CN101254458A - Ion exchange chromatography fixed phase, preparing method and application of the same - Google Patents
Ion exchange chromatography fixed phase, preparing method and application of the same Download PDFInfo
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- CN101254458A CN101254458A CNA2007101687317A CN200710168731A CN101254458A CN 101254458 A CN101254458 A CN 101254458A CN A2007101687317 A CNA2007101687317 A CN A2007101687317A CN 200710168731 A CN200710168731 A CN 200710168731A CN 101254458 A CN101254458 A CN 101254458A
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- ion
- exchange chromatography
- hydroxyapatite
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- silica gel
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Abstract
A stationary phase for ion-exchange chromatography is provided. The stationary phase comprises a chromatographic matrix superficially coated with hydroxyapatite. The stationary phase for ion-exchange chromatography has good biocompatibility and bioaffinity and higher mechanical strength. The stationary phase for ion-exchange chromatography is prepared by the following steps: activating an inorganic chromatographic matrix material such as silica gel or Zr/Mg composite oxide, immersing the matrix material into an oversaturated calcium/phosphorus solution, standing for deposition at 36.5 plus or minus 0.5 DEG C to obtain hydroxyapatite coating, washing with water twice, and drying to obtain the hydroxyapatite-coated stationary phase for ion-exchange chromatography in the protein separation. The method is also used to prepare a coating on the inner wall of a capillary tube to reduce the non-specific adsorption of proteins and simultaneously to provide an ion-exchange function for improvement of separation selectivity.
Description
Technical field
The present invention relates to fixedly phase and its production and application of ion-exchange chromatography.
Background technology
Ion-exchange is requisite clastotype in the high performance liquid chromatography (HPLC), and it is widely used in biomedical sector, as amino acid analysis, and the separating of peptide and protein.When using the ion-exchange chromatography isolated protein, the power of the ion capacity of the ion-exchanger on the charge density of separated protein and isoelectric point value and chromatographic column size decision reserve capability.
The fixing composition by its matrix of ion-exchange chromatography roughly is divided into organic and inorganic matrix two big classes.The organic substrate filler has good chemical stability, can use in the pH value scope widely, and the ability alkali cleaning has power of regeneration preferably.But their mechanical strengths are not high, and the pore structure more complicated is easy to generate swelling or shrinkage phenomenon in elution process, these effects limit the organic substrate filler in ion-exchange chromatography, further use.At present, the inorganic matrix filler that comprises silica gel, zirconium dioxide is research and the main flow used.Silica gel has the pore structure and the specific area of excellent mechanical intensity, control easily, it is unstable in alkaline medium (pH＞8), and stronger interaction takes place amino easy and protein after the silicon hydroxyl deprotonation on surface, thereby be unsuitable for Separation of Proteins.The zirconium dioxide chemical stability is good, and the surface exists abundant lewis acid site, can realize the separation of alkaline protein after to its modification with lewis base.Its shortcoming is exactly that specific area is little, and the aperture is difficult to control.
Hydroxyapatite also is a kind of host material that can be used for liquid chromatography stuffing, and main component is a calcium hydroxy phosphate, Ca10 (PO4) 6 (OH) 2.Hydroxyapatite belongs to hexagonal, two kinds of different crystal faces are arranged on the crystal, two kinds of adsorption sites have been formed with different characterization of adsorptions, promptly be positioned at the site and the cationic site of absorption that is positioned on the PO43-ion of the adsorpting anion on the Ca2+ ion, therefore have the characteristic of weak anionic and weak cation exchange.In addition, hydroxyapatite is the basis of animal skeleton, has good biocompatibility, so it can be as the ion-exchange chromatography filler of Separation of Proteins.This application starts from people's such as Tiselius in 1956 report.Nineteen eighty-three, the HPLC chromatographic column that commercial hydroxyapatite is filled has appearred.This hydroxyapatite is made up of tiny flat crystal, and the shape size differences is big, and broken easily under the pressure of phase that flows, thereby post is imitated, the permeability and the stability of pillar are not fine, has limited being extensive use of of it.
Recent two decades comes, and in order to improve the mechanical strength of hydroxyapatite, improves separating property, the expansion scope of application, and people have proposed various approach and have remedied the frangible shortcoming of hydroxyapatite crystal.Be the preparation ball shaped hydroxy-apatite on the one hand, as secondary granulation technology, spray pyrolysis etc.But these spheric granules are hydroxyapatite crystallites to form by the physical action accumulation, and restriction is received in the improvement of mechanical strength.The ball shaped hydroxy-apatite of the homogeneous phase method preparation that Chinese workers Institute of Metallurgical Technology of the Chinese Academy of Sciences proposes has that particle diameter is controlled, the characteristics of stable performance, suitablely fills out the HPLC chromatographic column that post forces down, flow velocity is fast.Be the compound of preparation hydroxyapatite on the other hand.There is research that the silica gel microball of phosphorylation is successively immersed substitute that calcium chloride and phosphoric acid solution obtain hydroxyapatite and is used for separating topoisomerase in the wheat embryo.Human dry processes such as Honda hydroxyapatite or ox bone ash coat poly compound, under the pattern of gradient elution, successfully separated five kinds of protein.Yet, because Irreversible Adsorption can take place between protein and the polyethylene, thereby cause protein recovery not high in hydrophobic effect.
Summary of the invention
The invention provides a kind of fixedly phase and its production and use of ion-exchange chromatography that hydroxyapatite simple, with low cost coats for preparing.
Technical scheme provided by the invention is: a kind of ion-exchange chromatography is phase fixedly, and the chromatography matrix material that is coated with hydroxyapatite by the surface constitutes.
Above-mentioned chromatography matrix material is silica gel or zirconium Magnesium coumpoud oxide.
The present invention also provides the fixedly preparation method of phase of above-mentioned ion-exchange chromatography: with silica gel microball or zirconium Magnesium coumpoud oxide microballoon in 36.5 ± 0.5 ℃, be soaked in the supersaturation calcium phosphorus solution 20-30 days, and obtained the chromatography matrix material that the surface is coated with hydroxyapatite and be fixedly phase of ion-exchange chromatography.
Above-mentioned silica gel microball through the activation of peracid, is soaked in the supersaturation calcium phosphorus solution earlier again.
Above-mentioned zirconium Magnesium coumpoud oxide is handled through pre-calcification earlier, is soaked in the supersaturation calcium phosphorus solution again.
Above-mentioned supersaturation calcium phosphorus solution can prepare by laxative remedy: with sodium chloride, and potassium chloride, sodium hydrogen phosphate, hydrochloric acid, calcium chloride are dissolved in the secondary water successively, and its final ion concentration is respectively [Na
+]=136.8mM, [K
+]=3.71mM, [Cl
-]=144.5mM, [Ca
2+]=3.10mM, [HPO
4 2-]=1.86mM; Regulating the pH value with trishydroxymethylaminomethane at last is 7.4.
The fixing separation that can be used for protein mutually of above-mentioned ion-exchange chromatography, not can with protein and other generation non-specific adsorption, can obtain the very high rate of recovery.
The present invention is with sodium chloride, potassium chloride, sodium hydrogen phosphate, calcium chloride, hydrochloric acid, trishydroxymethylaminomethane, water is raw material, adopt bionics techniques, coat hydroxyapatite coating layer, obtain fixedly phase of a kind of novel ion-exchange chromatography on inorganic matrix chromatograph packing material surface.This filler can be realized the separation of protein owing to have good biocompatibility and biological affinity and higher mechanical strength under HPLC.In addition, this method also can be used for preparing the hydroxyapatite coating layer capillary, reduces the non-specific adsorption of protein and capillary tube inner wall, improves separation selectivity.The inventive method is simple to operate, with low cost.
Description of drawings
The hydroxyapatite that Fig. 1 makes in the present invention for protein coats the fixing separation of going up mutually of zirconium Magnesium coumpoud oxide ion-exchange chromatography.1 is bovine serum albumin(BSA) among the figure; 2 is trypsase; 3 is lysozyme; 4 is ribonuclease A; 5 is cytochrome c.
The hydroxyapatite that Fig. 2 makes in the present invention for protein coats the fixedly phase chromatographic stationary separation of going up mutually of silica gel ion-exchange chromatography.1 is bovine serum albumin(BSA) among the figure; 2 is trypsase; 3 is ribonuclease A; 4 is cytochrome c; 5 is lysozyme.
The specific embodiment
Embodiment 1: hydroxyapatite coats zirconium Magnesium coumpoud oxide the ion-exchange chromatography fixedly preparation of phase and the separation of protein
(1) preparation of supersaturation calcium phosphorus solution: analytically pure sodium chloride, potassium chloride, sodium hydrogen phosphate, hydrochloric acid, calcium chloride is water-soluble successively, and its ion concentration is respectively [Na
+]=136.8mM, [K
+]=3.71mM, [Cl
-]=144.5mM, [Ca
2+]=3.10mM, [HPO
4 2-]=1.86mM.It is 7.4 in 5 ℃ of preservations that the solution for preparing is regulated the pH value with trishydroxymethylaminomethane.
(2) deposition growing of hydroxyapatite coating layer: after the zirconium Magnesium coumpoud oxide usefulness 1M naoh treatment, be washed till neutrality, 160 ℃ following dry 8 hours.Then, immerse successively in 0.5M disodium phosphate soln and the saturated aqua calcis, clean up back 120 ℃ of dryings.It is dipped in the 500mL supersaturation calcium phosphorus solution, keeping temperature is 36.5 ± 0.5 ℃ again, takes out after immersion a period of time, and secondary water is rinsed well, 120 ℃ of drying for standby.
(3) separation of protein: after 600 ℃ of processing of above-mentioned material, in the 150mm that packs into * 4.6mmi.d. chromatographic column, be the phase that flows, separated five kinds of protein (as Fig. 1) under the gradient mode with PBS.
Embodiment 2: hydroxyapatite coats silica gel microball the ion-exchange chromatography fixedly preparation of phase and the separation of protein
After the acid treatment of 5 μ m silica gel microballs usefulness 1M salt, be washed till neutrality, 160 ℃ following dry 8 hours.It is dipped in the supersaturation calcium phosphorus solution of 500mL, and keeping temperature is 36.5 ± 0.5 ℃, takes out after immersion a period of time, and secondary water is rinsed well, 120 ℃ of dryings.Above-mentioned material is packed in 150mm * 4.6mmi.d. chromatographic column, is the phase that flows with PBS, has separated five kinds of protein (as Fig. 2) under the gradient mode.
Embodiment 3: hydroxyapatite coating layer preparation capillaceous
Get capillary, use the 1M sodium hydroxide solution successively, secondary water, 1M hydrochloric acid, secondary water flushing 1 hour, back 120 ℃ of dryings 4 hours under logical nitrogen.Under the gravity effect supersaturation calcium phosphorus solution is injected capillary continuously, keeping temperature is 36.5 ± 0.5 ℃, deposits 14 days, prepares the capillary of hydroxyapatite coating layer.
Claims (7)
1. the fixing phase of an ion-exchange chromatography is characterized in that: this fixing chromatography matrix material that is coated with hydroxyapatite by the surface constitutes.
2. ion-exchange chromatography according to claim 1 is phase fixedly, it is characterized in that: the chromatography matrix material is silica gel or zirconium Magnesium coumpoud oxide.
3. the fixing preparation method of phase of claim 1 or 2 described ion-exchange chromatographies, it is characterized in that: with silica gel microball or zirconium Magnesium coumpoud oxide microballoon in 36.5 ± 0.5 ℃, be soaked in the supersaturation calcium phosphorus solution 20-30 days, and obtained the chromatography matrix material that the surface is coated with hydroxyapatite and be fixedly phase of ion-exchange chromatography.
4. preparation method according to claim 3 is characterized in that: silica gel microball through the activation of peracid, is soaked in the supersaturation calcium phosphorus solution earlier again.
5. preparation method according to claim 3 is characterized in that: the zirconium Magnesium coumpoud oxide is handled through pre-calcification earlier, is soaked in the supersaturation calcium phosphorus solution again.
6. according to claim 3 or 4 or 5 described preparation methods, it is characterized in that: supersaturation calcium phosphorus solution is formulated by sodium chloride, potassium chloride, sodium hydrogen phosphate, calcium chloride, hydrochloric acid and trishydroxymethylaminomethane: sodium chloride, potassium chloride, sodium hydrogen phosphate, hydrochloric acid and calcium chloride are dissolved in the secondary water successively, and its final ion concentration is respectively [Na
+]=136.8mM, [K
+]=3.71mM, [Cl
-]=144.5mM, [Ca
2+]=3.10mM, [HPO
4 2-]=1.86mM; Regulating the pH value with trishydroxymethylaminomethane at last is 7.4.
7. the fixing application in Separation of Proteins of claim 1 or 2 described ion-exchange chromatographies.
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| CNA2007101687317A CN101254458A (en) | 2007-12-11 | 2007-12-11 | Ion exchange chromatography fixed phase, preparing method and application of the same |
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| CNA2007101687317A CN101254458A (en) | 2007-12-11 | 2007-12-11 | Ion exchange chromatography fixed phase, preparing method and application of the same |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103191705A (en) * | 2013-04-15 | 2013-07-10 | 武汉大学 | Preparation method of open tubular capillary electrochromatographic column |
| CN102006927B (en) * | 2008-04-18 | 2013-07-24 | Hoya株式会社 | Coated particles, method of producing coated particles and adsorption apparatus |
| CN108079976A (en) * | 2017-12-07 | 2018-05-29 | 辽宁科技大学 | Nanometer hydroxyapatite/diallyl dimethyl ammoniumchloride/material silica gel composite preparation method |
| CN109174010A (en) * | 2018-08-31 | 2019-01-11 | 深圳市迪莫斯环保科技有限公司 | One kind is except modified formaldehyde silica gel material and preparation method thereof |
| CN108079977B (en) * | 2017-12-07 | 2020-10-16 | 辽宁科技大学 | Preparation method of nano hydroxyapatite/polyhexamethylene guanidine hydrochloride/silica gel composite material and solid phase extraction method |
| CN112973806A (en) * | 2021-02-09 | 2021-06-18 | 珠海高新区维得力生物工程有限公司 | Chromatographic separation filler applied to fructo-oligosaccharide, preparation process and chromatographic separation device |
-
2007
- 2007-12-11 CN CNA2007101687317A patent/CN101254458A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102006927B (en) * | 2008-04-18 | 2013-07-24 | Hoya株式会社 | Coated particles, method of producing coated particles and adsorption apparatus |
| CN103191705A (en) * | 2013-04-15 | 2013-07-10 | 武汉大学 | Preparation method of open tubular capillary electrochromatographic column |
| CN103191705B (en) * | 2013-04-15 | 2014-09-17 | 武汉大学 | Preparation method of open tubular capillary electrochromatographic column |
| CN108079976A (en) * | 2017-12-07 | 2018-05-29 | 辽宁科技大学 | Nanometer hydroxyapatite/diallyl dimethyl ammoniumchloride/material silica gel composite preparation method |
| CN108079976B (en) * | 2017-12-07 | 2020-10-16 | 辽宁科技大学 | Preparation method of nano hydroxyapatite/poly (diallyldimethylammonium chloride)/silica gel composite material |
| CN108079977B (en) * | 2017-12-07 | 2020-10-16 | 辽宁科技大学 | Preparation method of nano hydroxyapatite/polyhexamethylene guanidine hydrochloride/silica gel composite material and solid phase extraction method |
| CN109174010A (en) * | 2018-08-31 | 2019-01-11 | 深圳市迪莫斯环保科技有限公司 | One kind is except modified formaldehyde silica gel material and preparation method thereof |
| CN112973806A (en) * | 2021-02-09 | 2021-06-18 | 珠海高新区维得力生物工程有限公司 | Chromatographic separation filler applied to fructo-oligosaccharide, preparation process and chromatographic separation device |
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Open date: 20080903 |