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SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof

A mixed-mode chromatography and stationary phase technology is applied to a hydrophobic/strong cation-exchange dual-functional mixed-mode chromatography stationary phase. In the field of preparation of the mixed-mode stationary phase, problems such as limited types can be solved, and the synthesis cost is low and the service life is long. , good separation effect

Inactive Publication Date: 2013-11-06
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few dual-function chromatographic packing materials specially designed for separating proteins, and the types are very limited

Method used

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  • SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof
  • SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof
  • SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] （1） Take 3.3g (about 13.7 mmol) of cystine and dissolve it in 150 mL of 0.5 mol / L Na 2 CO 3 buffer solution, then adjust the pH to 11.0, add it to a 250 mL three-necked flask, and slowly add 3 mL (about 13.7 mmol) of TM560 dropwise under ice-bath stirring. 65°C, reacted for 24 hours, the reaction was completed, the solution was light yellow, cooled to room temperature, adjusted the pH of the reaction solution to 5.5 with glacial acetic acid, added 2 g of silica gel, reacted at 90°C for 2 hours, filtered, and washed three times with water, methanol, Wash with acetone and methanol, and dry the obtained solid under vacuum at 50° C. for 8 hours to obtain a cystine-bonded silica gel derivative. Take 5.0 mL of ninhydrin ethanol solution, add a small amount of cystine silica gel derivative, heat at 90°C for 5 min, and find that the solution turns purple, indicating that cystine has been successfully bonded to the silica gel surface.

[0047] (2) Disperse 2.0 g of cystine-bo...

Embodiment 2

[0055] The chromatographic filler prepared in Example 1 was used to pack the column, and then the five standard proteins were separated under the strong cation exchange mode. Separation conditions:

[0056] Mobile phase: Liquid A: 20 mmol / L KH 2 PO 4 (pH 6.5); B liquid: 20 mmol / L KH 2 PO 4 + 1.0 mol / L NaCl (pH 6.5), linear gradient elution, 0→100%B, 30 min; flow rate 1.0 mL / min, for five proteins including myoglobin, RNase B, RNase A, cytochrome c, and lysozyme good separation (as figure 2 1, 2, 3, 4, and 5 shown are myoglobin, RNase B, RNase A, cytochrome c, and lysozyme, respectively).

Embodiment 3

[0058] The chromatographic filler prepared in Example 1 was used to pack the column, and then the seven standard proteins were separated in the hydrophobic mode. Separation conditions:

[0059] Mobile phase: Liquid A: 20 mmol / L KH 2 PO 4 (pH 7.0) + 3.0 mol / L (NH 4 ) 2 SO 4 (pH 7.0); Solution B: 20 ​​mmol / L KH 2 PO 4 (pH 7.0), linear gradient elution, 0→100%B, 30 min, flow rate of 1.5 mL / min, achieved cytchrome c, myoglobin, RNase A, lysozyme, OVA, α-amylase, insulin and other seven proteins good separation (eg image 3 1, 2, 3, 4, 5, 6, and 7 shown are cytchrome c, myoglobin, RNase A, lysozyme, OVA, α-amylase, insulin, respectively).

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Abstract

The invention discloses a strong cation exchange / hydrophobic mixed-mode chromatograph stationary phase, wherein X is -OCH3 or -OCH2CH3; R is shown in the specification, wherein n is equal to 1-5, or R is shown in the specification, wherein n is equal to 1-6; or R is PEG (Polyethylene Glycol) 200-1000. A preparation method comprises the following steps of: bonding cystine on the activated silica gel surface with hydroxyl groups by a silane coupling agent, then using DTT to open cystine disulfide bonds on the silica gel surface and form sulfydryl silica gel, then using H2O2 to oxidize the sulfydryl groups into sulfonic acid groups and form silica-gel derivatives bonded with the sulfydryl groups, and finally reacting with fatty alcohol (or aromatic alcohol or PEG and the like) to obtain the hydrophobic / strong cation exchange chromatograph stationary phase. The stationary phase can realize effective separation of proteins under the hydrophobic mode and the strong cation exchange mode, and one chromatographic column filled with a double-function separating medium can replace two common strong cation exchange / hydrophobic chromatographic columns to carry out separation and purification on the proteins.

Description

technical field [0001] The present invention relates to a high-performance liquid chromatography stationary phase for protein separation, specifically a hydrophobic / strong cation exchange dual-function mixed-mode chromatography stationary phase, and the invention also relates to the preparation method and application of the mixed-mode stationary phase . Background technique [0002] With the development of biotechnology and life sciences, both the production of recombinant protein drugs and the research of proteomics depend on the efficient and rapid separation technology of proteins. The analysis of complex samples puts forward higher and higher requirements for separation science, and the development of new high-efficiency separation materials, separation modes and high-sensitivity detection methods is one of the effective ways to solve this problem. [0003] Multidimensional liquid chromatography (MDLC) is a key technology for the separation and analysis of complex sampl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/286
Inventor 白泉赵开楼
Owner NORTHWEST UNIV
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