CN1831526A - Gradient separation material - Google Patents
Gradient separation material Download PDFInfo
- Publication number
- CN1831526A CN1831526A CN 200510054456 CN200510054456A CN1831526A CN 1831526 A CN1831526 A CN 1831526A CN 200510054456 CN200510054456 CN 200510054456 CN 200510054456 A CN200510054456 A CN 200510054456A CN 1831526 A CN1831526 A CN 1831526A
- Authority
- CN
- China
- Prior art keywords
- gradient
- separation material
- porosint
- gradient separation
- polyamino
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
A gradient separation material is cylinder - shaped cellular material fixed with molecule containing both of positive and negative electric charge group, its pH gradient Scope is 0 - 14 or any section of 0 - 14, the hole diameter of cellular material is 0.000000001 to 0.00001 metre . The method for preparing gradient separation material is also disclosed.
Description
Technical field
The present invention relates to a kind of gradient separation material.
The invention still further relates to the preparation method of above-mentioned material.
Background technology
Finishing of the Human Genome Project indicated the beginning of protein groups engineering.Present two-dimentional polyacrylamide gel electrophoresis (2D PAGE) (document 1:O ' Farrell, P.H.J Biol.Chem., 1975,250,4007-4021.) be the major technique of protein groups research, the widespread use of 2D PAGE has benefited from realization (the document 2:Bjellqvist of immobilization pH gradient (IPG), B.Ek K., Righetti P.G., Gianazza, E., G rg, A., Westermeier, R., Postel, W.J Biochem Biophys Methods 1982,6,317-339.), the introducing of IPG has improved applied sample amount and has improved the reappearance of electrophoresis method (document 3:G rg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R., Weiss, W.Electrophoresis 2000,21,1037-1053.).Capillary Electrophoresis (CE) method has solved the Joule heat problem that produces in the electrophoresis process effectively, greatly improved the efficient of electrophoresis separating method, make Capillary Electrophoresis become the important laboratory facilities of life science day by day, wherein capillary isoelectric focusing (CIEF) is to separate the especially strong method of peptide, protein of ampholyte, needs to add carrier ampholyte (CAs) and set up the pH gradient in the CIEF experiment.Yet the use of carrier brings a series of problem: subsequent treatment is loaded down with trivial details, CAs costs an arm and a leg, at low wavelength strong absorption is arranged---reduce detection sensitivity ... everything has all limited promoting the use of of isoelectric focusing method.
In order to overcome the problems that CAs brings, people have developed brine electrolysis (document 4:Huang, T., Wu, X.-Z., Pawliszyn, J.Anal.Chem.2000,72,4758-4761.), thermic pH gradient (document 5:Lochmuller, C.H., Breiner, S.J.J Chromatogr.1989,480,293-300.), self-focusing (document 6:Sova, O.J.Chromatogr.1985,320,15-22.), method (document 7:Rilbe, H.J.Chromatogr, 1978 such as fluidised form focusing, 159,193-205.).Yet these methods still have the not high shortcoming of instability, resolution.
Summary of the invention
The purpose of this invention is to provide a kind of is the gradient separation material of skeleton with stable inertia porous mass, material takes column or chip form to adapt to different requirements, the group of the different electric charges of band yin, yang ion is arranged on the surface of material, therefore utilize this material can realize the gradient separations of different mode: to comprise ion-exchange, isoelectric focusing etc.
Another purpose of the present invention provides a kind of method for preparing above-mentioned gradient separation material.
For achieving the above object, gradient separation material provided by the invention is, is fixed with the molecule that contains positive and negative electric charge group simultaneously on the porosint of column or sheet, and the pH gradient scope is 0～14 or wherein any one section; The aperture of this porosint is 10
-9To 10
-5Rice.
Described porosint is poly-(methyl) acrylate, silica gel, Lauxite, silicon dioxide, zirconium dioxide or titania.
The described molecule that contains positive and negative electric charge group simultaneously is a carrier ampholyte, as the many carboxyls of polyamino, the many phosphorus of polyamino (phosphine) acid, the many sulfonic acid of polyamino or the many sulfuric acid of polyamino.
The method of the above-mentioned gradient separation material of preparation provided by the invention, key step is:
A) to feed volume ratio be the carrier ampholyte of 1-10% to the porosint that will have a reactive group, in ℃ down reaction 2-24 hour of room temperature to 80;
B) material of step a preparation places the solution of different pH values, and its two ends add DC voltage, form supported pH gradient.
The present invention has following advantage:
1. utilize porous material as matrix, can greatly guarantee carrying out smoothly of mass transport process.
2. porosint matrix has rigid characters, can tolerate the mechanical pressure of certain limit.
3. when in this material, carrying out gradient separations (as isoelectric focusing), need not to add in addition carrier ampholyte, have monitor compatibility flexibly.
Description of drawings
Fig. 1 is the synthetic synoptic diagram of material in the one embodiment of the invention.Symbology among the figure:
Carboxyl, aldehyde radical, acyl chlorides, imino group, isosulfocyanate radical or the like;
R
1, R
2, R
3: amino, carboxyl, pyridine radicals, pyrrole radicals, pyrimidine radicals, purine radicals, phosphate radical, sulfonate radical, sulfate radical etc.;
P: pepsin, e: elastoser, d: detection window, t: trypsase.
Fig. 2 is the sem photograph of porosint in the one embodiment of the invention.
Fig. 3 is the graph of pore diameter distribution of porosint in the one embodiment of the invention.
Fig. 4 is the integral post form of novel gradient parting material in the one embodiment of the invention.
Fig. 5 is the chip form of novel gradient parting material in the one embodiment of the invention.
Fig. 6 is two amino acid whose isoelectric focusing (CIEF) spectrogram in the one embodiment of the invention.Its condition is: concentration 2mg/mL; Buffer solution PBS (5mmol/L, pH7.2); Voltage 10kV; Detect wavelength 210nm.
Fig. 7 is the gradient separations spectrogram of protein mixture in the one embodiment of the invention.A among the figure: elastoser, b: haemoglobin, c: insulin, d: ovalbumin, e: casein.Protein concentration 0.2mg/mL; Separate column length 25cm (effectively long 20cm); Buffer solution NH
4Ac (25mmol/L contains the 10%v/v acetonitrile, 0.1%v/v TFA, pH 3.9); Voltage 10kV; Detect wavelength 214nm.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail.
Capillary column (Hebei Yongnian sharp foreign chromatogram device company limited, 50 μ m I.D., 375 μ m O.D.), carrier ampholyte Pharmalyte (pH3.0-10.0, BioChemika, Switzerland), the biomacromolecule component is a haemoglobin, be the biochemical institute in Shanghai product, other (its reagent name of the present invention) reagent are pure for analyzing.
Capillary electrophoresis apparatus is the TriSep of Unimicro Technology
TM-2000GV type comprises a Data Module wavelengthtunable ultraviolet-visible detecting device, the continuous adjustable high-voltage DC power supply of CEC control module; Data aggregation and spectrogram are handled and are adopted according to the Echrom98 of Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S. workstation and Echrom98 V2.0 software.
Synthetic route as shown in Figure 1.Selection is with methacrylate glycol ester (EDMA, ethylenedimethacrylate) as the poly (glycidyl methacrylate) (GMA of crosslinking chemical, glycidylmethacrylate) as poriness integral post matrix, capillary tube inner wall is in advance through the silane reagent modification with two key functional groups, and monomer polymerization therein also is fixed on the capillary wall by chemical bond.The glycidyl of GMA specifically is that such porosint in-situ polymerization is made into integral post and is applied to separating experiment in kapillary as the carrier ampholyte molecule that active reactive group is used for deriving and fixedly contains the many carboxyls of polyamino.Take by weighing GMA 100mg, EDMA 80mg, be dissolved in 400mg pore-foaming agent cyclohexanol/1, in the mixed solvent of 4-butylene glycol (mass ratio 3/2).Add 6mg initiating agent azobis isobutyronitrile (AIBN), ultrasonic degas 5 minutes is cooled to 4 ℃.Get above-mentioned solution with syringe and inject kapillary, two ends are sealed with silicon rubber, place 60 ℃ of water-bath reactions 12 hours.
In order to improve the reactivity of epoxy radicals, adopt water+ethanol mixed solvent in the experiment, as specifying, it is water+ethanol preparation of 1: 1 that following solution all uses volume ratio.The integral post that polymerization finishes is taken out, washed polymkeric substance 5 minutes with ethanolic solution, so that remove unnecessary initiating agent, unreacted monomer and pore-foaming agent.The carrier ampholyte (Ampholine) of volume ratio 2% is injected kapillary, silicon rubber sealing two ends, 60～80 ℃ were reacted 2～8 hours, and ethanol is cleaned and is got final product.Perhaps continuous 6 hours pumps of 1M pentanediamine aqueous solution are crossed integral post, with the matrix epoxy reaction with manual pump.Clean unreacted amine with distilled water, pump into 10% glutaraldehyde solution reaction 12 hours.Behind the clean unreacted glutaraldehyde of distilled water, 2% carrier ampholyte (isoelectric point is the polyamino polycarboxylic acid mixing solution of 0-14) is injected kapillary, leave standstill reaction 2～24 hours under the room temperature, the sodium borohydride of 25mmol/L or bromine sodium borohydride are at room temperature reacted got final product in 1～24 hour.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 0～14.
Experimental result:
Obtain porous novel gradient parting material, its scanning electron microscope such as Fig. 2, pore diameter distribution is shown in Fig. 3, and it takes the integral post of Fig. 4, the chip form of Fig. 5 in appearance.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 0～14.
Porosint is the preparation method of silica gel
The trimethoxy aminopropyl silane of 50% volume ratio was 0 ℃ of hydrolysis 12 hours, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, at room temperature reacted 24 hours with 25% glyoxal or butanedial or glutaraldehyde, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, the feeding volume ratio is 1% carrier ampholyte (isoelectric point is the sour mixed solutions of the many phosphorus of the polyamino of 3-11 (phosphine)), leave standstill reaction 24 hours under the room temperature, the sodium borohydride of 25mmol/L or bromine sodium borohydride are at room temperature reacted got final product in 2 hours.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 3～10.
Embodiment 3
Porosint is the preparation method of Lauxite
The urea liquid of the formalin of 25% volume ratio and 50% volume ratio was 40 ℃ of reactions 12 hours, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, reacted 10 hours down at 70 ℃ with 10% oxirane or epoxypropane, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, feed the carrier ampholyte (isoelectric point is the many sulfuric acid mixed solutions of the polyamino of 2-4) of volume ratio 2%, leave standstill reaction 12 hours under the room temperature, the sodium borohydride of 25mmol/L or bromine sodium borohydride are at room temperature reacted got final product in 4 hours.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 1～3.
Embodiment 4
Porosint is the preparation method of silicon dioxide
Solid silicone soaked 24 hours with concentrated hydrochloric acid, clean and 80～100 ℃ of dryings 12 hours with chloroform, reacted 4 hours down at 70 ℃ with the epichlorokydrin of drying, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, feed volume ratio 4% carrier ampholyte (isoelectric point is many sulfonic acid of polyamino mixed solution of 4-6), 80 ℃ of reactions got final product in 4 hours.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 3～5.
Embodiment 5
Porosint is the preparation method of zirconium dioxide
The solid Zirconium dioxide powder soaked 24 hours with concentrated hydrochloric acid, clean and 80～100 ℃ of dryings 12 hours with chloroform, reacted 4 hours down at 50 ℃ with the epichlorokydrin of drying, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, feed volume ratio 5% carrier ampholyte (isoelectric point is many sulfonic acid of polyamino mixed solution of 6-13), 80 ℃ of reactions got final product in 4 hours.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 5～8.
Embodiment 6
Porosint is the preparation method of titania
The solid titania powder soaked 24 hours with concentrated hydrochloric acid, clean and 80～100 ℃ of dryings 12 hours with chloroform, reacted 4 hours down at 0～60 ℃ with the epichlorokydrin of drying, water, methyl alcohol, ethanol or acetonitrile washed 10 minutes, feed volume ratio 10% carrier ampholyte (isoelectric point is the many sulfuric acid mixed solutions of the polyamino of 7-12), 80 ℃ of reactions got final product in 4 hours.This parting material is placed the solution of different pH values, add DC voltage at the material two ends, can form supported pH gradient in this gradient separation material, its scope is that pH is between 8～11.
Embodiment 7
From the porosint of above-mentioned each embodiment preparation, choose one section evenly, the separating medium (integral post or chip form) in non-cracking, no cavity, be the H of 10mmol/L with concentration
2SO
4Handled last distilled water flushing pillar 5 minutes 2 hours.Be full of the kilnitamin sample solution therein, add the voltage of 2～8kV/cm, carry out isoelectric focusing and while image data.
Experimental result:
As shown in Figure 6, only differ a methylene on valine, the leucine molecular structure, its isoelectric point is respectively 5.97,5.98, and difference only is 0.01 pH unit, can well be separated by these two amino acid of porous medium of the present invention.
Difference from Example 4 is: the sample that this experiment is chosen is the protein mixed solution, after utilizing voltage (2kV/5sec) or pressure (0.1MPa/5sec) sample introduction, take the mode of gradient ion exchange that sample is separated, eluent is a 20mmol phosphate, its pH value from 3 to 9.
Experimental result:
As shown in Figure 7, the protein of 5 different Acidity of Aikalinitys such as elastoser, haemoglobin, insulin, ovalbumin, casein, different molecular weight has obtained fabulous separation.
Relevant comparative example
Hochstrasser (document: Hochstrasser D., Augsburger, V., Funk, M., Appel, R., Pellegrini, C., Muller, A.F.Electrophoresis 1986,7,505-511.) wait a kind of carrier ampholyte immobilization pH gradient (CAs-IPG) technology of introducing, its method is carrier ampholyte to be added in third rare amide solution carry out polymerization, utilizes this gel to carry out the isoelectric focusing experiment then.Because carrier ampholyte just adds in the solution simply, and it is immobilized to be unrealized,, cause risk of short-circuits so under high-tension situation, from plastic tube, overflow easily.
Claims (5)
1. gradient separation material, this gradient separation material are to be fixed with the molecule that contains positive and negative electric charge group simultaneously on the porosint of column or sheet, and the pH gradient scope is 0～14 or wherein any one section; The aperture of this porosint is 10
-9To 10
-5Rice.
2. the gradient separation material of claim 1 is characterized in that, porosint is poly-(methyl) acrylate, silica gel, Lauxite, silicon dioxide, zirconium dioxide or titania.
3. the gradient separation material of claim 1 is characterized in that, the molecule that contains positive and negative electric charge group simultaneously is a carrier ampholyte.
4. claim 1 or 3 degree parting material is characterized in that, carrier ampholyte is the many carboxyls of polyamino, the many phosphorus of polyamino (phosphine) acid, the many sulfonic acid of polyamino or the many sulfuric acid of polyamino.
5. method for preparing the described gradient separation material of claim 1, key step is:
A) to feed volume ratio be the carrier ampholyte of 1-10% to the porosint that will have a reactive group, in ℃ down reaction 2-24 hour of room temperature to 80;
B) material of step a preparation places the solution of different pH values, and its two ends add DC voltage, form supported pH gradient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510054456 CN1831526A (en) | 2005-03-07 | 2005-03-07 | Gradient separation material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510054456 CN1831526A (en) | 2005-03-07 | 2005-03-07 | Gradient separation material |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1831526A true CN1831526A (en) | 2006-09-13 |
Family
ID=36993941
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510054456 Pending CN1831526A (en) | 2005-03-07 | 2005-03-07 | Gradient separation material |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1831526A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101806787A (en) * | 2010-04-02 | 2010-08-18 | 北京化工大学 | Alkali-resistance high-efficiency liquid phase chromatography integral post and preparation method thereof |
| CN102993229A (en) * | 2012-12-19 | 2013-03-27 | 宁波工程学院 | Amphoteric electrolyte-modified hybrid silica gel material and solid-phase extraction method thereof |
| CN101294930B (en) * | 2007-04-27 | 2013-08-14 | 杨春 | Integral immobilization pH gradient production method and application thereof |
| CN103864967A (en) * | 2012-12-11 | 2014-06-18 | 中国科学院大连化学物理研究所 | Polymer particles modified by amphoteric carrier and application thereof in pretreatment of protein sample |
| CN109675344A (en) * | 2018-12-26 | 2019-04-26 | 上海交通大学 | Solidify the capillary isoelectric focusing hydrophily integral post and preparation method of pH gradient |
-
2005
- 2005-03-07 CN CN 200510054456 patent/CN1831526A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101294930B (en) * | 2007-04-27 | 2013-08-14 | 杨春 | Integral immobilization pH gradient production method and application thereof |
| CN101806787A (en) * | 2010-04-02 | 2010-08-18 | 北京化工大学 | Alkali-resistance high-efficiency liquid phase chromatography integral post and preparation method thereof |
| CN101806787B (en) * | 2010-04-02 | 2012-01-04 | 北京化工大学 | Alkali-resistance high-efficiency liquid phase chromatography integral post and preparation method thereof |
| CN103864967A (en) * | 2012-12-11 | 2014-06-18 | 中国科学院大连化学物理研究所 | Polymer particles modified by amphoteric carrier and application thereof in pretreatment of protein sample |
| CN103864967B (en) * | 2012-12-11 | 2016-03-23 | 中国科学院大连化学物理研究所 | Pharmalyte modify polymer beads and apply in protein example pre-treatment |
| CN102993229A (en) * | 2012-12-19 | 2013-03-27 | 宁波工程学院 | Amphoteric electrolyte-modified hybrid silica gel material and solid-phase extraction method thereof |
| CN102993229B (en) * | 2012-12-19 | 2015-06-03 | 宁波工程学院 | Amphoteric electrolyte-modified hybrid silica gel material and solid-phase extraction method thereof |
| CN109675344A (en) * | 2018-12-26 | 2019-04-26 | 上海交通大学 | Solidify the capillary isoelectric focusing hydrophily integral post and preparation method of pH gradient |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0354984A2 (en) | Capillary tube with reduced protein interactions and controllable electroosmotic flow | |
| Gübitz et al. | Recent progress in chiral separation principles in capillary electrophoresis | |
| Gübitz et al. | Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography | |
| Schaller et al. | Separation of antidepressants by capillary electrophoresis with in-line solid-phase extraction using a novel monolithic adsorbent | |
| Kato et al. | Enantiomeric separation of amino acids and nonprotein amino acids using a particle‐loaded monolithic column | |
| US8182730B2 (en) | Method for forming a concentrating device | |
| CN1831526A (en) | Gradient separation material | |
| Huang et al. | Preparation and evaluation of molecularly imprinted polymers based on 9‐ethyladenine for the recognition of nucleotide bases in capillary electrochromatography | |
| CN1185836A (en) | Protein separations by capillary electrophoresis using amino acid-containing buffers | |
| CN1242262C (en) | Method for sedparating bialogical macromolecule by using two-dimensinal or multi-dimensinal capillary electrophoresis and its used interface | |
| EP0452055B1 (en) | Surfaces with reduced protein interactions | |
| US7815783B2 (en) | Multi-compartment filter and method of filtering using same | |
| Zhu et al. | Macroporous polyacrylamide‐based monolithic column with immobilized pH gradient for protein analysis | |
| US5006313A (en) | Halogenated surface with reduced protein interaction | |
| Yang et al. | Repeatedly usable immobilized pH gradient in a monolithic capillary column | |
| CN101294930B (en) | Integral immobilization pH gradient production method and application thereof | |
| CN1668367A (en) | Method for pH-biased isoelectric trapping separation | |
| Yan et al. | Capillary electrochromatographic separation of ionizable compounds with a molecular imprinted monolithic cationic exchange column | |
| CN1588041A (en) | Capillary liquid phase chromatographic column and its preparing method | |
| Liao et al. | Rapid separation and determination of metronidazole benzoate and other antiprotozoal drugs by pressurized capillary electrochromatography | |
| Liang et al. | Separation, identification, and interaction of heparin oligosaccharides with granulocyte‐colony stimulating factor using capillary electrophoresis and mass spectrometry | |
| CN107297201B (en) | 4, 5-imidazole dicarboxylic acid modified organic-silica gel hybrid monolithic column and application | |
| CN107297087B (en) | Histidine-modified organic-silica gel hybrid monolithic column and preparation method and application thereof | |
| CN1719244A (en) | A kind of Capillary Electrophoresis multi dimension device | |
| CN1317051C (en) | Size exclusion chromatography method for separating biomacromolecule of preparation type transverse electric field |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |