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A novel reversed-phase/weak cation-exchange mixed-mode chromatographic stationary phase and its preparation method

A mixed-mode chromatography and weak cation exchange technology, applied in the field of chromatographic separation research, to achieve the effects of easy-to-obtain reaction raw materials, mild reaction conditions, and high yield

Active Publication Date: 2020-04-17
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a novel reversed-phase / weak cation exchange mixed-mode chromatographic stationary phase. Due to the existence of a dual separation mechanism, in the separation of complex protein samples, peptides with similar chemical properties can be further separated, and It can solve the problem of irreversible adsorption caused by too strong peptide hydrophobicity, thereby improving the identification coverage of the proteome; at the same time, the preparation method of the stationary phase provided by the invention has the advantages of good reaction selectivity, high yield, and simple reaction conditions.

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  • A novel reversed-phase/weak cation-exchange mixed-mode chromatographic stationary phase and its preparation method
  • A novel reversed-phase/weak cation-exchange mixed-mode chromatographic stationary phase and its preparation method
  • A novel reversed-phase/weak cation-exchange mixed-mode chromatographic stationary phase and its preparation method

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Effect test

Embodiment 1

[0052] Example 1, preparation of reversed phase / weak cation exchange mixed mode chromatographic stationary phase

[0053] (1) Take 30g spherical silica gel (particle size is 3μm, pore size is ), dispersed in 5mL / g hydrochloric acid solution (20wt%), ultrasonicated for half an hour, and shaken overnight on a rotary mixer. The silica gel was washed with deionized water until neutral, dried overnight at 110°C, and placed in a desiccator for later use.

[0054] (2) Disperse 15 g of pretreated silica gel microsphere matrix in 45 mL of dry toluene, add 15 mL of vinyltrichlorosilane, and react in a water bath at 40 °C at a magnetic stirring speed of 100 rpm for 24 h under nitrogen protection. After the reaction, wash with toluene, methanol and dichloromethane respectively, and dry at 80°C for 6h.

[0055] (3) Disperse the reacted silicon spheres in 100mL of methanol:water (V:V=1:2), add 15g of cysteine ​​and 0.15g of azobisisobutyronitrile, and magnetically Stir the reaction for ...

Embodiment 2

[0062] Embodiment 2, separation polycyclic aromatic hydrocarbon mixture

[0063] Using the reverse phase / weak cation exchange mixed mode chromatographic stationary phase prepared in Example 1, the stationary phase was packed into a 4.6×150mm stainless steel chromatographic column by the high-pressure homogenization method, and 16 kinds of polycyclic aromatic hydrocarbon mixtures were carried out in the reverse phase mode. separate. As a comparison, under the same chromatographic conditions, 16 polycyclic aromatic hydrocarbon mixtures were separated using a Shiseido reverse-phase chromatographic column (model: CAPCELL PAKC18MGII S3).

[0064] Chromatographic conditions: the sample concentration is 10 μg / mL, the sample loading volume is 10 μL, and the flow rate is 0.5 mL / min. Phase A: water; phase B: acetonitrile; gradient: 0-5min 60%-100%B, 5-45min 100%B. The detection wavelength is 254nm.

[0065] The separation chromatogram is as image 3 As shown (peak 1 represents napht...

Embodiment 3

[0066] Embodiment 3, separation polypeptide mixture

[0067] Using the reverse phase / weak cation exchange mixed-mode chromatographic stationary phase prepared in Example 1, the stationary phase was loaded into a 4.6×150mm stainless steel chromatographic column by the high-pressure homogenization method, and the mixture of 6 polypeptides was separated under different pH conditions .

[0068] The chromatographic conditions are: the sample concentration is 0.5 μg / μL, the sample loading volume is 20 μL, and the flow rate is 0.5 mL / min. Phase A: 98% water (20mM ammonium formate, pH 4.5, 6.0, 7.5) ~ 2% acetonitrile; phase B: 98% acetonitrile ~ 2% water; gradient: 0 ~ 30min2% ~ 95% B. The detection wavelength is 214nm.

[0069] The separation chromatogram is as Figure 4 , when the pH is low, the reversed-phase / weak cation exchange mixed-mode chromatographic stationary phase behaves as a reversed-phase separation mechanism, and cannot completely separate the six polypeptides; when...

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Abstract

The invention discloses a novel reversed-phase / weak cation exchange mixed mode chromatographic stationary phase and a preparation method thereof. The reversed-phase / weak cation exchange mixed mode chromatographic stationary phase has a structural formula shown in a formula I. The preparation method for the reversed-phase / weak cation exchange mixed mode chromatographic stationary phase comprises the following steps: 1) performing a silane coupling reaction on a vinyl silane coupling agent and silicon hydroxyl at the surface of a spherical silica gel to obtain a compound represented by a formulaII; 2) performing a thiol-ene click chemistry reaction on the compound represented by the formula II and cysteine to obtain a compound represented by a formula III; and 3) performing a reaction on the compound represented by the formula III and octadecyl isocyanate via a reaction of amino and an isocyanate group to obtain the chromatographic stationary phase. According to the invention, the reversed-phase / weak cation exchange mixed mode chromatographic stationary phase is bonded with hydrophilic groups, and the hydrophobicity is weakened, so that the problem of irreversible adsorption causedby too strong hydrophobicity of peptide fragments can be solved, and the qualitative and quantitative analysis results of proteome are more accurate.

Description

technical field [0001] The invention relates to a novel reverse phase / weak cation exchange mixed-mode chromatographic stationary phase and a preparation method thereof, belonging to the field of chromatographic separation research. Background technique [0002] With the development of proteomics research towards deep coverage qualitative identification and large-scale quantitative analysis, new requirements are put forward for proteomics technology. One of the core technologies of proteomics research is the separation method, and the combination of liquid chromatography separation and mass spectrometry detection has become a routine method in proteomics research. Faced with the extreme complexity of biological samples and the increasing requirements for the accuracy, sensitivity, selectivity, speed, automation and reproducibility of the analysis system itself, the traditional single-mode reversed-phase chromatography separation method can no longer meet the requirements. Re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/287B01J20/30B01D15/32
CPCB01D15/327B01J20/287B01J2220/52B01J2220/54B01J2220/80
Inventor 钱小红张养军周珊珊
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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