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Method for packing separation column and nozzle needle integrated capillary column and manufacturing stopper

A technology of capillary column and manufacturing method, which is applied in the fields of chromatography and mass spectrometry, can solve the problems of capillary separation column filler leakage, cumbersome manufacturing steps, and low sample load, and achieve high column efficiency, simple filling process, and low manufacturing cost. Effect

Inactive Publication Date: 2010-03-10
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a disadvantage of this capillary column is that when there is no mobile phase in the capillary column, the packing of the "arch bridge" plug formed at the conical mouth of the capillary will be rearranged and lost, resulting in leakage of the packing in the capillary separation column
Some researchers have integrated silica gel monolithic columns and polystyrene monolithic columns with electrospray nozzles to form an integrated capillary column, which has achieved better separation effects. However, the manufacturing steps of this integrated capillary column are very cumbersome. And due to the inherently low sample loading of monolithic columns, their application is limited
[0004] In summary, in order to ensure that the capillary separation column has sufficient sample loading capacity, a capillary separation column filled with chromatographic packing should be used. In order to minimize the extra-column band broadening, a capillary separation column integrated with an electrospray nozzle should be used. However, the current problem is that there is no very effective capillary column packing and plugging method for the integration of separation column and electrospray needle

Method used

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  • Method for packing separation column and nozzle needle integrated capillary column and manufacturing stopper
  • Method for packing separation column and nozzle needle integrated capillary column and manufacturing stopper
  • Method for packing separation column and nozzle needle integrated capillary column and manufacturing stopper

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Packing of the capillary column integrated with the separation column and the electrospray nozzle: use 5mL centrifuge tube 15 to prepare 2mL-5mg / mL-5μm C 18 Reversed-phase filler-methanol homogenate, with magnet 17 placed inside; put the centrifuge tube into the homogenization tank 18, and the homogenization tank is placed on the magnetic stirrer 19, and the speed of 200r / min is maintained; the inner diameter is 75 μm, the tapered 15μm inner diameter, 12cm length capillary column 12 presses figure 1 Insert it into the homogenate solution 16 as shown, and seal the homogenate tank with the sealing screw 13; open the nitrogen pressure reducing valve, and adjust the nitrogen pressure to 0.1MPa; under the nitrogen pressure, the filler slowly moves toward the tapered mouth of the capillary column for 2 hours Finally, the filler is filled with the capillary column. During this process, gradually increase the nitrogen pressure to 1MPa; remove the capillary column from the homog...

Embodiment 2

[0024] Production of a capillary plunger integrated with a separation column and a spray needle: Put a stainless steel tube with a length of 3 cm, an inner diameter of 0.5 mm, and an outer diameter of 1.6 mm on the capillary column filled in Example 1, and use a micro tee 33 to seal the capillary column 34 is connected to the liquid chromatography pump, and one end of the micro-tee is connected to a shunt capillary with an inner diameter of 250 μm and 50 cm; the mobile phase of the liquid chromatography pump is kept at 0.01 mL / min; Sinter the packing at the conical port of the capillary to form a sintered plug with a length of 0.25 mm; reduce the flow rate of the mobile phase to zero, and remove the capillary column.

Embodiment 3

[0026]The capillary column and stopper integrated with the separation column and spray needle prepared according to implementation 1 and 2 are used for actual sample analysis, and the results are as follows Figure 5 Show. As a comparison, under the same conditions, the same sample was analyzed using a self-assembled plug capillary column, the results are as follows Figure 6 Show. Comparing the two figures, it can be seen that the sintered plug capillary column with integrated separation column and injection needle prepared by the present invention has better separation effect.

[0027] Chromatographic conditions: mobile phase A, 0.1% formic acid-2% acetonitrile, mobile phase B, 0.1% formic acid-80% acetonitrile; within 90min, mobile phase B increases linearly from 5% to 50%, and within 10min, mobile phase B increases linearly by 100% %, flow rate 200nL / min. Mass spectrometry conditions: m / z=400-1600 full scan, select 6 strongest peaks for secondary scan after a full scan ...

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Abstract

The invention relates to a method for packing a separation column and a nozzle needle integrated capillary column and manufacturing a stopper, which comprises the following steps: preparing a chromatography packing into a homogenate with a certain concentration; reversely pressing the homogenate into the capillary column to pack by nitrogen under a certain pressure, wherein one end of the capillary column is a tapered taper; sintering the chromatography packing at the mouth of the taper to form the stopper; and during the process of sintering, using a stainless steel tube to control the lengthof the stopper and protect the chromatography packing at the back of the stopper. The method for packing the separation column and the nozzle needle integrated capillary column and manufacturing thestopper is simple and fast to operate without special equipment and high in success ratio. The capillary column packed according to the method has high separation efficiency and good separation reproducibility; and the stopper manufactured according to the invention has small volume, no sample adsorption and no post-column effect. The method for packing the separation column and the nozzle needleintegrated capillary column and manufacturing the stopper has very good application value in the fields of protein sciences, micro environments and biological sample analyses based on capillary liquidchromatography-electrospray ionization mass spectrometry technology.

Description

technical field [0001] The invention relates to a capillary column packing and stopper manufacturing method in which a separation column and a spray needle are integrated, and belongs to the field of chromatography and mass spectrometry analysis. Background technique [0002] In recent years, with the rapid development of proteomics research, microcapillary liquid chromatography-electrospray ionization mass spectrometry (nano-LC-ESI-MS) has been widely used. Microcapillary liquid chromatography uses a separation column with an inner diameter of 20 to 300 microns, and the corresponding mobile phase flow rate is generally tens to hundreds of nanoliters per minute. The components eluted from the capillary separation column are ionized by the electrospray nozzle (conical tube with an inner diameter of more than ten microns), and then introduced into the mass spectrometer through an electric field. For liquid chromatography separation columns (including capillary liquid chromato...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/56G01N30/96
Inventor 钱小红谭峰张养军王京兰蔡耘
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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