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An organic-inorganic hybrid monolithic column and its preparation and application

A monolithic, inorganic technology, applied in the field of protein and peptide separation chromatography stationary phase, can solve the problems of chemically unstable silanol non-specific adsorption, intolerant to extreme pH conditions, small specific surface area, etc., to improve mechanical strength and chemical The effect of stability, improving chromatographic peak shape and increasing peak capacity

Active Publication Date: 2017-02-08
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The organic polymer monolithic column is simple to prepare, has a variety of types, and has a wide pH range, but the pore size distribution is uneven and the specific surface area is small, so the column efficiency is low
Silica monolithic column has high mechanical strength, uniform skeleton distribution, large specific surface area, and high column efficiency, but the preparation reproducibility is poor, it is not resistant to extreme pH conditions, and the non-specific adsorption of silanol leads to peak tailing problems
Organic-inorganic hybrid monolithic column combines the advantages of organic polymer monolithic column and organic-inorganic hybrid monolithic column, and has the advantages of simple preparation, various types, large specific surface area, and high column efficiency, so it is more and more widely used; However, the problems of chemical instability and non-specific adsorption of silanol still need to be solved[1]

Method used

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  • An organic-inorganic hybrid monolithic column and its preparation and application
  • An organic-inorganic hybrid monolithic column and its preparation and application
  • An organic-inorganic hybrid monolithic column and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Such as figure 1 The organic-inorganic hybrid monolithic column was prepared as shown. Firstly, pre-treat the quartz capillary (100μmi.d.): rinse with 1M HCl solution for 2h, rinse with water to be neutral; pass through 4%HF (40%HF diluted 10 times with water), react at 35°C for 3h; rinse with 1MNaOH solution Rinse the capillary with water for 2 hours to make it neutral; rinse it with methanol, and dry it with nitrogen at 120°C for later use. Then weigh 24 mg of cetyltrimethylammonium chloride and 22 mg of ethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer F127 (mass ratio 24:22) and dissolve them in 200 μL of methanol, Add 25 μL of n-octyltriethoxysilane and 25 μL of bis(3-trimethoxysilylpropyl)amine (volume ratio 1:1), and finally add 20 μL of water, mix well, and quickly pour into the pretreated quartz capillary (100μm.d.), sealed, and reacted at 40°C for 12h. The resulting monolithic column was washed sequentially with methanol, water, 80% A...

Embodiment 2

[0027] The organic-inorganic hybrid monolithic column prepared in Example 1 was used as a separation column, and a nanoliter liquid chromatography-ultraviolet system was used for reversed-phase chromatographic separation of standard protein mixtures. Chromatographic separation conditions are as follows: Separation column: 100μmi.d.×23cm; sample: standard protein mixture (RNase A, Cyto C, Lysozyme, BSA each 0.2mg / mL); injection 0.1min; flow rate: 1μL / min; mobile phase A: 2%ACN+98%H 2 O+0.1%TFA; mobile phase B: 98%ACN+2%H 2 O+0.1%TFA; Gradient: 0-5-10-30-40min, 0%-0%-40%-80%-80%B; UV detection wavelength 214nm.

[0028] Such as figure 2 As shown, the four standard protein mixtures achieved baseline separation with good reproducibility, symmetrical peak shape, average half-peak width of 3.75s, and peak capacity of 377.

Embodiment 3

[0030] The organic-inorganic hybrid monolithic column prepared in Example 1 was used as a separation column, and a nanoliter liquid chromatography-ultraviolet system was used for reversed-phase chromatographic separation of proteins in E.coli complex samples. The chromatographic separation conditions are as follows: separation column: 100μmi.d.×23cm; sample: E.coli protein sample (including urea), 1.5mg / mL; injection 0.33min; flow rate: 1μL / min; mobile phase A: 2%ACN +98%H 2 O+0.1%TFA; mobile phase B: 98%ACN+2%H 2 O+0.1%TFA; Gradient: 0-10-30-70-90-100min, 0%-0%-20%-40%-80%-80%B; UV detection wavelength 214nm.

[0031] Such as image 3 Shown, 500ngE.coli protein sample has reached efficient separation. It can be seen that the organic-inorganic hybrid monolithic column has a good application prospect for actual samples.

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Abstract

The present invention relates to an organic-inorganic hybrid monolithic column for protein and peptide separation and a preparation method thereof. The organic-inorganic hybrid monolithic column uses alkyltrimethyl(eth)oxysilanes and amino-bridged trimethyl(eth)oxysilanes as silanization precursors, and is prepared by an alkali-catalyzed sol-gel method. . Based on amine bridged trimethyl(eth)oxysilane, this material has the following advantages: (1) the Si-C bond on the skeleton structure can improve the mechanical strength and chemical stability of the material; (2) the amine group on the skeleton structure It can inhibit the non-specific adsorption of residual silanol groups, thereby improving the chromatographic peak shape and peak capacity of protein and peptide separation; (3) amine group is a weakly charged polar group, which can improve the selectivity of protein and peptide separation .

Description

technical field [0001] The invention relates to a chromatographic stationary phase for separating proteins and peptides, in particular to a novel organic-inorganic hybrid monolithic column and its preparation and application in the separation of proteins and peptides. Background technique [0002] Proteomics is one of the core contents of life science research in the post-genome era. However, proteins and their enzymatic hydrolysis products in biological samples are extremely complex, so they usually need to be separated before detection to reduce the complexity of the sample. Liquid chromatography has the advantages of high separation efficiency and good reproducibility, and is currently a widely used separation technique. Among them, the chromatographic stationary phase is the core component of liquid chromatography, mainly including packed columns and monolithic columns. Due to the advantages of fast mass transfer and low back pressure, the monolithic column has attract...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01D15/22B01J20/286B01J20/30G01N30/60
Inventor 张丽华梁玉吴慈朱旭东杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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