CN102527357A - SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof - Google Patents
SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a strong cation exchange/hydrophobic mixed-mode chromatograph stationary phase, wherein X is -OCH3 or -OCH2CH3; R is shown in the specification, wherein n is equal to 1-5, or R is shown in the specification, wherein n is equal to 1-6; or R is PEG (Polyethylene Glycol) 200-1000. A preparation method comprises the following steps of: bonding cystine on the activated silica gel surface with hydroxyl groups by a silane coupling agent, then using DTT to open cystine disulfide bonds on the silica gel surface and form sulfydryl silica gel, then using H2O2 to oxidize the sulfydryl groups into sulfonic acid groups and form silica-gel derivatives bonded with the sulfydryl groups, and finally reacting with fatty alcohol (or aromatic alcohol or PEG and the like) to obtain the hydrophobic/strong cation exchange chromatograph stationary phase. The stationary phase can realize effective separation of proteins under the hydrophobic mode and the strong cation exchange mode, and one chromatographic column filled with a double-function separating medium can replace two common strong cation exchange/hydrophobic chromatographic columns to carry out separation and purification on the proteins.
Description
Technical field
The present invention relates to fixedly phase of a kind of high performance liquid chromatography that is used for Separation of Proteins, specifically a kind of hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase, the invention still further relates to this mixed mode fixedly preparation method and the purposes of phase.
Background technology
Along with biotechnology and development of life science, the production of recombinant protein drug still is the efficiently isolation technics fast that the research of proteomics all depends on protein.The analysis of complex sample has proposed increasingly high requirement to separation science, is one of effective way that addresses this problem and develop new and effective parting material, clastotype and highly sensitive detection method.
Multidimensional liquid chromatogram (MDLC) is the key technology of complex sample compartment analysises such as proteomics.A common root chromatogram column can only utilize a kind of clastotype that large biological molecule is carried out separation and purification, like anti-phase (RPLC), hydrophobic (HIC), ion-exchange (IEC) and affinity (AFC) etc.So the structure of two-dimensional liquid chromatography (2DLC) just needs two diverse chromatographic columns of active force character at present." mixed mode chromatogram " (Mixed mode chromatography is to utilize protein and fixing mutually multiple interaction force separates between the aglucon [the kind river rising in Ningxia and flowing into central Shaanxi of Yao etc., chemical industry journal, 2007,58:2169-2177 MMC); Zhao G F,
Et al.,
J Biotech, 2009,144: 3 – 11; Mc Laughlin,
Et al.,
Chem Rev, 1989,89:309-319].The separating medium that mixes retention mechanism can provide two or more active forces to be used for the separation of solute.Compare with single chromatographic isolation pattern, MMC has higher selectivity and adsorbance.Some scholars adopt cyanogen bromide method crosslinked on agarose fatty amine and aromatic amine, and the hydrophobic synthetic interaction is phase fixedly.Owing to have amido on the part, make this hydrophobic fixing certain ion-exchange character that has mutually, therefore think that the separation of protein is the common result of electrostatic interaction and hydrophobic interaction.1986, the anion exchange that Regnier group has synthesized anion exchange/HC mixed mode first fixedly phase [Kennedy L A,
Et al, J Chromatogr A,1986,359:73-84]; Subsequently Horvath group reported same result [Melander W R,
Et al,
J Chromatogr,1989,469:3-27].Though MMC has anti-phase and ion-exchange mixed mode [Apfelthaler E, et al, J now
Chromatogr. A,2008,1191:171-181], anti-phase and hydrophilic mixed mode [Liu X,
Et al. J Chromatogr. A,2008,1191:83-89], hydrophilic and ion-exchange mixed mode [Strege M A,
Et al., Anal Chem,2000,72:4629-4633] etc. type, but MMC is main with a kind of active force still; Another kind of power is auxilliary; So mixing the chromatograph packing material of retention mechanism is the master with a kind of clastotype all, i.e. a kind of separation of clastotype is better, and the another kind of deviation of dividing; Therefore the two dimensional separation that can not be used for protein example effectively can not be referred to as two-dimentional chromatograph packing material.
Though the commercialization of mixed mode chromatographic column, like the Capto MMC and the Capto adhere of GE company, the HEA of Pall company, PPA and MEP chromatographic column etc. all are to be main with ion-exchange, hydrophobic effect is auxilliary.So; Up to now; Do not find that as yet any mixed chromatogram filler can carry out two-dimentional liquid phase separation with two kinds of diverse functions (like IEC and HIC) to protein respectively, and can both obtain the separating effect that reaches when separating with certain single clastotype.Inventor research group had synthesized a kind of have simultaneously new and effective chromatography separation media [Ke Congyu etc., Science Bulletin, 2008,53:614 ~ 616 hydrophobic and weak cation exchange bifunctional group in 2009; Geng XD,
Et al., J Chromatogr A,2009,1216:3553-3562].Under high salt concn show as the character of HC, can be used as the HC separating medium and use; And under the low salt concn condition, show as the character of ion-exchange chromatography, can be used as the ion-exchange chromatography separating medium to use, and furtherd investigate protein under hydrophobic and two kinds of patterns of ion-exchange the mixing retention mechanism [Liu P,
Et al., J Chromatogr. A,2009,1216:7497 – 7504].Compare with corresponding unit reason chromatograph packing material, show unique selectivity, improved separating power to a certain extent.On this basis, utilize the valve handoff technique, adopt a two-dimentional chromatographic column to make up online two-dimensional liquid chromatography piece-rate system with WCX-HIC, be referred to as single-column-two-dimensional liquid chromatography separation technology [Geng XD,
Et al., J Chromatogr A,2009,1216:3553-3562].
Among the 2DLC of bibliographical information, how to adopt RPLC in second dimension.But because strong hydrophobicity and organic environment, most protein can lose activity even sex change.And HIC and IEC separation condition are gentle, can keep natural structure and the biologically active of large biological molecule better, IEC cooperate the separation and purification of suitable protein of HIC [Ascenjo J A, et al.,
J Mol Recog.,2004,17:236-247].Above-mentioned IEC-HIC single-column two dimension chromatogram has farthest guaranteed the biologically active of whole albumen, has realized the fast separating and purifying of active whole albumen.
Single-column two dimension chromatogram new technology is at the early-stage at present, and the realization of this technology mainly depends on the exploitation and the preparation of difunctional chromatograph packing material.But the difunctional chromatograph packing material that designs for isolated protein specially also seldom, and kind is very limited.Different with the two-dimentional chromatogram of routine is, utilizes the difunctional separating medium of this IEC/HIC, can on a root chromatogram column, accomplish HIC-IEC or IEC-HIC two dimension chromatogram.The excellent menu of this single-column two dimension chromatographic column is present; Can realize replacing the new method of the quick protein isolate of two ion-exchanges commonly used and HC post with this chromatographic column; This will reduce the quantity of the needed separating medium of separation and purification of biological macromolecule greatly; Thereby reduce the production cost of large biological molecule, particularly recombinant protein medicine greatly, the separation and purification of large biological molecule is had broad application prospects.
Summary of the invention
The object of the present invention is to provide a kind of novel hydrophobic/strong cation exchange mixed mode chromatographic stationary phase, to satisfy the high efficiency separation requirement that under ion-exchange pattern and hydrophobic pattern, realizes respectively protein.
Another object of the present invention is to provide the preparation method of above-mentioned chromatographic stationary phase.
For realizing above-mentioned purpose, the present invention adopts technical scheme to be:
Hydrophobic/strong cation exchange (HIC/SAC) mixed mode chromatographic stationary phase shown in the structural formula (I),
Wherein X is-OCH
3Or-OCH
2CH
3;
R is
, wherein n=1 ~ 5; Or
, wherein n=1 ~ 6; Or PEG200-1000.
Said fixing through being that bridge is bonded on the activated silica gel surface that the surface has hydroxyl with cystine with the silane coupler, with DTT the cystine disulfide bond on silica gel surface is opened again and formed sulfhydrylation silica gel, and then use H
2O
2With sulfhydryl oxidase is that sulfonic group forms sulfonic group bonded silica gel derivative, obtain with reaction such as aliphatic alcohol, aromatic alcohols or PEG more at last hydrophobic/the strong cation exchange chromatographic stationary mutually.Its preparation method may further comprise the steps:
(1) cystine and silane coupler reaction back add silica gel react cystine bonding silica derivative thing;
(2) with cystine bonding silica derivative thing and DDT react cysteine bonded silica gel derivative;
(3) cysteine bonded silica gel derivative and strong sulfuric acid response are obtained sulfonic group bonded silica gel derivative;
(4) react hydrophobicly with aliphatic alcohol, aromatic alcohols or PEG sulfonic group bonded silica gel derivative/strong cation exchange mixed mode chromatographic stationary is mutually.
Specifically, the preparation method is following:
(1) cystine of getting 1 part of weight is dissolved in the alkaline buffer solution of certain volume, regulates then about pH to 11.0, and ice bath stirs down, slowly splashes into the silane coupler of 0.5～1 part of weight; Dropwise, continue ice bath and stir 30 min, be warming up to 60～80 ℃ then, reacted 12～24 hours; Reaction finishes, and it is light yellow that solution is, and is cooled to room temperature, transfers reactant liquor PH to 4.0～7.0 with glacial acetic acid; The silica gel that adds 0.5～1 part of weight, 80～95 ℃ were reacted 1.5～4 hours down, filter; Use three water, methyl alcohol, acetone, methanol wash successively, 50 ℃ of vacuum drying of gained solid 5～15 hours can obtain cystine bonding silica derivative thing.This step bonding reaction can be expressed as:
Used cushioning liquid is the Na of 0.5 mol/L
2CO
3Cushioning liquid, the amount of the required cushioning liquid of every gram cystine is 50～70 mL; Used silane coupler has following structure:
Wherein X is-OCH
3Or-OCH
2CH
3Used silica gel is the full multi-hole blangel bead, and particle diameter is 3～40 μ m, and the aperture is 50～300, and through 1:1 hydrochloric acid activation 3～7 hours, is washed till neutrality then, 100～160 ℃ of vacuum drying 10～24 hours.
(2) the cystine bonding silica derivative thing of 1 part of weight of said step (1) preparation being scattered in pH is in 7～8 the cushioning liquid; The DDT that adds 0.1～0.4 part of weight; Stirring at room reaction 1～3 hour is filtered, and respectively washs twice with three water, methyl alcohol successively; 50 ℃ of vacuum drying of gained solid 5～15 hours can obtain cysteine bonded silica gel derivative.This step reaction can be expressed as:
Used cushioning liquid is the Tris-HCl cushioning liquid of 1 mol/L, and the amount of the required cushioning liquid of every gram cystine bonding silica derivative thing is 10～20 mL.
(3) the cysteine bonded silica gel derivative that said step (2) is prepared is scattered in the H of methyl alcohol and 25～35%
2O
2Mixed solution in, stir down and splash into an amount of concentrated sulfuric acid, make reactant liquor be faintly acid (pH is 3 to 6), in 20～35 ℃ of reactions 10～24 hours down, product filtration.Respectively wash twice with three water, methyl alcohol successively, 50 ℃ of vacuum drying of gained solid 5～15 hours can obtain sulfonic group bonded silica gel derivative.This step oxidation reaction can be expressed as:
Oxidation reaction is with the H of methyl alcohol and 25～35%
2O
2The mixed solution of (volume ratio 1:2～5) adds an amount of concentrated sulfuric acid again, and to make it to be faintly acid be reaction medium, and the required reactant liquor volume of every gram cysteine bonded silica gel derivative is 30～40 mL.
(4) the sulfonic group bonded silica gel derivative with 1 part of weight of said step (3) preparation is scattered in the organic solvent; Add the aliphatic alcohol (or aromatic alcohols or PEG etc.) of 1.5～3 parts of weight, the DIC of 1.5～3 parts of weight, the DMAP of 0.1～0.3 part of weight; Stirring at room 24～48 hours, product filters, and respectively washs twice with three water, methyl alcohol successively; 50 ℃ of vacuum drying of gained solid 5～15 hours can obtain the difunctional mixed mode chromatographic stationary of hydrophobic/strong cation exchange phase.This step reaction can be expressed as:
Wherein X is-OCH
3Or-OCH
2CH
3;
R is
, wherein n=1 ~ 5; Or
, wherein n=1 ~ 6; PEG200 (400,600,800 or 1000).
Used organic solvent is carrene or N, dinethylformamide, and the amount of the required organic solvent of every gram sulfonic group bonded silica gel derivative is 25～40mL.
Advantage of the present invention and good effect:
(1) adopt simple method to synthesize the difunctional mixed mode chromatographic stationary of the hydrophobic/strong cation exchange phase that is used for Protein Separation first, this fixing stable, synthetic mutually cost is low, long service life, good separating effect.
(2) experiment shows the difunctional mixed mode chromatographic stationary of this hydrophobic/strong cation exchange phase, can under ion-exchange and hydrophobic two kinds of patterns, realize respectively the effective of mixed protein separated.
(3) chromatographic column with a this difunctional separating medium of filling can replace two strong cation exchange commonly used and HC post that protein is carried out separation and purification, just can realize IEC-HIC or HIC-IEC two dimension chromatographic isolation with a root chromatogram column.
Description of drawings
Fig. 1 is the IR spectrogram of the difunctional mixed mode chromatographic stationary of the hydrophobic/strong cation exchange phase of embodiment of the invention preparation;
Fig. 2 is the difunctional mixed mode chromatographic stationary of hydrophobic/strong cation exchange of the embodiment of the invention preparation separate colors spectrogram to five kinds of protein under the ion-exchange pattern;
Fig. 3 is the difunctional mixed mode chromatographic stationary of hydrophobic/strong cation exchange of the embodiment of the invention preparation separate colors spectrogram to seven kinds of protein under hydrophobic pattern;
Fig. 4 carries out the separate colors spectrogram for using embodiment 1 prepared chromatograph packing material dress post to eight kinds of albumen.
The specific embodiment
A kind of difunctional mixed mode chromatographic stationary of hydrophobic/strong cation exchange phase that is used for Separation of Proteins of the present invention can realize respectively the effective of mixed protein separated under ion-exchange and hydrophobic two kinds of patterns.Below in conjunction with embodiment and accompanying drawing, the present invention is further specified.Should be appreciated that embodiment only limits to explain the present invention rather than to qualification of the present invention.
(1)Get 3.3g (about 13.7 mmol) cystine and be dissolved in the Na of 150 mL, 0.5 mol/L
2CO
3In the cushioning liquid, regulate pH to 11.0 then, add in the 250 mL three-neck flasks, ice bath stirs down; Slowly splash into 3 mL (about 13.7 mmol) TM560, dropwise, continue ice bath and stir 30 min, be warming up to 65 ℃ then; Reacted 24 hours, reaction finishes, and it is light yellow that solution is, and is cooled to room temperature; Transfer reactant liquor pH to 5.5 with glacial acetic acid, add 2 g silica gel, 90 ℃ were reacted 2 hours down, filter; Use three water, methyl alcohol, acetone, methanol wash successively, 50 ℃ of vacuum drying of gained solid 8 hours can obtain cystine bonding silica derivative thing.Get ethanol solution of ninhydrin 5.0 mL, add a small amount of cystine silica derivative thing, 90 ℃ of heating 5 min find the solution purple, explain that cystine successfully is bonded on the silica gel surface.
(2) getting 2.0 g cystine bonding silica derivative things, to be scattered in 30 mL pH be in 8 the Tris-HCl cushioning liquid; Add 0.2 g DDT; Stirring at room reaction 1.5 hours is filtered, and respectively washs twice with three water, methyl alcohol successively; 50 ℃ of vacuum drying of gained solid 10 hours can obtain cysteine bonded silica gel derivative.Get cysteine bonded silica gel derivative a little, add in the 5 mL ethanol solution of ninhydrin, it is yellow that solution is, 90 ℃ of heating 5 min are yellow thin out, explain that cystine has been reduced into sulfydryl; Sulfydryl is identified that its experimental procedure is with sodium nitroprussiate in addition:
Solution: I. 1.5 g sodium nitroprussiates are dissolved in 5 mL, 2 mol/L hydrochloric acid solutions and the 95 mL methyl alcohol, add 10mL 25% Ammonia, filter, and promptly get;
II. 2 g Cymags are dissolved in 5ml water, are diluted to 100 mL with methyl alcohol.
Get on a little cysteine bonded silica gel derivative evenly is laid on as the clear glass, earlier the spray solution I, spray the solution II again, the result finds that the silica gel surface is pale red, explain that there is the sulfydryl existence on the silica gel surface.
(3) get 100 mL three-neck flasks, add 2.0 g cysteine bonded silica gel derivatives successively, methyl alcohol 15 mL, 35% H
2O
250 mL splash into an amount of concentrated sulfuric acid under stirring, and make reactant liquor be faintly acid (pH is 3); Reacted 24 hours down at 30 ℃; Product filters, and respectively washs 50 ℃ of vacuum drying of twice gained solid 8 hours with three water, methyl alcohol successively, can obtain sulfonic group bonded silica gel derivative.Get a little sulfonic group bonded silica gel derivative set by step (2) experimentize with sodium nitroprussiate, redfree occurs as a result, explain that the silica gel surface do not had the sulfydryl existence, and is oxidized.
(4) get 100 mL three-neck flasks, add 2.0 g sulfonic group bonded silica gel derivatives successively, 50 mL DMF; 4 mL phenmethylols, 2 mL DIC, 0.3 g DMAP; Stirring at room 36 hours, product filters, and respectively washs twice with three water, methyl alcohol successively; 50 ℃ of vacuum drying of gained solid 10 hours can obtain hydrophobic/strong cation exchange mixed mode chromatographic stationary phase.Gained is fixing to be tested with FTIS, and test result is as shown in Figure 1, at 1205cm
-1There is a strong wide absworption peak at the place, at 1078cm
-1The place has and to absorb a very strong peak, overlaps together in these two peaks, explains that the silica gel surface contains-SO
3 -Simultaneously at 794 cm
-1There is individual spike at the place, and stronger, explains that phenyl ring exists; At 1631 cm
-1There is individual spike at the place, explains that carbonyl exists; At 3486 cm
-1There is the absworption peak of individual broad at the place, explains that imido grpup exists, and comprehensive above information explains that institute's synthetic filling aglucon contains-SO simultaneously
3 -And phenyl ring.
Phenmethylol is changed to PEG400 to the inventor and normal propyl alcohol repeats above-mentioned experiment, is easy to obtain hydrophobic/strong cation exchange mixed mode chromatographic stationary phase.
Embodiment 2
Use embodiment 1 prepared chromatograph packing material to adorn post, under the strong cation exchange pattern, five kinds of standard proteins are separated then.Separation condition:
Flowing phase: A liquid: 20 mmol/L KH
2PO
4(pH 6.5); B liquid: 20 mmol/L KH
2PO
4+ 1.0 mol/L NaCl (pH 6.5), linear gradient elution, 0 → 100%B, 30 min; Flow velocity is 1.0 mL/min, and five kinds of protein such as myoglobin, RNase B, RNase A, cytochrome c, lysozyme have been realized good separation (as shown in Figure 21,2,3,4,5 are respectively myoglobin, RNase B, RNase A, cytochrome c, lysozyme).
Use embodiment 1 prepared chromatograph packing material to adorn post, under hydrophobic pattern, seven kinds of standard proteins are separated then.Separation condition:
Flowing phase: A liquid: 20 mmol/L KH
2PO
4(pH 7.0)+3.0 mol/L (NH
4)
2SO
4(pH 7.0); B liquid: 20 mmol/L KH
2PO
4(pH 7.0); Linear gradient elution; 0 → 100%B; 30 min, flow velocity are 1.5 mL/min, and seven kinds of protein such as cytchrome c, myoglobin, RNase A, lysozyme, OVA, α-amylase, insulin have been realized good separation (as shown in Figure 31,2,3,4,5,6,7 are respectively cytchrome c, myoglobin, RNase A, lysozyme, OVA, α-amylase, insulin).
Embodiment 4
Use the chromatograph packing material dress post of embodiment 1 preparation; Eight kinds of albumen are separated; First dimension is separated under the strong cation exchange pattern, collects undivided component under the strong cation exchange pattern, carries out the hydrophobic pattern of second dimension and separates; As shown in Figure 4, separate back eight kinds of albumen through the hydrophobic pattern of second dimension and obtained to separate fully.Separation condition:
Flowing phase: A liquid: 20 mmol/L KH
2PO
4(pH 7.0); B liquid: 20 mmol/L KH
2PO
4(pH 7.0)+3.0 mol/L (NH
4)
2SO
4(pH 7.0), gradient mode are shown in Fig. 4 dotted line, and flow velocity is 1.0 mL/min, to 1, and HSA; 2, BSA; 3, insulin; 4, myoglobin; 5, RNase B; 6, α-chymotrypsin; 7, cytochrome c; 8, eight kinds of protein such as lysozyme have been realized good separation.
Claims (10)
1. hydrophobic/strong cation exchange mixed mode chromatographic stationary the phase shown in the structural formula (I),
Wherein X is-OCH
3Or-OCH
2CH
3;
R is
, wherein n=1 ~ 5; Or
, wherein n=1 ~ 6; Or PEG200-1000.
Claim 1 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method, may further comprise the steps:
(1) cystine and silane coupler reaction back add silica gel react cystine bonding silica derivative thing;
(2) with cystine bonding silica derivative thing and DDT react cysteine bonded silica gel derivative;
(3) cysteine bonded silica gel derivative and strong sulfuric acid response are obtained sulfonic group bonded silica gel derivative;
(4) with sulfonic group bonded silica gel derivative and aliphatic alcohol, aromatic alcohols or PEG, and DIC, DMAP react hydrophobicly/strong cation exchange mixed mode chromatographic stationary phase.
According to claim 2 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method, it is characterized in that may further comprise the steps:
(1) cystine of getting 1 part of weight is dissolved in the alkaline buffer solution, regulates pH to 11.0, and ice bath stirs down; The silane coupler that adds 0.5～1 part of weight, ice bath stirring reaction 30 min are warming up to 60～80 ℃ of reactions 12～24 hours then; Reaction finishes, and transfers reactant liquor pH to 4.0～7.0 with glacial acetic acid, adds the silica gel of 0.5～1 part of weight; 80～95 ℃ were reacted 1.5～4 hours down, and filtration, washing, drying can obtain cystine bonding silica derivative thing;
(2) the cystine bonding silica derivative thing of 1 part of weight being scattered in pH is in 7～8 the cushioning liquid, adds the DDT of 0.1～0.4 part of weight, stirring at room reaction 1～3 hour, and filtration, washing, drying can obtain cysteine bonded silica gel derivative;
(3) cysteine bonded silica gel derivative is scattered in the H of methyl alcohol and 25～35%
2O
2Mixed solution in, stir down and splash into the concentrated sulfuric acid, make reactant liquor be faintly acid, reacted 10～24 hours down at 20～35 ℃, product filtration, washing, drying can obtain sulfonic group bonded silica gel derivative;
(4) the sulfonic group bonded silica gel derivative with 1 part of weight is scattered in the organic solvent; The aliphatic alcohol, aromatic alcohols or the PEG that add 1.5～3 parts of weight; 1.5 the DIC of～3 parts of weight; 0.1 the DMAP of～0.3 part of weight, stirring at room 24～48 hours, product filtration, washing, drying get final product hydrophobicly/strong cation exchange mixed mode chromatographic stationary phase.
According to claim 3 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method, it is characterized in that: the said alkaline buffer solution of step (1) is the Na of 0.5mol/L
2CO
3Cushioning liquid, the amount of the required cushioning liquid of every gram cystine is 50～70mL.
According to claim 3 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method, it is characterized in that: the said silane coupler structure of step (1) is following:
Wherein X is-OCH
3Or-OCH
2CH
3
According to claim 3 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method; It is characterized in that: used silica gel is the full multi-hole blangel bead in the step (1); Particle diameter is 3～40 μ m, and the aperture is 50～300, and passes through the 1:1 hydrochloric acid activation 3～7 hours; Be washed till neutrality then, 100～160 ℃ of vacuum drying 10～24 hours.
According to claim 3 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method; It is characterized in that: used cushioning liquid is the Tris-HCl cushioning liquid of 1mol/L in the step (2), and the amount of the required cushioning liquid of every gram cystine bonding silica derivative thing is 10～20mL.
According to claim 3 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method, it is characterized in that: the oxidation reaction in the step (3) is with the H of methyl alcohol and 25～35%
2O
2The mixed solution of volume ratio 1:2～5 adds the concentrated sulfuric acid again, and to make it to be faintly acid be reaction medium, and the required reactant liquor volume of every gram cysteine bonded silica gel derivative is 30～40mL.
According to claim 3 said hydrophobic/the difunctional mixed mode chromatographic stationary of strong cation exchange phase preparation method; It is characterized in that: used organic solvent is carrene or N in the step (4); Dinethylformamide, the amount of the required organic solvent of every gram sulfonic group bonded silica gel derivative is 25～40mL.
Claim 1 described hydrophobic/application of the difunctional mixed mode chromatographic stationary of strong cation exchange in separation and purification of protein.
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| CN103203225A (en) * | 2013-04-16 | 2013-07-17 | 西北大学 | Weak cation exchange/hydrophobic bifunctional hybrid-mode chromatographic stationary phase as well as preparation method and application thereof |
| CN105148881A (en) * | 2014-06-06 | 2015-12-16 | 中国科学院大连化学物理研究所 | Liquid chromatographic stationary phase and preparation method thereof |
| CN105561960A (en) * | 2016-01-14 | 2016-05-11 | 重庆大学 | High-performance liquid chromatography filler with switchable chiral selectivity and preparing method thereof |
| CN107694539A (en) * | 2016-08-08 | 2018-02-16 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | A kind of new anti-phase/weak cation exchange mixed mode chromatographic stationary phases and preparation method thereof |
| CN108218954A (en) * | 2016-12-09 | 2018-06-29 | 中国科学院大连化学物理研究所 | A kind of separation method of protein |
| CN108236934A (en) * | 2016-12-23 | 2018-07-03 | 西北大学 | It is a kind of using cholic acid as the preparation and application of the mixed mode chromatograph packing material of aglucon |
| CN109338348A (en) * | 2018-10-24 | 2019-02-15 | 河南恒润昌环保科技有限公司 | A kind of without phosphorus water-based metal silane finish and preparation method thereof |
| CN111389383A (en) * | 2020-02-26 | 2020-07-10 | 广东省测试分析研究所（中国广州分析测试中心） | Zwitterion mixed mode chromatographic stationary phase and preparation method and application thereof |
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