CN112892505A - Surface charge modified reversed phase chromatographic separation material, preparation and application thereof - Google Patents
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- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
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Abstract
The invention relates to a preparation method of a surface charge modified reversed phase chromatographic separation material for separating protein and peptide fragments. The method is characterized in that a material with rich silicon hydroxyl on the surface is used as a substrate, hydrophobic group modification of a monomolecular layer is firstly carried out, and then controllable modification of surface charge modification is carried out on the surface of the monomolecular layer based on the reaction of five-membered azacyclosilane and silicon hydroxyl. The invention has the following advantages: (1) the preparation method has good universality, and silica gel particles, silica gel monolithic columns and organic-silicon hybrid monolithic columns with surfaces rich in silicon hydroxyl can be prepared by the method; (2) according to the invention, five-membered azacyclo-silane is adopted to react with silicon hydroxyl, so that the surface of the material has secondary amine groups, the nonspecific adsorption of the silicon hydroxyl can be effectively shielded, and the chromatographic peak pattern is improved; (3) the method can control the surface charge density by controlling the reaction solvent and the reaction time.
Description
Technical Field
The invention relates to a reverse phase chromatographic separation material used for separating peptide fragments and proteins, in particular to a preparation method of a surface charge modified reverse phase chromatographic separation material and high-efficiency separation of the peptide fragments and the proteins.
Background
The analysis strategy of proteomics is divided into top-down proteomics and bottom-up proteomics, and the analysis objects are complete protein and peptide fragments respectively. However, since the constitution of both proteins and peptides in a biological sample is very complicated, a high-resolution separation means is required before detection. The liquid chromatography plays an important role in the research of proteomics due to the advantages of high resolution, compatibility with mass spectrum detection and the like. In liquid chromatography, the separation material is the core, wherein silica gel matrix and organic-silicon hybrid matrix are widely applied to chromatographic separation due to the advantages of strong pore diameter adjustability, good mechanical stability and the like, but have serious problem of non-specific adsorption of residual silicon hydroxyl. The non-specific adsorption of the silicon hydroxyl in the reverse phase chromatography mode can cause serious tailing problems of peptide fragments and proteins in the separation process. The invention develops a preparation method of a surface charge modified reversed phase chromatographic separation material aiming at the problem and is used for separating proteins and peptide fragments.
Disclosure of Invention
The invention aims to provide a preparation method of a surface charge modified reverse phase chromatographic separation material, which solves the problem of non-specific adsorption of the traditional silica gel matrix and organic-silicon hybridization matrix separation materials on samples at present and is used for separating proteins and peptide fragments.
In order to achieve the purpose, the invention adopts the technical scheme that:
using a material with a surface rich in silicon hydroxyl as a substrate, and firstly carrying out hydrophobic group modification on a monomolecular layer; followed by charge modification at its surface. The method comprises the following specific steps:
(1) monolayer hydrophobic group modification
Firstly, preparing a reaction solution, wherein the reaction solution consists of a silanization reagent and an anhydrous organic solvent, and the content of the silanization reagent in the reaction solution is 5-60%. The anhydrous organic solvent comprises one or more of benzene, toluene, dichloromethane, dimethyl sulfoxide, diethyl ether and tetrahydrofuran; the silylation agent includes one or more of dimethyl alkyl chlorosilanes, diethyl alkyl chlorosilanes, and alkyl dimethylamine dimethylsilanes.
And then dispersing the material with rich silicon hydroxyl on the surface in the reaction solution for reaction or introducing the reaction solution into the monolithic column matrix for reaction. The reaction temperature is 0-120 ℃, and the reaction time is 1-24 h. After the reaction, the reaction solution is washed with an anhydrous organic solvent.
(2) Modification of surface charge
Firstly, preparing a reaction solution, wherein the reaction solution consists of pentabasic azacyclo-silane and an anhydrous organic solvent, and the content of a silanization reagent in the reaction solution is 5-60% (v/v). The anhydrous organic solvent comprises one or more of benzene, toluene, dichloromethane, dimethyl sulfoxide, diethyl ether and tetrahydrofuran; the pentabasic azacyclic silanes include one or more of N- (2-aminoethyl) -2,2, 4-trimethyl-1-aza-2-silacyclopentane, N-methyl-aza-2, 2, 4-trimethylsilane, N-N-butyl-aza-2, 2-dimethoxysilapentane, N-tert-butyl-aza-2, 2-dimethoxysilapentane, 2-diethoxysilane, and N-methyl-aza-2, 2-dimethoxysilapentane. And then dispersing the material modified by the hydrophobic group of the monomolecular layer in a reaction solution for reaction or introducing the reaction solution into the monolithic column matrix for reaction. The reaction temperature is 0-120 ℃, and the reaction time is 1-24 h.
After the reaction, the reaction solution was washed with an anhydrous organic solvent and acetonitrile in this order.
(3) And separating the protein or peptide sample by using the prepared capillary column as a separation column and adopting a liquid chromatography system.
The invention has the following advantages:
(1) the preparation method has good universality, and silica gel particles, silica gel monolithic columns and organic-silicon hybrid monolithic columns with surfaces rich in silicon hydroxyl can be prepared by the method;
(2) according to the invention, five-membered azacyclo-silane is adopted to react with silicon hydroxyl, so that the surface of the material has secondary amine groups, the nonspecific adsorption of the silicon hydroxyl can be effectively shielded, and the chromatographic peak pattern is improved;
(3) the method can control the surface charge density by controlling the reaction solvent and the reaction time.
Drawings
FIG. 1 surface charge modified C18Preparation method of reverse phase silica gel particle material
FIG. 2 surface charge modified C4Preparation method of reversed-phase organic-silicon hybrid column
FIG. 3 surface charge modified C4Preparation method of reversed-phase organic-silicon hybrid column
FIG. 4 uses prepared surface charge modified C18Chromatogram for separating BSA enzymolysis peptide fragment by using reverse phase organic-silicon hybrid column
FIG. 5 modified with prepared surface Charge C4Chromatogram for separating five standard protein mixtures by reversed phase organic-silicon hybrid column (five times of parallel analysis)
Detailed Description
The process provided by the present invention is described in detail below by way of examples, but is not intended to be limiting in any way.
Example 1
As shown in FIG. 1, surface charge modified C was prepared according to the following scheme18Reverse phase silica gel particulate material
(1) Silica gel surface activation: 1g of silica gel is dispersed in 30ml of 6M hydrochloric acid and refluxed at 100 ℃ for 3 hours with stirring. After the reflux is completed, the mixture is filtered by a sand core funnel, washed to be neutral by distilled water, washed by absolute ethyl alcohol for three times, and finally dried in vacuum at 50 ℃.
(2)C18Bonding of monolayer hydrophobic groups: the activated and dried silica gel particles were dispersed in 30ml of 20% dimethyloctadecylchlorosilane in toluene (v/v) and the reaction was stirred at 120 ℃ under reflux for 24 h. After the reaction, the mixture was filtered through a sand core funnel, washed with anhydrous toluene, acetone and methanol in this order, and then vacuum-dried at 50 ℃.
(3) Modification of surface charge: will modify C18The radical silica gel particles were dispersed in 30ml of a 20% solution of N-methyl-aza-2, 2, 4-trimethylsilane in dichloromethane (v/v) and stirred at 25 ℃ for 24 h. After the reaction is finished, performing suction filtration by using a sand core funnel, sequentially washing by using anhydrous dichloromethane, dichloromethane and methanol, and then drying in vacuum at 50 ℃ to obtain the surface charge modified C18Separating the material by reverse phase chromatography.
Example 2 surface modification of silica gel monolith column
As shown in FIG. 2, surface charge modified C was prepared according to the following scheme18/C4Reversed phase silica gel monolithic column:
(1) preparation of silica gel monolithic column matrix:
0.2400g of PEG and 0.1800g of urea were weighed out and dissolved in 2ml of 0.01M HAc, and 1.12ml of TMOS was slowly added dropwise thereto. Under the condition of ice-water bath, stirring vigorously for 40 minutes to form clear and transparent sol. Slowly introducing the sol solution into the pretreated 75-micron capillary, sealing two ends of the capillary with silicon rubber, placing the capillary in a water bath, and reacting for 24h at 40 ℃. Then transferring the capillary column to a reaction kettle filled with deionized water, placing the reaction kettle in an oven, directly heating to 120 ℃, and reacting for 3 hours. And (3) introducing a large amount of deionized water and methanol into the obtained capillary monolithic column in sequence by using a high performance liquid chromatography pump so as to remove the template agent and the unreacted reagent in the material. The capillary monolith was then placed in a 50 ℃ oven and allowed to drain slowly. And finally, placing the monolithic column in a muffle furnace, programming the temperature from room temperature to 200 ℃ at the speed of 1 ℃/min, calcining for 25 hours at the temperature, and cooling to room temperature for later use.
(2)C18/C4Bonding of monolayer hydrophobic groups: and (3) introducing 20% of dimethyl octadecyl chlorosilane (or dimethyl butyl chlorosilane) in toluene solution (v/v) into the silica gel monolithic column, and reacting for 24h at the temperature of 60 ℃. After completion of the reaction, the reaction mixture was washed with toluene.
(3) Modification of surface charge: to modify C18/C4A solution of 20% N-methyl-aza-2, 2, 4-trimethylsilane in dichloromethane (v/v) was passed through a column of the group silica gel monolith and stirred at 25 ℃ for 24 h. After the reaction is finished, washing the mixture by dichloromethane and acetonitrile in sequence to obtain the surface charge modified C18/C4Separating the material by reverse phase chromatography.
Example 3
As shown in FIG. 3, surface charge modified C was prepared as follows18/C4Reversed phase organic-silicon hybrid column
(1) Preparation of an organic-silicon hybrid material matrix: 0.1580g of PEG and 0.1800g of urea were weighed out separately and dissolved in 2mL of 0.01M HAc, and a mixed silanization solution of 640. mu.L of TMOS and 160. mu.L of LBTME was slowly added dropwise thereto. Under the condition of ice-water bath, stirring vigorously for 40 minutes to form clear and transparent sol. Slowly introducing the sol solution into the pretreated capillary, sealing two ends of the capillary with silicon rubber, placing the capillary in a water bath, and reacting for 24h at 25 ℃. Then transferring the capillary column to a reaction kettle filled with deionized water, placing the reaction kettle in an oven, directly heating to 120 ℃, and reacting for 3 hours. And (3) introducing a large amount of deionized water and methanol into the obtained capillary monolithic column in sequence by using a high performance liquid chromatography pump so as to remove the template agent and the unreacted reagent in the material. The capillary monolith was then placed in a 50 ℃ oven and allowed to drain slowly. And finally, placing the monolithic column in a muffle furnace, programming the temperature from room temperature to 200 ℃ at the speed of 1 ℃/min, calcining for 25 hours at the temperature, and cooling to room temperature for later use. It is an organo-silicon hybrid monolithic column without surface charge modification;
(2)C18/C4bonding of monolayer hydrophobic groups: transfer 200. mu.L of octadecylDimethylamine dimethylsilane (or butyl dimethylamine dimethylsilane) is dissolved in 800 mu l of anhydrous toluene, and the reaction liquid is slowly and continuously introduced into a capillary silica gel hybrid monolithic column which is dried in advance and reacts for 24h at the temperature of 60 ℃. After the reaction was complete, the capillary monolith was washed with toluene and dichloromethane.
(3) Modification of surface charge: 200 mu L N-methyl-aza-2, 2, 4-trimethylsilane was transferred and dissolved in 800. mu.l of anhydrous dichloromethane, and C was slowly and continuously passed through the reaction mixture18/C4Reacting for 24 hours at 25 ℃ in a functionalized capillary silica gel hybrid monolithic column. After the reaction is finished, washing the capillary monolithic column by using dichloromethane and acetonitrile in sequence to obtain the surface charge modified C18/C4A reversed phase organo-silicon hybrid column.
Example 4
Surface charge modified and non-surface charge modified C of example 318The organic-silicon hybrid monolithic column is used for reversed phase chromatographic separation of the BSA trypsin enzymolysis peptide segment. The chromatographic separation conditions were as follows: separating the column: 75 μm i.d.. times.25 cm; sample preparation: BSA enzymolysis peptide segment; sample introduction is carried out for 2 min; flow rate: 500 nL/min; mobile phase A: 2% ACN + 98% H2O + 0.1% FA; mobile phase B: 98% ACN + 2% H2O + 0.1% FA; gradient: 0-10-50-50.1-60 min, 0-40-80% B; the ultraviolet detection wavelength is 214 nm.
As shown in fig. 4, the organic-silicon hybrid column after charge modification has narrower peak width and more symmetrical peak shape for BSA enzymatic peptide separation, which indicates that nonspecific adsorption of silicon hydroxyl groups is significantly reduced through charge modification.
Example 5
C surface Charge modification of example 34The reversed phase organic-silicon hybrid column is used as a separation material, adopts a nano-liter liquid chromatography-ultraviolet system and is used for the reversed phase chromatographic separation of a standard protein mixture. The chromatographic separation conditions were as follows: separating the column: 75 μm i.d.. times.25 cm; sample preparation: standard protein mixtures (RNase A, Cyto C, Lysozyme, BSA each 0.01 mg/mL); sample introduction is carried out for 1 min; flow rate: 500 nL/min; mobile phase A: 2% ACN + 98% H2O + 0.1% TFA; mobile phase B: 98% ACN + 2% H2O+0.1％TFA；Gradient: 0-10-50-60 min, 0-80% B; the ultraviolet detection wavelength is 214 nm.
As shown in fig. 5, the four standard protein mixtures achieved baseline separation with better reproducibility and symmetrical peak patterns.
Claims (9)
1. A method for preparing a surface charge modified reversed phase chromatographic separation material is characterized by comprising the following steps: using a material with a surface rich in silicon hydroxyl as a substrate, and firstly carrying out hydrophobic group modification on a monomolecular layer; then carrying out surface charge modification based on the reaction of pentabasic azacyclo-silane and silicon hydroxyl;
obtaining the reverse phase chromatographic separation material.
2. The method of claim 1, wherein:
the material with rich silicon hydroxyl on the surface comprises one or more than two of silica gel particles, silica gel monolithic columns and organic-silicon hybrid monolithic columns.
3. The method of claim 2, wherein:
the particle size range of the silica gel particles is 100nm-10 mu m;
the silica gel monolithic column and the organic-silicon hybrid monolithic column are polymerized in a capillary with the inner diameter of 5-500 mu m.
4. The method of claim 1, wherein:
the hydrophobic group of the monolayer is modified, and the modified group comprises one or more than two of butyl, octyl, phenyl, dodecyl and octadecyl.
5. The production method according to any one of claims 1 to 4, wherein:
the monolayer hydrophobic group modification is to disperse a material with rich silicon hydroxyl on the surface in a reaction solution for reaction or introduce the reaction solution into an integral column matrix for reaction;
the reaction solution consists of a silylation agent and an organic solvent: the solvent comprises one or more of benzene, toluene, dichloromethane, dimethyl sulfoxide, diethyl ether and tetrahydrofuran; the silanization reagent comprises one or more than two of dimethyl alkyl chlorosilanes, diethyl alkyl chlorosilanes and alkyl dimethylamine dimethylsilane;
the content of the silanization reagent in the reaction solution is 5-60%; the reaction temperature is 0-120 ℃, and the reaction time is 1-24 h.
6. The method of claim 1, wherein:
the modification of the surface charge is to disperse the material modified by the hydrophobic group of the monomolecular layer in a reaction solution for reaction or to continuously introduce the reaction solution into the monolithic column matrix for reaction;
the reaction solution consists of five-membered azacyclosilane and a solvent: the solvent comprises one or more of benzene, toluene, dichloromethane, dimethyl sulfoxide, diethyl ether and tetrahydrofuran; the pentabasic azacyclo-silane comprises one or more than two of N- (2-aminoethyl) -2,2, 4-trimethyl-1-aza-2-silacyclopentane, N-methyl-aza-2, 2, 4-trimethylsilane, N-N-butyl-aza-2, 2-dimethoxy silapentane, N-tert-butyl-aza-2, 2-dimethoxy silapentane, 2-diethoxy silapentane and N-methyl-aza-2, 2-dimethoxy silapentane.
7. The method of claim 6, wherein:
the content of pentabasic azacyclo-silane in the reaction solution is 5-60% (v/v); the reaction temperature is 0-120 ℃; the reaction time is 1-24 h.
8. A surface charge-modified reverse phase chromatographic separation material prepared by the preparation method of claims 1 to 7.
9. Use of the reverse phase chromatographic separation material of claim 8, wherein: the reverse phase chromatographic separation material can be applied to the reverse phase chromatographic separation of peptide fragments and/or proteins in biological samples.
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| CN115814773A (en) * | 2022-12-07 | 2023-03-21 | 纳谱分析技术（苏州）有限公司 | Modified silica gel microsphere and preparation method and application thereof |
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| WO2011012019A1 (en) * | 2009-07-31 | 2011-02-03 | 中国科学院大连化学物理研究所 | Separation material based on silica gel having copolymerization reaction on surface and preparation method thereof |
| US20130319086A1 (en) * | 2009-08-04 | 2013-12-05 | Waters Technologies Corporation | High purity chromatographic materials comprising an ionizable modifier |
| CN107519850A (en) * | 2016-06-22 | 2017-12-29 | 中国科学院大连化学物理研究所 | Preparation and material and application for the hybridisation silica gel integral material of chromatographic isolation |
| CN109865507A (en) * | 2017-12-05 | 2019-06-11 | 中国科学院大连化学物理研究所 | A kind of novel silica gel stromal surface method of modifying and its application |
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| 刘国诠 等编著: "《生物工程下游技术——细胞培养、分离纯化、分析检测》", 31 July 1993, 化学工业出版社 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115814773A (en) * | 2022-12-07 | 2023-03-21 | 纳谱分析技术（苏州）有限公司 | Modified silica gel microsphere and preparation method and application thereof |
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