of the main body part of the quartz capillary.

[0020] In step (3), the method for processing the beveled opening can be cutting or grinding.

[0021] In step (4), the chromatographic packing is one of the following or a combination of the following three: silica gel medium packing, soft gel medium packing, or polymer chromatographic packing, and the solvent is methanol, acetonitrile, or isopropanol. The concentration of the chromatographic packing in the suspension is 1 - 100 mg / mL; the particle size of the chromatographic packing is 0.1 - 3 μm.

[0022] In step (4), the suspension is made to enter the quartz capillary from one end of the beveled opening by applying air pressure or hydraulic pressure to the suspension.

[0023] In step (4), the suspension is stirred or ultrasonically treated during the process of making the suspension enter the quartz capillary from one end of the beveled opening.

[0024] Advantages of the present invention:

[0025] By processing the inlet end into a beveled opening, the cross-sectional area of the inlet end is significantly increased, and the direction of the air pressure or hydraulic pressure acting on the cross-sectional area forms an acute angle with the internal channel of the capillary, so that it is very difficult for the packing to accumulate and block the inlet at the capillary inlet. It solves the problem that the inlet end blockage during the filling process of a narrow inner diameter chromatographic column with an inner diameter of 5 - 50 μm can cause the interruption of the chromatographic packing when the inlet end of the chromatographic packing is ground into a beveled opening with sandpaper. Description of the Drawings

[0026] Figure 1 Figure for the start of etching the capillary tip with hydrofluoric acid.

[0027] Figure 2 Figure for the completion of etching the capillary tip with hydrofluoric acid.

[0028] Figure 3 Figure for filling the chromatographic packing into the quartz capillary.

[0029] Markings in the figure: 1 is the quartz capillary, 2 is hydrofluoric acid, 3 is a single packing particle dispersed in the chromatographic packing suspension, 4 is an agglomerated particle formed by multiple packing particles in the chromatographic packing suspension, 5 is the chromatographic column formed by the regular arrangement of the chromatographic packing inside the capillary, 6 is the solvent for dispersing the chromatographic packing, and 7 is the rotating magnetic stirrer. Detailed implementation method

[0030] The present invention provides a method for fabricating a narrow-inner-diameter integrated injection needle capillary chromatographic column, including the following steps:

[0031] (1) Provide a quartz capillary with an integrated tip formed during processing;

[0032] (2) Immerse the integrated tip of the quartz capillary in step (1) into hydrofluoric acid for etching to expand the inner diameter at the integrated tip;

[0033] (3) Process the other end of the quartz capillary etched in step (2) relative to the integrated tip to form an inclined mouth, and the angle of the inclined mouth is 10° - 40°;

[0034] (4) Disperse the chromatographic packing in a solvent to obtain a suspension, insert one end of the quartz capillary with an inclined mouth polished in step (3) into the suspension, so that the suspension enters the quartz capillary from the inclined mouth end, and the chromatographic packing accumulates in the inner cavity of the quartz capillary to obtain a chromatographic column.

[0035] Among them, in step (1), the processing can be carried out by pulling with a laser microelectrode puller, or by heating with a flame to soften the capillary and then pulling, or by polishing with sandpaper.

[0036] In the present invention, the inner diameter of the main body part of the quartz capillary can be selected to be 5 - 50 μm;

[0037] In step (2), the concentration of the hydrofluoric acid is 20% - 50% by mass percentage, and the volume is 1 μL - 100 mL.

[0038] In step (2), when the integrated tip of the quartz capillary is immersed in hydrofluoric acid for etching, the immersion depth is 1 μm - 1 cm. After the inner diameter of the tip is etched to the required size, the quartz capillary is taken out of the hydrofluoric acid, and the etching ends immediately.

[0039] Specifically, in step (2), before the integrated tip of the quartz capillary is immersed in hydrofluoric acid, the other end of the quartz capillary relative to the integrated tip is connected to an injection pump, and water is continuously pumped into the quartz capillary through the injection pump at a flow rate of 10 - 1000 nL / min.

[0040] In step (4), the chromatographic packing is one of the following or a combination of the following three: silica gel medium packing, soft gel medium packing, or polymer chromatographic packing, and the solvent is methanol, acetonitrile, or isopropanol. The concentration of the chromatographic packing in the suspension is 1 - 100 mg / mL; the particle size of the chromatographic packing is 0.1 - 3 μm.

[0041] In step (4), the suspension is made to enter the quartz capillary from the beveled end by applying air pressure or hydraulic pressure to the suspension.

[0042] In step (4), the suspension is stirred or sonicated during the process of making the suspension enter the quartz capillary from the beveled end.

[0043] The specific manufacturing steps are as follows:

[0044] Step 1: Fix the quartz capillary 1 with a tip on a three - dimensional translation stage, connect the other end of the quartz capillary 1 except the tip to an injection pump, and make the injection pump continuously pump water into the quartz capillary.

[0045] Step 2: Move the three - dimensional translation stage to insert the tip of the quartz capillary 1 1 μm - 1 cm below the liquid level of the hydrofluoric acid 2 for etching (as shown); the mass percentage concentration of the hydrofluoric acid 2 is 20% - 50%. Figure 1 shown); the mass percentage concentration of the hydrofluoric acid 2 is 20% - 50%.

[0046] Step 3: After etching to the required inner and outer diameters of the tip (as shown), the inner diameter of the integrated tip after etching is 3 - 15 μm and is not greater than the inner diameter of the main body part of the quartz capillary; move the three - dimensional translation stage to make the quartz capillary 1 leave the liquid level of the hydrofluoric acid 2, and then rinse the inside and outside of the quartz capillary 1 with water. Figure 2 shown), the inner diameter of the integrated tip after etching is 3 - 15 μm and is not greater than the inner diameter of the main body part of the quartz capillary; move the three - dimensional translation stage to make the quartz capillary 1 leave the liquid level of the hydrofluoric acid 2, and then rinse the inside and outside of the quartz capillary 1 with water.

[0047] Step 4: Grind the other end of the quartz capillary with sandpaper into a bevel as shown. Figure 3 shown.

[0048] Step 5: Insert the quartz capillary into the chromatographic packing suspension. The chromatographic packing suspension is magnetically stirred using a magnetic stirrer 7. There are dispersed single packing particles 3 and aggregated particles 4 formed by multiple packing particles in the chromatographic packing suspension. By applying air pressure to the chromatographic packing suspension, the suspension enters the quartz capillary, and the solvent 6 of the suspension flows out from the etched capillary tip, and the chromatographic packing accumulates in the capillary and is regularly arranged to form a chromatographic column 5.Figure 3 )。

[0049] The present invention uses specific capillaries to conduct tests through the above method. Capillaries with inner diameters of 30 and 20 μm in the main part of the quartz capillary are used for testing respectively. The bevel angles at the inlet end are 10°, 20°, 40°, and the unprocessed bevel (i.e., 90°) respectively. The particle size of the chromatographic packing is 1.7 μm. It is prepared into a suspension with a concentration of 30 mg / mL with isopropanol, and 5 capillaries are processed respectively by pneumatic drive and magnetic stirring (rotation speed of 500 rpm) to test their packing success rate. Here, success means that the capillary chromatographic column can be packed to 5 cm without blocking the inlet end, and failure means that the inlet end is blocked before the chromatographic column is packed to 5 cm, and the chromatographic packing cannot enter the capillary, resulting in the interruption of packing. The results are shown in Table 1 below.

[0050] Table 1

[0051]

[0052]

[0053] Judging from the results in Table 1, the inlet end is blocked before the capillary with the unprocessed bevel is packed to 5 cm, and the chromatographic packing cannot enter the capillary, resulting in the interruption of packing. For the capillaries with bevels of different angles prepared by the preparation method of the present invention, the success rate is above 60%. Especially when the bevel angle at the inlet end is 10°, the packing success rate is 100%.

Claims

1. A manufacturing method of a narrow-inner-diameter integrated injector capillary chromatographic column, characterized in that It includes the following steps: (1) Provide a quartz capillary with an integrated tip; (2) Immerse the integrated tip of the quartz capillary in step (1) into hydrofluoric acid for etching to expand the inner diameter at the integrated tip; (3) Process the other end of the quartz capillary after etching in step (2) relative to the integrated tip to form an inclined mouth, and the angle of the inclined mouth is 10°-40°; (4) Disperse the chromatographic packing in a solvent to obtain a suspension, insert one end with the inclined mouth of the quartz capillary after grinding in step (3) into the suspension, so that the suspension enters the quartz capillary from the inclined mouth end, and the chromatographic packing accumulates in the inner cavity of the quartz capillary to obtain a chromatographic column.

2. The manufacturing method of the narrow-inner-diameter integrated injector capillary chromatographic column according to claim 1, characterized in that, In step (1), the processing method of the integrated tip is any one of the following: pulling with a laser microelectrode puller, pulling after heating and softening, and grinding with sandpaper.

3. The manufacturing method of the narrow-inner-diameter integrated injector capillary chromatographic column according to claim 1, characterized in that In step (2), the mass percentage concentration of the hydrofluoric acid is 20%-50%; When the integrated tip of the quartz capillary is immersed in hydrofluoric acid for etching, the immersion depth is 1μm-1cm. After the inner diameter at the tip is etched to the required size, take out the quartz capillary from the hydrofluoric acid, and the etching ends immediately.

4. The manufacturing method of the narrow inner diameter integrated spray needle capillary chromatographic column according to claim 1, characterized in that, In step (2), before the integrated tip of the quartz capillary is immersed in hydrofluoric acid, first connect a syringe pump to the other end of the quartz capillary relative to the integrated tip, and continuously pump water into the quartz capillary through a liquid driving device.

5. The manufacturing method of the narrow inner diameter integrated injector capillary chromatographic column according to claim 1, characterized in that, The inner diameter of the main body part of the quartz capillary is 5-50μm; In step (2), the inner diameter of the integrated tip after etching is 3-15μm and does not exceed the inner diameter of the main body part of the quartz capillary.

6. The manufacturing method of the narrow inner diameter integrated injector capillary chromatographic column according to claim 1, characterized in that, In step (3), the processing method of the inclined mouth is cutting or grinding.

7. The manufacturing method of the narrow inner diameter integrated injector capillary chromatographic column according to claim 1, characterized in that, In step (4), the chromatographic packing is at least one of the following: (1) Silica gel medium packing; (2) Soft gel medium packing; (3) Polymer chromatographic packing; The solvent is methanol, acetonitrile or isopropanol.

8. The manufacturing method of the narrow inner diameter integrated injector capillary chromatographic column according to claim 1, characterized in that, In step (4), the concentration of the chromatographic packing in the suspension is 1-100mg / mL; The particle size of the chromatographic packing is 0.1-3μm.

9. The manufacturing method of the narrow inner diameter integrated spray needle capillary chromatographic column according to claim 1, characterized in that, In step (4), apply air pressure or hydraulic pressure to the suspension to make the suspension enter the quartz capillary from the inclined mouth end.

10. The manufacturing method of the narrow inner diameter integrated injector capillary chromatographic column according to claim 1, characterized in that, In step (4), stir or ultrasonically treat the suspension during the process of making the suspension enter the quartz capillary from the inclined mouth end.

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