CN101224414A - Multifunctional separation medium with tetrazole as functional group and preparing method thereof - Google Patents

Multifunctional separation medium with tetrazole as functional group and preparing method thereof Download PDF

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CN101224414A
CN101224414A CNA2007100189051A CN200710018905A CN101224414A CN 101224414 A CN101224414 A CN 101224414A CN A2007100189051 A CNA2007100189051 A CN A2007100189051A CN 200710018905 A CN200710018905 A CN 200710018905A CN 101224414 A CN101224414 A CN 101224414A
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tetrazolium
alpha
separation medium
hydroxy
glucan
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卫引茂
雷根虎
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Northwest University
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Abstract

The invention discloses a multi-purpose separation medium and a preparation method thereof, wherein, tetrazole is the functional group of the medium. More particularly, a separation medium with novel structure is prepared by linking the tetrazole on the surface of silica gel, dextran and agarose gel separation group. The invention has dual purposes of being applied to ion-exchange chromatography and metal chelate chromatography. When the medium is applied to the ion-exchange separation of proteins, the separation of proteins is highly selective, easy to regenerate and quick to separate and both the quality and recovery rate of the activity of the proteins are high. When the medium is applied to the metal chelate chromatography of proteins, the separation of proteins is highly selective, small in metal ion loss and quick to separate and both the quality and recovery rate of activity of the proteins are high. The invention can be applied to the quick separation and purification of genetic engineering products and plasma proteins.

Description

It with the tetrazolium multifunctional separation medium of functional group and preparation method thereof
Technical field
The invention belongs to the chromatographic separation technology field, being specifically related to the tetrazolium is multifunctional separation medium of functional group and preparation method thereof.
Background technology
Chromatographic separation technology is one of common method that large biological molecule separates in the bioengineering field.In the method, separating medium is the critical material in the whole isolation technics, and its consumption accounts for about 50% of production cost.
At present, the pattern that is used for the large biological molecule chromatographic isolation mainly contains: reverse-phase chromatography, hydrophobic interaction chromatograph, ion-exchange chromatography, affinity chromatography (comprising immobilized metal ion afinity chromatography) and exclusion chromatography.Ion-exchange chromatography (IEC) is a method wherein commonly used.As everyone knows, according to the fixing charged character that goes up ion-exchange group mutually, IEC is fixing, and the cation exchange that can be divided into is mutually fixed mutually with anion-exchange chromatography; According to the dissociation degree of cation exchange groups, it is fixing mutually with weak sun/weak anionic exchange chromatography to be divided into strong sun/reinforcing yin essence ion again.At present, the cation exchange groups that goes up mutually of weak cation crossover fixation comprises carboxyl and phosphate.Protein not only depends on character and the quantity of its surface charging lotus in such fixing reservation of going up mutually, and relevant with the fixing dissociation degree that goes up carboxyl or phosphate mutually.So, when setting up the separation condition of protein, can select suitable pH value to make protein obtain Selective Separation.But along with proteomics, metabolism group and engineered development, existing fixedly phase factor poor selectivity can not satisfy the needs that complex biological sample is separated.Therefore, the function separating medium of development property still is the challenge that faces in the chromatogram research.
Immobilized metal ion afinity chromatography is to rely on fixing part and the transition metal ions chelating of going up mutually, then protein again with metal ion-chelant, according to the power of chelation power large biological molecule is carried out the method for Selective Separation, in the haemocyanin separation, be widely used.In the method, influence separation selectivity ground factor and mainly contain: ligand structure, species of metal ion and chromatogram flow phase are formed.At present, the fixing ligand species of going up mutually mainly contains: (i) bidentate ligand, and as salicylic aldehyde (salicylaldehyde), ammonia hydroxy amide acid (aminohydroxamic acid) or oxine (8-hydroxy-quinoline); (ii) tridentate ligand, as o-phosphoserine (ortho-phosphoserine), pyridine diamines (dipicolylamine), cis or trans-carboxyl-methylproline (cis-or trans-carboxy-methylproline), amino oxalic acid (Iminodiacetic acid) and N-(2-picolyl) amion acetic acid (N-(2-pyridylmethyl) aminiacetate); (iii) tetradentate ligands is as nitrilotriacetic acid (N-(2-pyridylmethyl) aminiacetate); (iv) five tooth parts, as N, N, N-three-(carboxymethyl) ethylene diamine (N, N, N-tris-(carboxy-methyl) ethylenediamine).The intensity that these parts combine with metal and different to the selectivity of protein.Simultaneously, these parts still exist the risk that metal ion runs off, and purposes is single.
Summary of the invention
The new multi-purpose chromatography separation media that one of purpose of the present invention provides a kind of long service life, is easy to regenerate, this separating medium both can be used as the ion-exchange separating medium and had used, and also can be used as the immobilized metal ion afinity chromatography separating medium and used.
Another object of the present invention provides the preparation method of above-mentioned multipurpose chromatography separation media.
Implementation procedure of the present invention is as follows:
With the tetrazolium is the multifunctional separation medium of functional group, has following general structure:
Figure S2007100189051D00021
Figure S2007100189051D00022
Represent silica gel, agarose or glucan; R 1Be O or NH; R 2Be H, C 1-C 4Alkyl, phenyl or benzyl.
The preparation method of above-mentioned multifunctional separation medium: silica gel and γ-glycidoxypropyltrietandysilane andysilane reaction, the silica gel of synthetic surface bonding epoxy radicals reacts under alkali or BFEE catalyst action with alpha-amido tetrazolium or Alpha-hydroxy tetrazolium then and promptly gets the tetrazolium multifunctional separation medium;
Described alpha-amido tetrazolium structure is as follows:
Figure S2007100189051D00031
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl; Described Alpha-hydroxy tetrazolium structure is as follows:
Figure S2007100189051D00032
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl.
Glucan or Ago-Gel and epichlorohydrin reaction, the glucan or the Ago-Gel of synthetic surface bonding epoxy radicals react under alkali or BFEE catalytic action with alpha-amido tetrazolium or Alpha-hydroxy tetrazolium then and promptly get the tetrazolium multifunctional separation medium;
Described alpha-amido tetrazolium structure is as follows:
Figure S2007100189051D00033
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl; Described Alpha-hydroxy tetrazolium structure is as follows:
Figure S2007100189051D00034
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl.
Silica gel and γ-glycidoxypropyltrietandysilane andysilane reaction, the silica gel of synthetic surface bonding epoxy radicals reacts with alpha-hydroxy nitrile under BFEE catalysis then, in the silica gel of bonding alpha-hydroxy nitrile, adds DMF, NaN 3And NH 4The Cl reaction promptly gets the tetrazolium multifunctional separation medium.
Glucan or Ago-Gel and epichlorohydrin reaction, the glucan or the Ago-Gel of synthetic surface bonding epoxy radicals, under BFEE catalysis, react then, in the glucan or Ago-Gel of bonding alpha-hydroxy nitrile, add DMF, NaN with alpha-hydroxy nitrile 3And NH 4The Cl reaction promptly gets the tetrazolium multifunctional separation medium.
The present invention is bonded in silica gel, glucan and Ago-Gel isolation medium surface with chemical method with tetrazolium, the separating medium that has prepared a kind of novel structure, do not use when this medium can be used as the ion-exchange separating medium during chelated metal ions, use if can be used as the immobilized metal ion afinity chromatography separating medium on the chelating behind the metal ion again.
Advantage of the present invention and good effect:
(1) multifunctional separation medium of the present invention has the dual-use function of using on ion-exchange chromatography and immobilized metal ion afinity chromatography;
(2) multifunctional separation medium of the present invention is used for the ion-exchange separation of protein, and the choosing of protein separates selecting property height, albumen quality and activity recovery height; The immobilized metal ion afinity chromatography that is used for protein, the choosing of protein separates selecting property height, and metal ion runs off little, and separating rate is fast, white matter amount and activity recovery height.At room temperature, the activity of lysozyme rate of recovery＞95%.Myoglobins, ribonucleic acid, Chymetin be former-mass recovery＞95% of A, cytochromes-C and lysozyme.Cu on the chelating 2+, used Gu continuously 2 days 2+The chelating amount loss of ion is less than 5%.This multifunctional separation medium can be widely used in the separation and purification of albumen, enzyme and Bio-engineering Products.
(3) multifunctional separation medium long service life of the present invention.The silica gel medium under the pH=2.0-8.0 situation, the glucan medium used 1000 hours under the pH=1.0-12.0 situation, after cleaning, separating property is not seen change.
(4) multifunctional separation medium of the present invention is easy to regeneration, needs only with the renewable use of 10 times of volume 1mol/L sodium chloride washing agent, and not resembling some commodity posts will regenerate with NaOH behind each sample introduction.
(5) urea of Bio-engineering Products extracts and also can directly go up sample, and step separation has reached higher degree.
Description of drawings
Fig. 1 is the ion-exchange chromatography separation graph of protein on alpha-amido phenylpropyl alcohol tetrazolium filler (silica matrix).
Fig. 2 is the ion-exchange chromatography separation graph of protein on Alpha-hydroxy benzene second tetrazolium filler (silica matrix).
Fig. 3 is that albumen is at Zn 2+Immobilized metal ion afinity chromatography figure on the alpha-amido phenylpropyl alcohol tetrazolium filler (silica matrix) of chelating
Fig. 4 is that albumen is at Zn 2+Immobilized metal ion afinity chromatography figure on the Alpha-hydroxy benzene second tetrazolium filler (silica matrix) of chelating
Fig. 5 is gene engineering product recombinant human interferon-'s a 8mol/L urea extract separation graph
The specific embodiment
The fixing mutually used primary raw material of synthetic multipurpose tetrazolium: macro porous silica gel (7 μ m, aperture 30nm), cross-linked agarose gel (Bio-sep Bio-technique Stock Co., Ltd. Xi'an Jiaotong University), sephadex (Shanghai monarch create thing science and technology fine chemical product), γ-glycidoxypropyltrietandysilane andysilane, acetaldehyde cyanhydrin, hydroxyacetonitrile, Alpha-hydroxy benzene acetonitrile, sodium azide and epoxychloropropane are the commercial goods.
The alpha-amido tetrazolium and the Alpha-hydroxy tetrazolium that use prepare as follows:
(1) preparation (R of alpha-amido tetrazolium 2Can select CH 2CH (CH 3) 2, CH (CH 3) 2, CH 3, C 6H 5, CH (CH 3) CH 2CH 3Or CH 2C 6H 5)
Figure S2007100189051D00051
The synthetic route of alpha-amido tetrazolium
Leucine, isoleucine, valine, alanine and phenylalanine with the Fmoc protection are initiation material; according to literature method (Huo Yanmin; Lei Genhu; Zheng Xiaohui; Wei Yinmao.Synthesis and characterization of the tetrazole analogues of α-amino acids.ChineseChemical Letters, 2006,17 (12); 1537-1539), the alpha-amido tetrazolium for preparing the Fmoc protection respectively.With the alpha-amido tetrazolium (2.5mmol) of Fmoc protection, the methanol solution of 100mL 20%-50% triethylamine joins the round-bottomed flask of 250mL, stirring at room 30min.Evaporating solvent after reaction finishes adds an amount of 0.5molL -1Hydrochloric acid till the dissolving of no solid, remove insoluble matter with petroleum ether extraction, collect the hydrochloric acid solution of tetrazolium, be neutralized to neutrality with NaOH solution, promptly adularescent tetrazolium solid is separated out.The solid-liquid mixture is put into refrigerator overnight, filter, collect solid, 60 ℃ of vacuum drying promptly get end product alpha-amido tetrazolium.
(2) preparation (R of Alpha-hydroxy tetrazolium 2Can select H, phenyl or methyl)
The synthetic route of Alpha-hydroxy tetrazolium
In the round-bottomed flask of 500mL, add alpha-hydroxy nitrile (5mmol), add the 200mL isopropyl alcohol again, 80 ℃ of heating for dissolving.Add 100mL water again, NaN 3(3.25g, 50mmol) and ZnBr 2(3.25g, 12.5mmol), reactant mixture refluxes at 80 ℃ and stirred 48 hours.Reaction finishes, and adds 35mL 3molL in reactant mixture -1HCl with 100mL ethyl acetate extraction (add an amount of NaCl saltout layering), tells organic layer, and aqueous layer is used the ethyl acetate extraction of 2 * 100mL again, merges organic layer, and evaporation obtains the tetrazolium solid.Use the silicagel column purified product, eluant, eluent is 1: 1: 1 methanol/ethyl acetate/benzinum.Use the Rotary Evaporators evaporating solvent, 30 ℃ of vacuum drying get light yellow product.
Embodiment 1: the alpha-amido tetrazolium is the preparation of the silica gel separating medium of part
Figure S2007100189051D00062
The alpha-amido tetrazolium is the preparation route of the silica gel separating medium of part
Take by weighing 2 gram macro porous silica gels, add 1: 1 hydrochloric acid of 30mL, ultrasonic 5min refluxed 3 hours 100 ℃ of following oil baths then, and reaction finishes, and filters, and is washed with distilled water to neutrality, at 130 ℃ of dry 4h.This silica gel is joined in the 100mL three-necked bottle, add the 35mL dry toluene, ultrasonic 5min after temperature is raised to 120 ℃, stirs down and drips 1.8mL γ-glycidoxypropyltrietandysilane andysilane, back flow reaction 12 hours with dropping funel.Reaction finishes, and filters, and washes 3 times with dry toluene, and acetone is washed 4 times.50 ℃ of vacuum drying are standby.
In the epoxy radicals silica gel of drying, add 30mL DMF, 0.5g alpha-amido phenylpropyl alcohol tetrazolium and 0.1mL pyridine, stirring at room 24h.Reaction finishes, and uses acetone, water washing successively, and vacuum drying promptly gets the tetrazolium multifunctional separation medium.
Embodiment 2: the alpha-amido tetrazolium is the preparation of agarose (or glucan) separating medium of part
Figure S2007100189051D00071
The alpha-amido tetrazolium is the preparation route of agarose (or glucan) separating medium of part
Get 10mL agarose (or glucan) in the 100mL three-necked bottle, add 50mL 5%NaOH and 5.0mL epoxychloropropane, reaction 12h.Reaction finishes, and with phosphate buffer (pH7.0) washing, obtains the agarose (or glucan) of surface bond epoxy radicals.In epoxy radicals agarose (or glucan), add 50mL DMF, 1.5g alpha-amido phenylpropyl alcohol tetrazolium and 1.0mL pyridine, stirring at room 24h.Reaction finishes, and uses phosphate buffer (pH7.0) washing successively, promptly gets tetrazolium agarose (or glucan) chromatography separation media.
Embodiment 3, be the preparation of the silica gel separating medium of part with the Alpha-hydroxy tetrazolium
Figure S2007100189051D00072
The Alpha-hydroxy tetrazolium is the preparation route of the silica gel separating medium of part
Take by weighing 2 grams, 7 μ m macro porous silica gels, add 30mL1: 1 hydrochloric acid, ultrasonic 5min refluxed 3 hours 100 ℃ of following oil baths then, and reaction finishes, and filters, and is washed with distilled water to neutrality, at 130 ℃ of dry 4h.This silica gel is joined in the 100mL three-necked bottle, add the 35mL dry toluene, ultrasonic 5min after temperature is raised to 120 ℃, stirs down and drips the 1.8mL γ-glycerine ether oxygen propyl-triethoxysilane that contracts with dropping funel, reacts 12 hours.Reaction finishes, and filters, and washes 3 times with dry toluene, and acetone is washed 4 times.50 ℃ of vacuum drying are standby.
In the epoxy radicals silica gel of drying, add 30mL 1.4-dioxane, 0.5g Alpha-hydroxy benzene second tetrazolium, 0.5mL boron trifluoride ether solution, stirring reaction 14h under the room temperature.Reaction finishes, and uses acetone, water washing successively, and vacuum drying gets fixedly phase of Alpha-hydroxy tetrazolium silica gel chromatograph separating medium.
Embodiment 4, be the preparation of agarose (or glucan) separating medium of part with the Alpha-hydroxy tetrazolium
The alpha-amido tetrazolium is the preparation route of agarose (or glucan) separating medium of part
Get 10mL agarose (or glucan) in the 100mL three-necked bottle, add 50mL 5%NaOH and 5.0mL epoxychloropropane, reaction 12h.Reaction finishes, and with phosphate buffer (pH7.0) washing, promptly gets the agarose (or glucan) of surface bond epoxy radicals.After the drying, add 50mL 1.4-dioxane, 1.5g Alpha-hydroxy benzene second tetrazolium, 1.5mL boron trifluoride ether solution, stirring reaction 14h under the room temperature.Reaction finishes, and uses acetone, water washing successively, gets Alpha-hydroxy tetrazolium agarose (or glucan) chromatography separation media.
Embodiment 5, solid-phase synthesis preparation are the silica gel separating medium of part with the Alpha-hydroxy tetrazolium
Solid-phase synthesis prepares the preparation route of tetrazole radical silica gel separating medium
Take by weighing 2 grams, 7 μ m macro porous silica gels, add 30mL1: 1 hydrochloric acid, ultrasonic 5min refluxed 3 hours 100 ℃ of following oil baths then, and reaction finishes, and filters, and is washed with distilled water to neutrality, at 130 ℃ of dry 4h.This silica gel is joined in the 100mL three-necked bottle, add the 35mL dry toluene, ultrasonic 5min after temperature is raised to 120 ℃, stirs down and drips the 1.8mL γ-glycerine ether propyl group-triethoxysilane that contracts with dropping funel, reacts 12 hours.Reaction finishes, and filters, and washes 3 times with dry toluene, and acetone is washed 4 times.50 ℃ of vacuum drying are standby.
In the epoxy radicals silica gel of drying, add 30mL 1.4-dioxane, 1.5mL 3-hydroxypropionitrile, 0.5mL boron trifluoride ether solution, stirring reaction 14h under the room temperature.Reaction finishes, and uses acetone, water washing successively, vacuum drying.In the silica gel of bonding acetaldehyde cyanhydrin, add 30mL water, 2.5gNaN 3And 1.0gNH 4Cl mixes reaction 30h for 80 ℃.Reaction finishes, and is washed with water to neutrality, and vacuum drying promptly gets Alpha-hydroxy tetrazolium silica gel separating medium.
Embodiment 6, solid-phase synthesis preparation are agarose (or glucan) separating medium of part with the alpha-amido tetrazolium
Figure S2007100189051D00101
Solid-phase synthesis prepares the preparation route of tetrazole radical agarose (or glucan) separating medium
Get the 10mL glucan in the 100mL three-necked bottle, add 50mL 5%NaOH and 5.0mL epoxychloropropane, reaction 12h.Reaction finishes, and with phosphate buffer (pH7.0) washing, promptly gets the glucan of surface bond epoxy radicals.After the drying, add 50mL 1.4-dioxane, 3.0mL 3-hydroxypropionitrile, 1.5mL boron trifluoride ether solution, stirring reaction 14h under the room temperature.Reaction finishes, and washes with water, in the silica gel of bonding acetaldehyde cyanhydrin, adds 50mL water, 5.0g NaN then 3With 2.0g NH 4Cl mixes reaction 30h for 30 ℃.Reaction finishes, and is washed with water to neutrality, and vacuum drying promptly gets 'alpha '-hydroxy acids tetrazolium glucan separating medium.
Embodiment 7:
Use the separating medium for preparing among the embodiment 1, the ion-exchange chromatography that is used for protein separates, the result as shown in Figure 1, this separating medium has good separating property to four kinds of mixed proteins as can be seen.
Chromatographic condition is: chromatographic column: the stainless steel column of 50 * 4.6mm; Mobile phase: A (equilibrium liquid), 20mmol/l potassium dihydrogen phosphate (pH 6.0), Mobile phase B (eluent): A+0.5mol/l NaCl (pH 6.0); Flow rate of mobile phase: 1.0ml/min; Linear gradient 20min, 100%A-100%B, 100%B prolong the 10min. standard protein: 1. myoglobins; 2. α-rotten albumen-A; 3. cytochromes-C; 4, lysozyme.
Embodiment 8:
Use the separating medium for preparing among the embodiment 3, the ion-exchange chromatography that is used for protein separates, the result as shown in Figure 2, this separating medium has good separating property to four kinds of mixed proteins as can be seen, and splitter is imitated higher.
Chromatographic condition is: chromatographic column: the stainless steel column of 50 * 4.6mm; Mobile phase: A (equilibrium liquid), 20mmol/l potassium dihydrogen phosphate (pH 7.5); Mobile phase B (eluent): A+0.5mol/l NaCl (pH 7.5); 3, flow rate of mobile phase: 3ml/min, 4, linear gradient 20min, 100%A-100%B, 100%B prolongs 10min; Standard protein: 1. myoglobins; 2. ribonuclease-a; 3. cytochromes-C; 4, lysozyme.
Embodiment 9:
Use the separating medium for preparing among the embodiment 1, according to literature method (WEI Yinmao *, Huang Xiaodong, et al.J.Liq.Chromatogr.﹠amp; Relat.Technol.2001,24 (19): 2983-2998) chelating Zn 2+, obtain chelating Zn 2+Alpha-amido phenylpropyl alcohol tetrazolium filler.This metal-chelating separating medium to the separation of protein as shown in Figure 3, this separating medium has good separating property to four kinds of mixed proteins as can be seen.
Chromatographic condition is: chromatographic column: the stainless steel column of 50 * 4.6mm; Mobile phase: A (equilibrium liquid), 50mmol/l potassium dihydrogen phosphate (pH6.0); Mobile phase B (eluent), A+1.0mol/l NaCl (pH 6.0); Flow rate of mobile phase: 1.0ml/min; Linear gradient 20min, linear gradient 20min, 100%A-100%B, 100%B prolongs the 10min. standard protein: 1.MyO, 2.RNase-A, 3.4.Cyt-C, 5.lys.
Embodiment 10:
Use the separating medium for preparing among the embodiment 3, according to literature method (WEI Yinmao *, Huang Xiaodong, et al.J.Liq.Chromatogr.﹠amp; Relat.Technol.2001,24 (19): 2983-2998) chelating Zn 2+, obtain chelating Zn 2+Alpha-hydroxy benzene second tetrazolium filler.This metal-chelating separating medium to the separation of protein as shown in Figure 4, this separating medium has good separating property to five kinds of mixed proteins as can be seen, and splitter is imitated higher.。
Chromatographic condition is: chromatographic column: the stainless steel column of 5.0 * 4.6cm; Mobile phase: A (equilibrium liquid), 50mmol/l potassium dihydrogen phosphate (pH6.0), Mobile phase B (eluent), A+0.5mol/l NaCl (pH 6.0); Flow rate of mobile phase: 1ml/min; Linear gradient 20min, linear gradient 20min, 100%A-100%B, 100%B prolong the 10min. standard protein: 1. myoglobins; 2. α-rotten albumen-A; 3 ribonuclease-as; 4. cytochromes-C; 5, lysozyme
Embodiment 11:
Use the separating medium for preparing among the embodiment 1, be used for the separation of gene engineering product recombinant human interferon-'s 8mol/L urea extract, the result as shown in Figure 5, interferon-has obtained good separation as can be seen.
Chromatographic condition is: fixing phase: Zn 2+The alpha-amido phenylpropyl alcohol tetrazolium silica gel of chelating; The stainless steel column of chromatographic column: 50 * 4.6mm; Mobile phase A: (equilibrium liquid), 50mmol/l potassium dihydrogen phosphate (pH 6.0), Mobile phase B (eluent), A+0.5mol/lNaCl (pH 6.0); Flow rate of mobile phase: 1ml/min linear gradient 20min, linear gradient 20min, 100%A-100%B, 100%B prolongs 10min. *Recombinant human interferon-behind the purifying, purity is 90%.

Claims (7)

1. be the multifunctional separation medium of functional group with the tetrazolium, have following general structure:
Figure S2007100189051C00011
Figure S2007100189051C00012
Represent silica gel, agarose or glucan; R 1Be O or NH; R 2Be H, C 1-C 4Alkyl, phenyl or benzyl.
2. multifunctional separation medium according to claim 1 is characterized in that: R 2Be hydrogen atom, methyl, isopropyl or isobutyl group.
3. multifunctional separation medium according to claim 1 is characterized in that: R 2Be phenyl or benzyl.
4. the preparation method of the described multifunctional separation medium of claim 1, it is characterized in that: silica gel and γ-glycidoxypropyltrietandysilane andysilane reaction, the silica gel of synthetic surface bonding epoxy radicals reacts under alkali or BFEE catalyst action with alpha-amido tetrazolium or Alpha-hydroxy tetrazolium then and promptly gets the tetrazolium multifunctional separation medium;
Described alpha-amido tetrazolium structure is as follows:
Figure S2007100189051C00013
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl; Described Alpha-hydroxy tetrazolium structure is as follows:
Figure S2007100189051C00014
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl.
5. the preparation method of the described multifunctional separation medium of claim 1, it is characterized in that: glucan or Ago-Gel and epichlorohydrin reaction, the glucan or the Ago-Gel of synthetic surface bonding epoxy radicals react under alkali or BFEE catalytic action with alpha-amido tetrazolium or Alpha-hydroxy tetrazolium then and promptly get the tetrazolium multifunctional separation medium;
Described alpha-amido tetrazolium structure is as follows:
Figure S2007100189051C00021
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl; Described Alpha-hydroxy tetrazolium structure is as follows:
Figure S2007100189051C00022
R 2Be H, C 1-C 4Alkyl, phenyl or benzyl.
6. the preparation method of the described multifunctional separation medium of claim 1, it is characterized in that: silica gel and γ-glycidoxypropyltrietandysilane andysilane reaction, the silica gel of synthetic surface bonding epoxy radicals, under BFEE catalysis, react then with alpha-hydroxy nitrile, in the silica gel of bonding alpha-hydroxy nitrile, add DMF, NaN 3And NH 4The Cl reaction promptly gets the tetrazolium multifunctional separation medium.
7. the preparation method of the described multifunctional separation medium of claim 1, it is characterized in that: glucan or Ago-Gel and epichlorohydrin reaction, the glucan or the Ago-Gel of synthetic surface bonding epoxy radicals, under BFEE catalysis, react then with alpha-hydroxy nitrile, in the glucan or Ago-Gel of bonding alpha-hydroxy nitrile, add DMF, NaN 3And NH 4The Cl reaction promptly gets the tetrazolium multifunctional separation medium.
CNA2007100189051A 2007-10-18 2007-10-18 Multifunctional separation medium with tetrazole as functional group and preparing method thereof Pending CN101224414A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103228328A (en) * 2010-11-29 2013-07-31 通用电气健康护理生物科学股份公司 Affinity chromatography matrix
CN105854850A (en) * 2016-05-16 2016-08-17 中国科学院兰州化学物理研究所 Tetraazacyclododecane chromatographic stationary phase as well as preparation method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103228328A (en) * 2010-11-29 2013-07-31 通用电气健康护理生物科学股份公司 Affinity chromatography matrix
CN105854850A (en) * 2016-05-16 2016-08-17 中国科学院兰州化学物理研究所 Tetraazacyclododecane chromatographic stationary phase as well as preparation method and application thereof
CN105854850B (en) * 2016-05-16 2019-02-01 中国科学院兰州化学物理研究所 Tetraazacyclododecanand base chromatographic stationary phases and its preparation method and application

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