CN102553549A - Preparation method of novel protein chiral immobile phase - Google Patents
Preparation method of novel protein chiral immobile phase Download PDFInfo
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- CN102553549A CN102553549A CN2011103278547A CN201110327854A CN102553549A CN 102553549 A CN102553549 A CN 102553549A CN 2011103278547 A CN2011103278547 A CN 2011103278547A CN 201110327854 A CN201110327854 A CN 201110327854A CN 102553549 A CN102553549 A CN 102553549A
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Abstract
The invention relates to a preparation method of a novel protein chiral immobile phase. A liquid chromatography chiral immobile phase is prepared by simultaneously bonding protein molecules to external surface of a macroporous immobile phase matrix and the internal surfaces of the pores; micromolecular chiral specimen can sufficiently utilize the bonded inner pore channels to act with protein molecules modified by the internal surfaces of the pores, thereby greatly improving column capacity and effectively improving separation; and the matrix can be silica gel or high polymer, and the protein serving as a chiral selector can be adjusted according to the separation selectivity. The novel protein immobile phase has the advantages of simple preparation method and high separation efficiency.
Description
[technical field]
The present invention relates to fixedly phase technical field of chirality liquid chromatogram, specifically, is a kind of preparation method of novel protein chiral stationary phase.
[background technology]
Chipal compounds is meant that molecular weight, molecular structure are identical, but about arrange the pair of opposite molecule, its physicochemical properties are almost completely identical, but possibly there are very big difference in their biochemistry and pharmacological action.For example have the thalidomide (thalidomide, neurosedyn) of sedation, its active ingredient is the R configuration, and the S configuration then has teratogenesis.In recent years, along with bioscience and bionic development, chiral resolution has become the focus that people pay close attention to.The method that is used for chiral separation at present mainly contains thin-layered chromatography, capillary electrophoresis, subcritical and supercritical fluid chromatography, gas chromatography and liquid chromatography etc.High performance liquid chromatography has become one of strong means of chiral resolution with its separating power efficiently.
The liquid chromatogram chiral resolution generally need be used special chromatographic stationary, i.e. chiral stationary phase (CSP), and its performance has determined the ability of chiral resolution.CSP is fixedly processing on the phase carrier by adopting a kind of chiral material chemical bonding or being coated with stain.The chiral stationary phase that can be used for the liquid chromatogram chiral resolution is of a great variety, and modal have type chiral stationary phases such as polysaccharide, protein and cyclodextrin.
The protein-Based Chiral Stationary Phase chiral selectivity is high, and separating power is strong, and chromatographic peak profile is good.Usually can under rp mode, accomplish separation, through the adjustment of chromatographic run parameter, like composition, ionic strength, pH value, organic modifiers and the column temperature etc. of buffer solution, the optimized Separation method, so its detachable compound distributes wide.For protein bonding mode, mainly contain absorption method and chemical bonding.1973, Stewart and Doherty were bonded to bovine serum albumin(BSA) (BsA) on the agarose and have successfully split D/L one tryptophan above that, have occurred the commercialization silica gel H PLC filler of bonding BSA and AGP the eighties in 20th century.The post that be to improve CSPs holds and stability, and people have carried out many trials, as with glutaraldehyde with the protein interlinkage, prepare CSPs with protein fragments or domain, change carrier framework etc.Because protein molecule itself has larger volume, no matter takes which kind of bonding curing mode, the CSP supported quantity that obtains is all lower.This also makes them on the limited filler in aperture, have considerably less chiral Recognition position, and column capacity is lower.
[summary of the invention]
The objective of the invention is to overcome the deficiency of prior art, a kind of preparation method of novel protein chiral stationary phase is provided,, improve column capacity through at fixing phase matrix outer surface and the internal surface of hole bonding protein molecule simultaneously of macropore.
The objective of the invention is to realize through following technical scheme:
One, silica matrix:
1, the preliminary treatment of silica gel
(1) alkalization
With pore size is that the macro porous silica gel of 30～200nm places dilute alkaline soln to alkalize, and water is washed till neutrality then, makes-the abundant activation of Si-OH.
(2) silanization modification
Under certain solvent condition (APTES: MeOH: H2O=2: 1: 7), mechanical agitation was reacted 24 hours, and amino fully is bonded on the macro porous silica gel with macro porous silica gel and 3-aminopropyl triethoxysilane (APTES) after the alkalization.
2, protein bonding
In the macro porous silica gel that above-mentioned preliminary treatment finishes, adding concentration is 15% glutaraldehyde solution, and it is covered fully, react 12 hours, make on the silica gel-NH2 is fully by the C=O replacement, and with the macro porous silica gel hydroformylation after the silanization modification, and with second distillation water washing neutrality extremely; To be dissolved in phosphate buffer (20ml then; PH=7.4) macro porous silica gel mixes after protein and the hydroformylation; Mechanical oscillation reaction 12～24 hours is repeatedly washed with redistilled water, can obtain the novel protein chiral stationary phase of silica matrix; The PBS of 0.02% Sodium azide under 4 ℃ of environment (50mmol/L, PH=7) the middle preservation.
Two, polyamide-based high polymer matrix:
1, hydroformylation
Polyamide is dissolved in the mixed solution of ethanol and isopropyl alcohol, adds 15% glutaraldehyde solution, the stirring at room reaction was washed with water to neutrality after 12 hours.
2, protein bonding
(20ml, the polyamide matrix after protein pH=7.4) and the modification mixes, and more than the mechanical oscillation reaction 12h, repeatedly washs with redistilled water, can obtain the novel protein chiral stationary phase of polymer substrate will to be dissolved in phosphate buffer.
Novel protein chiral stationary phase preparation method according to the invention is at fixing phase matrix outer surface and the internal surface of hole bonding protein molecule simultaneously of macropore; Little molecular chiral sample can make full use of the macropore internal diameter path behind the bonding, with the protein molecule effect of internal surface of hole modification.The matrix of using can be macro porous silica gel and the polyamide-based high polymer of macropore, can also be for macropore amino bonded silica gel and with other macroporous polymer of amino active group.Not only can be on matrix a certain albumen of bonding, can also be as required on matrix bonding multiple protein simultaneously.
Compare with existing protein-Based Chiral Stationary Phase preparation method, good effect of the present invention is:
(1) the present invention has made full use of the macropore internal diameter of big pore matrix, at fixing phase matrix outer surface and the internal surface of hole bonding protein molecule simultaneously of macropore, greatly improves column capacity, effectively improves and separates.
(2) universality is high, and matrix can be macro porous silica gel, amino bonded silica gel, polyamide-based high polymer and other macroporous polymer with amino active group; Can be on matrix one or more protein of bonding.
(3) simple to operate, the scope of application is wide, and the chiral stationary phase of preparation can be used for liquid chromatogram compartment analysis, capillary electric chromatogram analysis and preparative liquid chromatography chiral separation.
[description of drawings]
Fig. 1 is a gained macro porous silica gel bonding protein-Based Chiral Stationary Phase sketch map among the embodiment 1
Fig. 2 is a gained macropore polyamide bond hop protein matter chiral stationary phase sketch map among the embodiment 2
Fig. 3 is an amino acid enantiomer separate colors spectrogram among the embodiment 1
[specific embodiment]
The preparation method's of a kind of novel protein-Based Chiral Stationary Phase of the present invention the specific embodiment below is provided.
Embodiment 1
Take by weighing the macro porous silica gel that the 5.0g particle diameter is 5 μ m, diameter of bore 100 nanometers place the 0.1mol/L dilute alkaline soln, and mechanical agitation is reacted half an hour, and water is washed till neutrality then; Take by weighing APTES=6ml, H2O=3ml, MeOH=21ml after three kinds of solution mixing, mixes with above-mentioned macro porous silica gel adding, and mechanical agitation reaction 24 hours uses the second distillation water washing to neutral again; Added 15% glutaraldehyde solution reaction 12 hours, and made it-NH2 fully replaces by C=O, and with the second distillation water washing to neutrality; The glycoprotein that will be dissolved in phosphate buffer places the macro porous silica gel after the hydroformylation, and mechanical oscillation reaction 12 hours is repeatedly washed with redistilled water, is positioned in the PBS of Sodium azide under 4 ℃ of environment to preserve.
It is the HPLC analytical column of 4.6 * 200mm that the top glycoprotein chiral stationary phase that obtains is filled specification, is 20mmol/L phosphate buffer (pH=4.5) in flowing phase, and flow velocity 1mL/min separates the corresponding isomers of glutamic acid under the room temperature.The result is as shown in Figure 3, and the corresponding isomers of glutamic acid obtains good separation.
Polyamide is dissolved in the mixed solution of ethanol and isopropyl alcohol, after treating to dissolve fully, adds 15% glutaraldehyde solution, the stirring at room reaction was washed with water to neutrality, vacuum drying after 12 hours.Matrix diameter of bore 70 nanometers that obtain.
To be dissolved in phosphate buffer (20ml, protein pH=7.4) places the polyamide matrix after the modification, mechanical oscillation fully reacts it, repeatedly washs with redistilled water, at last products therefrom is slowly poured in the n-hexane, separates out deposition, filters drying.
The above is two kinds of occupation modes of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the present invention's design; Can also make some this and advance and retouch, these improvement and retouching also should be regarded as in protection scope of the present invention.
Claims (5)
1. the preparation method of a novel protein chiral stationary phase; It is characterized in that: macropore fixedly phase matrix outer surface and internal surface of hole simultaneously the bonding protein molecule be prepared into the liquid chromatogram chiral stationary phase; Little molecular chiral sample can make full use of the endoporus path behind the bonding; Protein molecule effect with internal surface of hole is modified greatly improves column capacity, effectively improves and separates;
2. the preparation method of novel protein chiral stationary phase as claimed in claim 1 is characterized in that: described macropore host material comprises: silica gel and high polymer such as polyamide-based;
3. the preparation method of novel protein chiral stationary phase as claimed in claim 1 is characterized in that: the external diameter of macropore particle matrix is the 5-50 micron, and the diameter of endoporus is the 30-200 nanometer;
4. the preparation method of novel protein chiral stationary phase as claimed in claim 1 is characterized in that: the protein that is used for fixing the phase surface modification comprises: bovine serum albumin(BSA), human serum albumins, the acid albumin of α 1-, egg albumin, cellobiohydrolase and other albumen;
5. novel protein chiral stationary phase as claimed in claim 1 is characterized in that: can be used for liquid chromatogram compartment analysis, capillary electric chromatogram analysis and preparative liquid chromatography chiral separation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105498706A (en) * | 2015-12-09 | 2016-04-20 | 广东研捷医药科技有限公司 | Preparation method for protein A immunoaffinity column |
| CN109569026A (en) * | 2018-01-11 | 2019-04-05 | 南开大学 | It prepares the chromatographic stationary phases that porous framework material is matrix and is used for chiral separation |
-
2011
- 2011-10-25 CN CN2011103278547A patent/CN102553549A/en active Pending
Non-Patent Citations (4)
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| 傅强等: "人血清蛋白键合固定相的合成及DL（±）氨基酸的手性HPLC分离", 《西北药学杂志》 * |
| 周静: "以大孔硅胶为基质的手性色谱填料的制备及应用", 《CNKI优秀硕士学位论文全文库》 * |
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| 郑翌等: "牛血清白蛋白手性毛细管整体柱的分离性能", 《华东理工大学学报(自然科学版)》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105498706A (en) * | 2015-12-09 | 2016-04-20 | 广东研捷医药科技有限公司 | Preparation method for protein A immunoaffinity column |
| CN105498706B (en) * | 2015-12-09 | 2019-07-23 | 广东研捷医药科技有限公司 | A kind of preparation method of albumin A immune affinity column |
| CN109569026A (en) * | 2018-01-11 | 2019-04-05 | 南开大学 | It prepares the chromatographic stationary phases that porous framework material is matrix and is used for chiral separation |
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Application publication date: 20120711 |