CN102527357B - 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
- Publication number
- CN102527357B CN102527357B CN 201210004100 CN201210004100A CN102527357B CN 102527357 B CN102527357 B CN 102527357B CN 201210004100 CN201210004100 CN 201210004100 CN 201210004100 A CN201210004100 A CN 201210004100A CN 102527357 B CN102527357 B CN 102527357B
- Authority
- CN
- China
- Prior art keywords
- hydrophobic
- cation exchange
- silica gel
- strong cation
- mixed mode
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Peptides Or Proteins (AREA)
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 a kind of Stationary Phase of HPLC for Separation of Proteins, specifically a kind of hydrophobic/the difunctional mixed mode chromatographic stationary phases of strong cation exchange, the invention still further relates to this mixed mode fixedly preparation method and the purposes of phase.
Background technology
Along with the development of biotechnology and life science, the production of recombinant protein drug or the research of proteomics all depend on the efficiently isolation technics fast of protein.The analysis of complex sample has proposed more and more higher requirement to separation science, and the efficient parting material of Development of Novel, clastotype and highly sensitive detection method are one of effective ways that addresses this problem.
Multidimensional liquid chromatogram (MDLC) is the key technology of the complex sample compartment analysises such as proteomics.A common root chromatogram column can only utilize a kind of clastotype to carry out separation and purification to large biological molecule, as 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, MMC) utilizes protein and [Yao kind river rising in Ningxia and flowing into central Shaanxi etc., Journal of Chemical Industry and Engineering, 2007,58:2169-2177 that fixedly between the phase aglucon, multiple interaction force separates; Zhao G F,
Et al.,
J Biotech, 2009,144: 3 – 11; Mc Laughlin,
Et al.,
Chem Rev, 1989,89:309-319].The separating medium of mixed-mode 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 selective and adsorbance.Some scholars adopt cyanogen bromide method crosslinked on agarose fatty amine and aromatic amine, and the hydrophobic synthetic interaction is phase fixedly.Due on part with amido, 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].Although 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 still take a kind of active force as main, another kind of power is auxiliary, so all take a kind of clastotype as main, i.e. a kind of separation of clastotype is better for the chromatograph packing material of mixed-mode retention mechanism, and another kind of minute deviation, can not effectively be used for the two dimensional separation of protein example, therefore can not be referred to as two-dimentional chromatograph packing material.
Although the commercialization of mixed mode chromatographic column, as Capto MMC and the Capto adhere of GE company, the HEA of Pall company, PPA and MEP chromatographic column etc. are all take ion-exchange as main, hydrophobic effect is auxiliary.So, up to now, find that not yet any mixed chromatogram filler can carry out Two-dimensional Liquid with two kinds of diverse functions (as IEC and HIC) to protein respectively and be separated, and the separating effect that reaches can obtain separating with certain single clastotype the time.Inventor research group in 2009 has synthesized and has a kind ofly had simultaneously hydrophobic and new and effective chromatography separation media [Ke Congyu etc., Science Bulletin, 2008,53:614 ~ 616 the weak cation exchange bifunctional group; 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 show as the character of ion-exchange chromatography under the low salt concn condition, 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 mixed-mode retention mechanism [Liu P,
Et al., J Chromatogr. A,2009,1216:7497 – 7504].Compare with corresponding unit reason chromatograph packing material, show the selective of uniqueness, improved to a certain extent separating power.On this basis, utilize the valve handoff technique, adopt a two-dimentional chromatographic column with WCX-HIC to build online two-dimensional liquid chromatography piece-rate system, be referred to as single-column-two-dimensional liquid chromatography separation technology [Geng XD,
Et al., J Chromatogr A,2009,1216:3553-3562].
In the 2DLC of bibliographical information, how to adopt RPLC in the second dimension.But due to strong hydrophobicity and organic environment, the most protein even sex change that can lose activity.And HIC and IEC separation condition are gentle, can keep better natural structure and the biologically active of large biological molecule, IEC coordinate 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 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 from the two-dimentional chromatogram of routine is, utilizes the difunctional separating medium of this IEC/HIC, can complete HIC-IEC or IEC-HIC two dimension chromatogram on a root chromatogram column.The excellent menu of this single-column two dimension chromatographic column is present, can realize replacing with this chromatographic column the new method of the quick protein isolate of two ion-exchanges commonly used and HC post, this will reduce the quantity of the needed separating medium of separation and purification of biological macromolecule greatly, thereby greatly reduce large biological molecule, the production cost of recombinant protein medicine particularly has broad application prospects to the separation and purification of large biological molecule.
Summary of the invention
The object of the present invention is to provide a kind of new hydrophobic/strong cation exchange mixed mode chromatographic stationary phases, to satisfy the efficient separation requirement that realizes respectively protein under ion-exchange pattern and hydrophobic pattern.
Another object of the present invention is to provide the preparation method of above-mentioned chromatographic stationary phases.
For achieving the above object, the present invention adopts technical scheme to be:
Shown in structural formula (I) hydrophobic/strong cation exchange (HIC/SAC) mixed mode chromatographic stationary phases,
Wherein X is-OCH
3Or-OCH
2CH
3;
R is
, n=1 ~ 5 wherein; Or
, n=1 ~ 6 wherein; Or PEG200-1000.
Described fixing by cystine being bonded to take silane coupler as bridge on the activated silica gel surface of surface with hydroxyl, then with DTT, the cystine disulfide bond of Silica Surface is opened and formed sulfhydrylation silica gel, and then use H
2O
2Be that sulfonic group forms sulfonic group bonded silica gel derivative with sulfhydryl oxidase, obtain fixedly phase of hydrophobic/strong cation exchange chromatography with the reaction such as aliphatic alcohol, aromatic alcohols or PEG more at last.Its preparation method comprises the following steps:
(1) add silica gel to react to get cystine bonding silica derivative thing after the reaction of cystine and silane coupler;
(2) cystine bonding silica derivative thing and DDT are reacted to get 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 sulfonic group bonded silica gel derivative and aliphatic alcohol, aromatic alcohols or PEG hydrophobic/strong cation exchange mixed mode chromatographic stationary phases.
Specifically, the preparation method is as follows:
(1) cystine of getting 1 part of weight is dissolved in the alkaline buffer solution of certain volume, then regulate pH to 11.0 left and right, under ice bath stirs, slowly splash into the silane coupler of 0.5～1 part of weight, dropwise, continue ice bath and stir 30 min, then be warming up to 60～80 ℃, reacted 12～24 hours, react complete, it is light yellow that solution is, be cooled to room temperature, transfer reactant liquor PH to 4.0～7.0 with glacial acetic acid, the silica gel that adds 0.5～1 part of weight, reacted under 80～95 ℃ 1.5～4 hours, filter, use successively water three times, methyl alcohol, acetone, methanol wash, 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:
Cushioning liquid used 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; Silane coupler used has following structure:
Wherein X is-OCH
3Or-OCH
2CH
3Silica gel used 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, then is washed till neutrality, 100～160 ℃ of vacuum drying 10～24 hours.
(2) the cystine bonding silica derivative thing of 1 part of weight of described step (1) preparation being scattered in pH is in 7～8 cushioning liquid, the DDT that adds 0.1～0.4 part of weight, stirring at room reaction 1～3 hour, filter, respectively wash 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:
Cushioning liquid used 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 of described step (2) preparation is scattered in the H of methyl alcohol and 25～35%
2O
2Mixed solution in, splash into the appropriate concentrated sulfuric acid under stirring, make reactant liquor be faintly acid (pH is 3 to 6), reacted under 20～35 ℃ 10～24 hours, product filters.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
2It is reaction medium that the mixed solution of (volume ratio 1:2～5) adds the appropriate concentrated sulfuric acid to make it to be faintly acid again, and the required reactant liquor volume of every gram cysteine bonded silica gel derivative is 30～40 mL.
The sulfonic group bonded silica gel derivative of the 1 part of weight that (4) described step (3) is prepared is scattered in organic solvent, add the DIC of the aliphatic alcohol (or aromatic alcohols or PEG etc.) of 1.5～3 parts of weight, 1.5～3 parts of weight, the DMAP of 0.1～0.3 part of weight, stirring at room 24～48 hours, product filters, respectively wash 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 phases of hydrophobic/strong cation exchange.This step reaction can be expressed as:
Wherein X is-OCH
3Or-OCH
2CH
3;
R is
, n=1 ~ 5 wherein; Or
, n=1 ~ 6 wherein; PEG200(400,600,800 or 1000).
Organic solvent used is carrene or DMF, 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 first to be used for Protein Separation hydrophobic/the difunctional mixed mode chromatographic stationary phases of strong cation exchange, this fixing stable, synthetic cost mutually is low, long service life, good separating effect.
(2) experiment shows the difunctional mixed mode chromatographic stationary phases of this hydrophobic/strong cation exchange, can realize respectively the effective of mixed protein separated under ion-exchange and hydrophobic two kinds of patterns.
(3) chromatographic column with a this difunctional separating medium of filling can replace two strong cation exchange commonly used and HC post to carry out separation and purification to protein, just can realize IEC-HIC or HIC-IEC two dimension chromatographic isolation with a root chromatogram column.
Description of drawings
Fig. 1 be embodiment of the present invention preparation hydrophobic/the IR spectrogram of the difunctional mixed mode chromatographic stationary phases of strong cation exchange;
Fig. 2 be embodiment of the present invention preparation hydrophobic/the difunctional mixed mode chromatographic stationary phases of strong cation exchange under the ion-exchange pattern to the separate colors spectrogram of five kinds of protein;
Fig. 3 be embodiment of the present invention preparation hydrophobic/the difunctional mixed mode chromatographic stationary phases of strong cation exchange under hydrophobic pattern to the separate colors spectrogram of seven kinds of protein;
Fig. 4 is for using the prepared chromatograph packing material dress post of embodiment 1 to carry out the separate colors spectrogram to eight kinds of albumen.
The specific embodiment
Of the present invention a kind of for Separation of Proteins hydrophobic/the difunctional mixed mode chromatographic stationary phases of strong cation exchange, 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 will be further described.Should be appreciated that embodiment only limits to illustrate the present invention rather than limitation of the invention.
(1)Get 3.3g (approximately 13.7 mmol) cystine and be dissolved in the Na of 150 mL 0.5 mol/L
2CO
3In cushioning liquid, then regulate pH to 11.0, add in 250 mL three-neck flasks, ice bath slowly splashes into 3 mL (approximately 13.7 mmol) TM560 under stirring, dropwise, continue ice bath and stir 30 min, then be warming up to 65 ℃, reacted 24 hours, react complete, it is light yellow that solution is, and is cooled to room temperature, transfers reactant liquor pH to 5.5 with glacial acetic acid, add 2 g silica gel, under 90 ℃, reaction is 2 hours, filters, and uses successively three water, methyl alcohol, acetone, methanol wash, 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, illustrate that cystine successfully is bonded on Silica Surface.
(2) getting 2.0 g cystine bonding silica derivative things, to be scattered in 30 mL pH be in 8 Tris-HCl cushioning liquid, add 0.2 g DDT, stirring at room reaction 1.5 hours, filter, respectively wash 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 5 mL ethanol solution of ninhydrin, it is yellow that solution is, 90 ℃ of heating 5 min are yellow thin out, illustrate that cystine has been reduced into sulfydryl; Separately with sodium nitroprussiate, sulfydryl is identified, its experimental procedure is:
Solution: I. 1.5 g sodium nitroprussiates are dissolved in 5 mL 2 mol/L hydrochloric acid solutions and 95 mL methyl alcohol, add 10mL 25% Ammonia, filter, and get final product;
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 clear glass, first spray solution I, spray the solution II again, found that Silica Surface is pale red, illustrate that Silica Surface has sulfydryl to exist.
(3) get 100 mL three-neck flasks, add successively 2.0 g cysteine bonded silica gel derivatives, methyl alcohol 15 mL, 35% H
2O
250 mL splash into the appropriate concentrated sulfuric acid under stirring, make reactant liquor be faintly acid (pH is 3), reaction is 24 hours under 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 and test with sodium nitroprussiate by step (2), redfree occurs as a result, illustrates that Silica Surface exists without sulfydryl, and is oxidized.
(4) get 100 mL three-neck flasks, add successively 2.0 g sulfonic group bonded silica gel derivatives, 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 phases.Gained is fixing to be tested with FTIS, test result as shown in Figure 1, at 1205cm
-1There is a strong wide absworption peak at the place, at 1078cm
-1There is the individual very strong peak that absorbs at the place, and these two peaks overlap together, illustrate that Silica Surface contains-SO
3 -Simultaneously at 794 cm
-1There is individual spike at the place, and stronger, illustrates that phenyl ring exists; At 1631 cm
-1There is individual spike at the place, illustrates that carbonyl exists; At 3486 cm
-1There is a wider absworption peak at the place, illustrates that imido grpup exists, and comprehensive above information illustrates 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 phases.
Use the prepared chromatograph packing material dress post of embodiment 1, then under the strong cation exchange pattern, five kinds of standard proteins are separated.Separation condition:
Mobile 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 myoglobin, RNase B, RNase A, five kinds of protein such as cytochrome c, lysozyme have been realized good separation (1,2,3,4,5 being respectively myoglobin, RNase B, RNase A, cytochrome c, lysozyme as shown in Figure 2).
Use the prepared chromatograph packing material dress post of embodiment 1, then under hydrophobic pattern, seven kinds of standard proteins are separated.Separation condition:
Mobile 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 is 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 (1,2,3,4,5,6,7 being respectively cytchrome c, myoglobin, RNase A, lysozyme, OVA, α-amylase, insulin as shown in Figure 3).
Embodiment 4
Use the chromatograph packing material dress post of embodiment 1 preparation, eight kinds of albumen are separated, the first dimension is separated under the strong cation exchange pattern, collect undivided component under the strong cation exchange pattern, carrying out the second hydrophobic pattern of dimension separates, as shown in Figure 4, obtained to separate fully through eight kinds of albumen after the second hydrophobic pattern separation of dimension.Separation condition:
Mobile 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 as shown in Fig. 4 dotted line, and flow velocity is 1.0 mL/min, to 1, HSA; 2, BSA; 3, insulin; 4, myoglobin; 5, RNase B; 6, α-chymotrypsin; 7, cytochrome c; Eight kinds of protein such as 8, lysozyme have been realized good separation.
Claims (10)
- Shown in structural formula (I) hydrophobic/strong cation exchange mixed mode chromatographic stationary phases,
Wherein X is-OCH 3Or-OCH 2CH 3;Be silica gel;
R is, n=1 ~ 5 wherein; Or , n=1 ~ 6 wherein.
- Claim 1 described hydrophobic/preparation method of strong cation exchange mixed mode chromatographic stationary phases, comprise the following steps:(1) add silica gel to react to get cystine bonding silica derivative thing after the reaction of cystine and silane coupler;(2) cystine bonding silica derivative thing and DTT are reacted to get 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, and DIC, DMAP react hydrophobicly/strong cation exchange mixed mode chromatographic stationary phases.
- 3. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 2 is characterized in that comprising the following steps:(1) cystine of getting 1 part of weight is dissolved in alkaline buffer solution, regulate pH to 11.0, under ice bath stirs, add the silane coupler of 0.5～1 part of weight, ice bath stirring reaction 30 min, then be warming up to 60～80 ℃ of reactions 12～24 hours, react complete, transfer reactant liquor pH to 4.0～7.0 with glacial acetic acid, the silica gel that adds 0.5～1 part of weight, reacted under 80～95 ℃ 1.5～4 hours, 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 cushioning liquid, adds the DTT 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, splash into the concentrated sulfuric acid under stirring, make reactant liquor be faintly acid, reacted under 20～35 ℃ 10～24 hours, 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 organic solvent, the aliphatic alcohol, the aromatic alcohols 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 phases.
- 4. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 3, it is characterized in that: the described 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.
- 5. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 3, it is characterized in that: the described silane coupler structure of step (1) is as follows:
Wherein X is-OCH 3Or-OCH 2CH 3 - 6. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 3, it is characterized in that: in step (1), silica gel used is the full multi-hole blangel bead, particle diameter is 3～40 μ m, the aperture is 50～300, and process 1:1 hydrochloric acid activation 3～7 hours, then be washed till neutrality, 100～160 ℃ of vacuum drying 10～24 hours.
- 7. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 3, it is characterized in that: in step (2), cushioning liquid used is the Tris-HCl cushioning liquid of 1mol/L, and the amount of the required cushioning liquid of every gram cystine bonding silica derivative thing is 10～20mL.
- 8. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 3, it is characterized in that: the oxidation reaction in step (3) is with the H of methyl alcohol and 25～35% 2O 2It is reaction medium that the mixed solution of volume ratio 1:2～5 adds the concentrated sulfuric acid to make it to be faintly acid again, and the required reactant liquor volume of every gram cysteine bonded silica gel derivative is 30～40mL.
- 9. the preparation method of hydrophobic/strong cation exchange mixed mode chromatographic stationary phases according to claim 3, it is characterized in that: in step (4), organic solvent used is carrene or N, dinethylformamide, the amount of the required organic solvent of every gram sulfonic group bonded silica gel derivative is 25～40mL.
- Claimed in claim 1 hydrophobic/application of strong cation exchange mixed mode chromatographic stationary phases in separation and purification of protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210004100 CN102527357B (en) | 2012-01-09 | 2012-01-09 | SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210004100 CN102527357B (en) | 2012-01-09 | 2012-01-09 | SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102527357A CN102527357A (en) | 2012-07-04 |
| CN102527357B true CN102527357B (en) | 2013-11-06 |
Family
ID=46336109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210004100 Expired - Fee Related CN102527357B (en) | 2012-01-09 | 2012-01-09 | SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102527357B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103203225B (en) * | 2013-04-16 | 2015-02-18 | 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 |
| CN105561960B (en) * | 2016-01-14 | 2018-01-09 | 重庆大学 | Changeable efficient liquid phase chromatographic stuffing of a kind of chiral selectivity and preparation method thereof |
| CN107694539B (en) * | 2016-08-08 | 2020-04-17 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Novel reversed phase/weak cation exchange mixed mode chromatographic stationary phase 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 |
| CN109338348B (en) * | 2018-10-24 | 2019-09-20 | 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 |
| CN111389383B (en) * | 2020-02-26 | 2022-08-09 | 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5277813A (en) * | 1988-06-17 | 1994-01-11 | S.A.C. Corporation | Shielded stationary phases |
| CN101413932A (en) * | 2007-10-17 | 2009-04-22 | 中国科学院大连化学物理研究所 | Two-phase integral column as well as preparation and application thereof |
| CN101829551A (en) * | 2010-05-12 | 2010-09-15 | 武汉大学 | Silica gel fixed phase of mixed model as well as preparation method and application thereof |
-
2012
- 2012-01-09 CN CN 201210004100 patent/CN102527357B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5277813A (en) * | 1988-06-17 | 1994-01-11 | S.A.C. Corporation | Shielded stationary phases |
| CN101413932A (en) * | 2007-10-17 | 2009-04-22 | 中国科学院大连化学物理研究所 | Two-phase integral column as well as preparation and application thereof |
| CN101829551A (en) * | 2010-05-12 | 2010-09-15 | 武汉大学 | Silica gel fixed phase of mixed model as well as preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102527357A (en) | 2012-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102527357B (en) | SCX/HIC (Strong Cation Exchange/Hydrophobic) mixed-mode chromatograph stationary phase and preparation method thereof | |
| Johansson et al. | Preparation and characterization of prototypes for multi-modal separation aimed for capture of positively charged biomolecules at high-salt conditions | |
| CN102614847B (en) | Amphoteric ion hydrophilic chromatographic stationary phase and preparation method thereof | |
| CN104096544B (en) | Taking amino benzoglyoxaline as the chromatography media of functional ligand and its preparation method | |
| Lu et al. | Evaluation of immunoglobulin adsorption on the hydrophobic charge-induction resins with different ligand densities and pore sizes | |
| CN101530782B (en) | Liquid phase chromatogram filler and method for synthesizing same | |
| Zhao et al. | Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation | |
| CN104645949B (en) | Affinity chromatography medium employing tetrapeptide as functional ligand and preparation method of affinity chromatography medium | |
| CN107126943A (en) | Phenyl boric acid modified silica-gel function chromatograph packing material, preparation method and application | |
| CN101829551A (en) | Silica gel fixed phase of mixed model as well as preparation method and application thereof | |
| CN103357390A (en) | Multi-layer structure bonded silica gel liquid chromatography packing and synthesis method thereof | |
| CN108114707A (en) | A kind of silica matrix hydrophobic amino acid bonded stationary phase and its preparation and application | |
| CN103551118A (en) | Column [5] aromatic bonding silica gel stationary phase as well as preparation method and application thereof | |
| CN105037283B (en) | Metal organic frame and its synthetic method and application of the one kind based on Cu (II) ion | |
| CN108114705A (en) | A kind of silica matrix basic amino acid bonded stationary phase and its preparation and application | |
| Phottraithip et al. | New hydrophobic charge-induction resin with 2-mercaptoimidazole as the ligand and its separation characteristics for porcine IgG | |
| CN101185881B (en) | Chromatogram medium for immunoglobulin class protein separation purification and preparation method thereof | |
| Wang et al. | Protein separation using a novel silica-based RPLC/IEC stationary phase modified with N-methylimidazolium ionic liquid | |
| CN104250298B (en) | A kind of Exenatide high efficiency separation and purification method | |
| CN103203225B (en) | Weak cation exchange/hydrophobic bifunctional hybrid-mode chromatographic stationary phase as well as preparation method and application thereof | |
| CN103120932A (en) | Preparation method and application of hydrophilic chromatographic stationary phase of cationic polysaccharide coating type | |
| CN111672486B (en) | Chromatography medium with aminobenzene (sulfonamide) pyridine as functional ligand and application thereof | |
| CN108114706A (en) | A kind of silica matrix taurine bonded stationary phase and its preparation and application | |
| CN104628892B (en) | Preparation method and use of 6-benzyl phenylethylamine derivative beta-cyclodextrin bonded SBA-15 | |
| Kale et al. | Characterization of superporous cellulose matrix for high‐throughput adsorptive purification of lysozyme |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131106 Termination date: 20150109 |
|
| EXPY | Termination of patent right or utility model |