CN103055832B - Chromatographic packing for separating water soluble polymer and protein and preparation method of same - Google Patents
Chromatographic packing for separating water soluble polymer and protein and preparation method of same Download PDFInfo
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- 238000012856 packing Methods 0.000 title claims abstract description 54
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 238000001814 protein method Methods 0.000 title description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 114
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 18
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- 238000000034 method Methods 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 125000003700 epoxy group Chemical group 0.000 claims description 12
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 10
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- 239000000243 solution Substances 0.000 claims description 10
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- 230000014759 maintenance of location Effects 0.000 description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 239000012472 biological sample Substances 0.000 description 6
- JSDXOWVAHXDYCU-VXSYNFHWSA-N cefminox Chemical compound S([C@@H]1[C@@](C(N1C=1C(O)=O)=O)(NC(=O)CSC[C@@H](N)C(O)=O)OC)CC=1CSC1=NN=NN1C JSDXOWVAHXDYCU-VXSYNFHWSA-N 0.000 description 6
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- GNWUOVJNSFPWDD-XMZRARIVSA-M Cefoxitin sodium Chemical compound [Na+].N([C@]1(OC)C(N2C(=C(COC(N)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)CC1=CC=CS1 GNWUOVJNSFPWDD-XMZRARIVSA-M 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
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- 229960003016 cefoxitin sodium Drugs 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 5
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 5
- 102000015790 Asparaginase Human genes 0.000 description 4
- 108010024976 Asparaginase Proteins 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 4
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- 229960003272 asparaginase Drugs 0.000 description 4
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 4
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- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical group CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 description 3
- 229930186147 Cephalosporin Natural products 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
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Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention discloses a hydrophilic silica gel chromatographic packing for separating a water soluble polymer and a protein and a preparation method of the hydrophilic silica gel chromatographic packing. The preparation method comprises the following steps: (A), activating silica gels to prepare activated silica gels; (B), carrying out epoxy modification on the activated silica gels so as to prepare epoxy-based modified silica gels; (C), introducing a hydrophilic polymer to the epoxy-based modified silica gels so as to prepare hydrophilic polymer modified silica gels; and (D), hydrolyzing the hydrophilic polymer modified silica gels so as to prepare the hydrophilic silica gel chromatographic packing for separating the water soluble polymer and the protein. The hydrophilic silica gel chromatographic packing disclosed by the invention has the advantages of larger pore volume, lower hydrophobicity, higher number of theoretical plates, and larger peak capacity.
Description
Technical Field
The invention relates to a chromatographic packing, in particular to a chromatographic packing for separating water-soluble polymers and proteins and a preparation method thereof.
Background
With the rapid development of chromatographic techniques, chromatographic packing materials are continuously updated and developed, and people have prepared various novel chromatographic packing materials of different types to solve various problems in separation science. Currently, the major problem faced in separation chemistry is the difficulty of separating complex biological samples. Because the composition of the biological sample is extremely complex and the content of a plurality of biological macromolecules in the biological sample is extremely low, the separation interference is large, the separation and purification steps are multiple, and the process is long; moreover, many biological macromolecules are very vulnerable to inactivation when an organic solvent is used as a mobile phase separation purification; therefore, the separation and purification technology of the biomacromolecule needs to be based on the above, and the selection of the proper chromatographic separation filler is the key point for overcoming the problems in the separation and purification process of the biomacromolecule and obtaining the high-efficiency separation and purification of the biomacromolecule.
Size exclusion chromatography is a rapid and simple separation technique developed in the early 60's of the 20 th century, also known as size exclusion chromatography or gel chromatography. The stationary phase is porous gel with certain pore size range, and is chromatographic technology for separating target molecule size. The separation does not depend on the acting force between a mobile phase and a fixed phase, and the separation is divided into gel permeation chromatography and gel filtration chromatography according to the type of the mobile phase. The commonly used fillers mainly comprise two main types of silica gel or polymer, and because the silica gel has larger internal water volume and more uniform pore diameter, the silica gel has better resolution than polymer gel, and is often used for separating biological samples such as protein, polypeptide, biological enzyme and the like with molecular weight ranging from thousands to nearly millions. However, the problems of poor hydrophilicity, poor stability and the like exist in the traditional process for preparing the size exclusion chromatography filler for separating biological macromolecules such as protein, polypeptide, biological enzyme and the like.
Therefore, providing a chromatographic packing with large pore volume, low hydrophobicity, high theoretical plate number and large peak capacity is becoming a hot point of research at home and abroad.
Disclosure of Invention
In view of the problems in the prior art, the present invention aims to provide a method for preparing a hydrophilic silica gel chromatographic packing for separating water-soluble polymers and proteins, wherein the chromatographic packing prepared by the method can be used as a plurality of separation and retention modes, can be used as a HILIC mode and a size exclusion mode respectively for separation and analysis of liquid chromatography, and is particularly suitable for water-soluble polymers and biological samples such as proteins, polypeptides, biological enzymes, etc.
In order to achieve the above object, the present invention provides a method for preparing a hydrophilic silica gel chromatography packing for separating a water-soluble polymer and a protein, comprising the steps of:
activating silica gel to prepare activated silica gel;
b, carrying out epoxy group modification on the activated silica gel to obtain epoxy group modified silica gel;
introducing a hydrophilic polymer to the epoxy-modified silica gel to prepare hydrophilic polymer-modified silica gel; and
and D, hydrolyzing the hydrophilic polymer modified silica gel to prepare the hydrophilic silica gel chromatographic packing for separating the water-soluble polymer and the protein.
According to the concept of the present invention, the a step includes: the silica gel is washed with distilled water, then soaked with hydrofluoric acid aqueous solution, then washed with distilled water to neutrality, finally washed with acetone and dried.
According to the concept of the present invention, the step B includes: taking boric acid buffer solution as a solvent, and carrying out modification reaction on the activated silica gel and an epoxy group silanization reagent under stirring and reflux, wherein the epoxy group silanization reagent is selected from gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane.
According to the concept of the present invention, the step C includes: and (2) carrying out modification reaction on the epoxy modified silica gel and the hydrophilic polymer under stirring and reflux, after the reaction is finished, carrying out suction filtration, and washing by using distilled water, tetrahydrofuran and acetonitrile in sequence.
According to the concept of the present invention, the step D includes: and (3) carrying out hydrolysis reaction on the hydrophilic polymer modified silica gel and a citric acid buffer solution, after the reaction is finished, carrying out suction filtration, washing with distilled water and methanol in sequence, and carrying out vacuum drying.
According to the concept of the invention, the mass concentration of the hydrofluoric acid aqueous solution is 0.08%, and the material-liquid ratio of the silica gel to the hydrofluoric acid aqueous solution is 1:8-1: 10.
According to the concept of the invention, the mass ratio of the silica gel to the epoxy group silanization reagent is 1:2-1:4, and the reaction time of the activated silica gel for epoxy group modification is 0.5-2 hours; the reaction temperature is 70-100 ℃.
According to the concept of the invention, the pH value of the boric acid buffer solution is 7.0-9.0, and the material-liquid ratio of the activated silica gel to the boric acid buffer solution is 1:8-1: 10.
According to the inventive concept, the hydrophilic polymer is soluble powder, cellulose or dextran.
According to the concept of the invention, the mass ratio of the silica gel to the soluble starch is 1:2-1:4, and the reaction time of modifying the hydrophilic polymer by using the epoxy modified silica gel is 20-24 hours; the reaction temperature is 70-100 ℃.
According to the concept of the invention, the pH value of the citrate buffer solution is 2.0-4.0, the material-liquid ratio of the silica gel to the citrate buffer solution is 1:8-1:10, the reaction time of the hydrophilic polymer modified silica gel for hydrolysis is 1-3 hours, and the reaction temperature is 70-90 ℃.
In order to achieve the purpose, the invention also provides a hydrophilic silica gel chromatographic packing for separating water-soluble polymers and proteins, which is prepared by the preparation method of any one of the above.
In the present invention, the ratio of the material to the liquid in each reaction step is a mass to volume ratio.
The invention is realized by the following measures: firstly, epoxy groups are bonded to the surface of porous silica gel with uniform particle size through the reaction of active silica hydroxyl on the surface of the silica gel and gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane to form epoxy group modified silica gel, then a layer of polymer is coated on the surface of the porous silica gel through the action of the silica hydroxyl on the surface of the porous silica gel and hydrophilic polymers (soluble starch, cellulose, glucan and the like), and finally the epoxy groups on the surface of the porous silica gel are hydrolyzed under an acidic condition to form diol groups, so that the diol group modified water-soluble polymer and the hydrophilic silica gel chromatographic packing for protein are obtained. The packing has a larger pore volume, lower hydrophobicity, a higher theoretical plate number and a larger peak capacity.
The invention adopts a molecular self-assembly mode to coat the hydrophilic polymer on the surface of the silica gel microsphere, and greatly enhances the stability of the polymer-coated diol primary color spectrum filler by synthesizing the polymer-coated diol primary color spectrum filler. The nonspecific adsorption of water-soluble polymers, proteins, biological enzymes, polypeptides and other biological macromolecules is extremely small; has excellent resolving power. In addition, the superior bonding phase surface eliminates other adsorption effects, maintains the biological activity of the sample, and is widely applied to separation of biomacromolecules.
Compared with the conventional chromatographic packing, the chromatographic packing prepared by the invention has the following advantages:
1. the hydrophilic polymer coated on the surface of the porous silica gel overcomes the problem that the filler has unsatisfactory hydrophilicity, and enhances the stability of the filler.
2. The filler has multiple retention mechanisms on a target object, can be used as two chromatographic separation modes of HILIC and size exclusion, and has extremely excellent separation capability on water-soluble polymers, protein, polypeptide, biological enzyme and other biological samples;
3. the mobile phase used by the filler is almost a pure water phase, and the separation condition is extremely mild, so that the high biological activity of a sample in the separation process is greatly ensured;
4. the hydrophilic silica gel chromatographic packing for separating water soluble polymers and proteins has a larger pore volume, lower hydrophobicity, a higher theoretical plate number and a larger peak capacity than the chromatographic packing of a conventional polymer matrix.
Drawings
FIG. 1 is a schematic diagram of the synthesis of hydrophilic silica gel chromatographic packing of the present invention for separating water-soluble polymers and proteins.
FIG. 2 is a schematic diagram showing the effect of the chromatographic packing prepared in example 1 of the present invention in separating cefminox sodium and related substances.
FIG. 3 is a schematic diagram showing the effect of the chromatographic packing prepared in example 1 of the present invention in separating cefoxitin sodium and related substances.
Fig. 4 is a schematic diagram of the effect of the chromatographic packing prepared in example 1 of the present invention in separating cefodizime sodium and related substances.
FIG. 5 is a schematic diagram showing the effect of separating asparaginase by using the chromatographic packing prepared in example 2 of the present invention.
FIG. 6 is a schematic diagram showing the effect of the chromatographic packing prepared in example 3 of the present invention in separating and analyzing insulin.
Detailed Description
The invention is further illustrated by the following figures and examples. However, the present invention is not limited to the following examples.
The invention provides a preparation method of a hydrophilic silica gel chromatographic packing for separating water-soluble polymers and proteins, which comprises the following steps:
activating silica gel to prepare activated silica gel;
b, carrying out epoxy group modification on the activated silica gel to obtain epoxy group modified silica gel;
introducing a hydrophilic polymer to the epoxy-modified silica gel to prepare hydrophilic polymer-modified silica gel; and
and D, hydrolyzing the hydrophilic polymer modified silica gel to prepare the hydrophilic silica gel chromatographic packing for separating the water-soluble polymer and the protein.
Wherein the step A comprises the following steps: the silica gel is washed with distilled water, then soaked with hydrofluoric acid aqueous solution, then washed with distilled water to neutrality, finally washed with acetone and dried.
Wherein the step B comprises the following steps: taking boric acid buffer solution as a solvent, and carrying out modification reaction on the activated silica gel and an epoxy group silanization reagent under stirring and reflux, wherein the epoxy group silanization reagent is selected from gamma- (2, 3-epoxypropoxy) propyl trimethoxy silane.
Wherein the step C comprises the following steps: and (2) carrying out modification reaction on the epoxy modified silica gel and the hydrophilic polymer under stirring and reflux, after the reaction is finished, carrying out suction filtration, and washing by using distilled water, tetrahydrofuran and acetonitrile in sequence.
Wherein the step D comprises: and (3) carrying out hydrolysis reaction on the hydrophilic polymer modified silica gel and a citric acid buffer solution, after the reaction is finished, carrying out suction filtration, washing with distilled water and methanol in sequence, and carrying out vacuum drying.
The specific synthetic steps are shown in figure 1. FIG. 1 is a schematic diagram of the synthesis of hydrophilic silica gel chromatographic packing of the present invention for separating water-soluble polymers and proteins.
The following are examples of the present invention for preparing hydrophilic silica gel chromatographic packing for separating water-soluble polymer and protein, but the concept of the present invention and its application raw materials/apparatuses are not limited thereto.
Example 1: preparation of hydrophilic silica gel chromatographic packing for separating water-soluble polymer and protein
(1) To a 500mL reaction vessel, 50g of silica gel was added, and 200mL of distilled water was added, stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 400mL of hydrofluoric acid aqueous solution with the mass ratio of 0.08%, stirring for 24 hours, then cleaning with distilled water to be neutral, finally adding 200mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) to a 500mL reaction vessel (equipped with condenser, mechanical stirrer, thermometer) was added 400mL of a pH =9.0 boric acid buffer solution (Buffer solution, purchased from Guangzhou Huayue instruments ltd), 50g of activated silica gel and 100mL of gamma- (2, 3-glycidoxy) propyltrimethoxysilane, and stirred at 80 ℃ for 30 minutes; obtaining epoxy group modified silica gel;
(3) adding 100g of soluble starch into a reaction vessel, and carrying out reflux stirring reaction at 97 ℃ for 20-22 hours; after the reaction is finished, cooling and refluxing are carried out for one night (about 12 hours); washing the prepared filler with 200mL of hot distilled water, 200mL of hot tetrahydrofuran and 200mL of hot acetonitrile in sequence, and filtering to obtain soluble starch modified silica gel;
(4) the soluble starch modified silica gel is placed in the reaction vessel again, and 500mL of citric acid buffer (pH3.0) is addedBuffer, available from Guangzhou and Inc.) was added to the reaction mixture, and the reaction was stirred under reflux at 70 ℃ for 1 hour; after the reaction is finished, cleaning the prepared filler with 400mL of distilled water and 400mL of methanol, filtering, and repeating the step once; and naturally airing the cleaned filler, and then drying the filler for 2 hours at 110 ℃ in vacuum to obtain the hydrophilic silica gel chromatographic filler for separating the water-soluble polymer and the protein.
Example 2: preparation of hydrophilic silica gel chromatographic packing for separating water-soluble polymer and protein
(1) To a 500mL reaction vessel, 50g of silica gel was added, and 200mL of distilled water was added, stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 400mL of hydrofluoric acid aqueous solution with the mass ratio of 0.08%, stirring for 24 hours, then cleaning with distilled water to be neutral, finally adding 200mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) to a 500mL reaction vessel (equipped with condenser, mechanical stirrer, thermometer) was added 450mL of boric acid buffer solution pH =9.0, 50g of activated silica gel and 150mL of gamma- (2, 3-glycidoxy) propyltrimethoxysilane, and the reaction was stirred at 80 ℃ for 30 minutes; obtaining epoxy group modified silica gel;
(3) adding 150g of glucan into a reaction container, and carrying out reflux stirring reaction at the temperature of 97 ℃ for 20-22 hours; after the reaction is finished, cooling and refluxing are carried out for one night (about 12 hours); washing the prepared filler with 200mL of hot distilled water, 200mL of hot tetrahydrofuran and 200mL of hot acetonitrile in sequence, and filtering; obtaining glucan modified silica gel;
(4) the resulting dextran-modified silica gel was placed in the reaction vessel again, and 400mL of pH3.0 citric acid buffer (A buffer solution, available from Guangzhou and Co., Ltd.), and reacted at 70 ℃ for 1 hour with stirring under reflux; after the reaction is finished, cleaning the prepared filler with 200mL of distilled water and 200mL of methanol, filtering, and repeating the step once; and naturally airing the cleaned filler, and then carrying out vacuum drying for 2 hours at 110 ℃ to obtain the hydrophilic silica gel chromatographic filler for separating the water-soluble polymer and the protein.
Example 3: preparation of hydrophilic silica gel chromatographic packing for separating water-soluble polymer and protein
(1) To a 500mL reaction vessel, 50g of silica gel was added, and 200mL of distilled water was added, stirred for 30 minutes, filtered, and the procedure was repeated twice. Then adding 400mL of hydrofluoric acid aqueous solution with the mass ratio of 0.08%, stirring for 24 hours, then cleaning with distilled water to be neutral, finally adding 200mL of acetone for washing, filtering, and drying at 110 ℃ for 12 hours to obtain activated silica gel;
(2) to a 500mL reaction vessel (equipped with condenser, mechanical stirrer, thermometer) was added 450mL of boric acid buffer solution pH =9.0, 50g of activated silica gel and 150mL of gamma- (2, 3-glycidoxy) propyltrimethoxysilane, and the reaction was stirred at 80 ℃ for 30 minutes; obtaining epoxy group modified silica gel;
(3) adding 150g of cellulose into a reaction vessel, and carrying out reflux stirring reaction at the temperature of 97 ℃ for 20-22 hours; after the reaction is finished, cooling and refluxing are carried out for one night (about 12 hours); washing the prepared filler with 200mL of hot distilled water, 200mL of hot tetrahydrofuran and 200mL of hot acetonitrile in sequence, and filtering to obtain cellulose modified silica gel;
(4) the resulting cellulose-modified silica gel was placed in a reaction vessel again, and 400mL of citric acid buffer (pH3.0) was addedA buffer solution, available from Guangzhou and Co., Ltd.), and reacted at 70 ℃ for 1 hour with stirring under reflux; after the reaction is finished, cleaning the prepared filler with 200mL of distilled water and 200mL of methanol, filtering, and repeating the step once; and naturally airing the cleaned filler, and then carrying out vacuum drying for 2 hours at 110 ℃ to obtain the hydrophilic silica gel chromatographic filler for separating the water-soluble polymer and the protein.
Example 4: hydrophilic silica gel chromatographic packing chromatographic column size exclusion separation for separating water soluble polymer and protein
The water-soluble polymer and the protein separated in the example 1 are packed into a column by using a hydrophilic silica gel chromatographic packing, the length of the column is 300mm, and the inner diameter of the column is 7.8 mm.
And (3) detecting cefminox sodium and related substances thereof by taking phosphate buffer solution (pH7.0) [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (61:39) ] -acetonitrile (95:5) as a mobile phase, wherein the flow rate is 0.8mL per minute, the detection wavelength is 254 nm. As shown in fig. 2, fig. 2 is a schematic diagram of the effect of the chromatographic packing prepared in example 1 of the present invention on separating cefminox sodium and related substances. Cefminox sodium is cephalosporin antibiotics, has a molecular weight of 667.67, and has a chromatogram with a retention time in min (minutes) on the abscissa and a response value in mV (millivolts) on the ordinate of a detection signal. As can be seen from the figure: the chromatographic peak of the cefminox sodium and the chromatographic peak of impurities thereof are separated to a great extent, so that the problems that the cefminox sodium and the impurities thereof are difficult to separate by adopting a conventional C18 column and the like are solved, and the filler has excellent separation performance.
Using phosphate buffer solution [0.01mol/L disodium hydrogen phosphate solution-0.01 mol/L sodium dihydrogen phosphate solution (66:34)]Acetonitrile (95:5) as mobile phase, flow rate of 0.8 mL/min, detection wavelength of 232nm, determination of cefoxitin sodium and its related substances. As shown in fig. 3, fig. 3 is a schematic diagram of the effect of the chromatographic packing prepared in example 1 of the present invention in separating cefoxitin sodium and related substances. Cefoxitin sodium is cephalosporin antibiotics, has molecular weight of 449.43, and has chromatogram with retention time in min (min) on abscissa and response value of detection signal in 10 on ordinate-3mV (millivolts). According to the chromatogram, it can be seen that: the cefoxitin sodium and impurities thereof can be well separated, and the sensitivity of separating impurity peaks is high, so that the filler is fully proved to have excellent chromatographic separation performance.
Using phosphoric acid buffer solution (pH =7.0) [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (61:39)]Acetonitrile (95:5) as mobile phase, flow rate 0.8mL per minute, detection wavelength 231nm, determination of cefodizime sodium and its related substances. As shown in fig. 4, fig. 4 is a schematic diagram of the effect of the chromatographic packing prepared in example 1 of the present invention in separating cefodizime sodium and related substances. The abscissa is retention time in min (minutes), and the ordinate is response value of detection signal in 10-3mV (millivolts). According to the chromatogram, it can be seen that: the cefodizime sodium and impurities thereof can be well separated, and the sensitivity of separating impurity peaks is high, thereby completely meeting the separation parameters specified by pharmacopoeia.
Example 5: hydrophilic silica gel chromatographic packing chromatographic column size exclusion separation for separating water soluble polymer and protein
The water-soluble polymer and the protein which are separated in the example 2 are filled into a column by using hydrophilic silica gel chromatographic packing, the length of the column is 300mm, and the inner diameter of the column is 7.8 mm. Asparaginase was assayed at a flow rate of 0.6mL per minute and a detection wavelength of 280nm using 0.1mol/L phosphate buffer (pH6.7) (sodium dihydrogen phosphate 6.0g, disodium hydrogen phosphate 20.2g, water 900mL, pH adjusted to 6.7 with phosphoric acid or sodium hydroxide solution, water 1000mL) as the mobile phase. As shown in FIG. 5, FIG. 5 is a schematic view showing the effect of separating asparaginase by using the chromatographic packing prepared in example 2 of the present invention. The abscissa is retention time in min (minutes), and the ordinate is response value of detection signal in 10-3mV (millivolts). As is evident from the figure: the chromatographic column can separate impurity peaks of asparaginase and completely accords with the regulations of pharmacopoeia.
Example 6: hydrophilic silica gel chromatographic packing chromatographic column size exclusion separation for separating globular protein chromatography
The globular protein separation chromatography in example 3 was carried out on a column packed with a hydrophilic silica gel chromatography packing material, the column having a length of 300mm and an inner diameter of 7.8 mm. Insulin and related high molecular substances thereof were measured using acetonitrile, trifluoroacetic acid, water =40:0.1:60 as a mobile phase, a flow rate of 1.0mL per minute, a detection wavelength of 210 nm. As shown in fig. 6, fig. 6 is a schematic diagram of the effect of the chromatographic packing prepared in example 3 of the present invention in separating and analyzing insulin. The abscissa is retention time in min (minutes), and the ordinate is response value of detection signal in 10-3mV (millivolts). As can be seen from the figure: the chromatographic column can obtain better insulin peak pattern and higher column effect.
Comparative example: the effect of the hydrophilic silica gel chromatographic packing chromatographic column for separating the globular protein is compared with that of other types of chromatographic columns.
The globular protein separation chromatography in example 3 was carried out on a column packed with a hydrophilic silica gel chromatography packing material, the column having a length of 300mm and an inner diameter of 7.8 mm. Using phosphoric acid buffer solution (pH =7.0) [0.005mol/L disodium hydrogen phosphate solution-0.005 mol/L sodium dihydrogen phosphate solution (61:39)]Acetonitrile (95:5) as mobile phase, flow rate 0.8mL per minute, detection wavelength 231nm, determination of cefodizime sodium and its related substances. And comparing the results with conventional C18 column, a column similar to other companies (e.g. Seiki technology)SEC-150) comparison, and found that cefodizime sodium and related impurities cannot be separated by using a conventional C18 column, the separation degree of the cefodizime sodium and the related impurities does not exceed 1.0, and Setaria is usedWhen the SEC-150 is used for separating the cefodizime sodium and related impurities, the separation degree is still not ideal enough, the target cefodizime sodium and the impurities cannot achieve baseline separation, and the response of impurity peaks is not ideal enough; the hydrophilic silica gel chromatographic packing chromatographic column for separating globular protein chromatography prepared by the method has the greatest advantage that the separation degree of the target cefodizime sodium and related impurities thereof can reach baseline separation, and the chromatographic packing is proved to have more excellent separation effect compared with the conventional C18 chromatographic column and chromatographic columns similar to other companies.
The size exclusion chromatography packing of the fully porous silica gel matrix has a larger pore volume, lower hydrophobicity, higher theoretical plate number and larger peak capacity. The stability of hydrophilic polymer coated diol based spectral fillers can be greatly enhanced by their synthesis (b.anspace, et al.j.chromatogr.,1988,443, 45). Also, the separation does not involve too much force, and the separation is generally very mild under aqueous conditions. In addition, the hydrophilic polymer coated on the silica gel has multiple retention mechanisms, the surface of the hydrophilic polymer is protected by the diol functional group and does not interact with protein, a chromatographic column does not accumulate a strongly adsorbed target substance, the recovery rate of the target substance is higher, side reaction is not easy to occur, the service life is long, and the mass recovery rate of most of proteins in the elution process is close to 100%. In addition, the hydrophilic polymer coated on the surface of the silica gel ensures the sufficient action of the filler and the target object, has higher resolution and can be operated at higher temperature.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, which is defined by the claims of the present application, and all simple, equivalent changes and modifications made according to the claims and the contents of the specification of the present application.
Claims (9)
1. A method for preparing hydrophilic silica gel chromatographic packing for separating water-soluble polymer and protein comprises the following steps:
activating silica gel to prepare activated silica gel;
b, carrying out epoxy group modification on the activated silica gel to obtain epoxy group modified silica gel;
coating a hydrophilic polymer on the epoxy-modified silica gel to prepare hydrophilic polymer-modified silica gel; and
d, hydrolyzing the hydrophilic polymer modified silica gel to prepare the hydrophilic silica gel chromatographic packing for separating the water-soluble polymer and the protein;
wherein,
the step A comprises the following steps:
washing silica gel with distilled water, soaking in hydrofluoric acid water solution, washing with distilled water to neutrality, washing with acetone and drying;
the step B comprises the following steps:
taking boric acid buffer solution as a solvent, and carrying out modification reaction on the activated silica gel and an epoxy group silanization reagent under stirring and reflux;
the step C comprises the following steps:
carrying out modification reaction on the epoxy modified silica gel and the hydrophilic polymer under stirring and reflux, after the reaction is finished, carrying out suction filtration, and washing with distilled water, tetrahydrofuran and acetonitrile in sequence;
the step D comprises the following steps:
and (3) carrying out hydrolysis reaction on the hydrophilic polymer modified silica gel and a citric acid buffer solution, after the reaction is finished, carrying out suction filtration, washing with distilled water and methanol in sequence, and carrying out vacuum drying.
2. The preparation method according to claim 1, wherein the hydrofluoric acid aqueous solution has a mass concentration of 0.08%, and the feed-to-solution ratio of the silica gel to the hydrofluoric acid aqueous solution is 1:8 to 1:10 in terms of mass-to-volume ratio in g/mL.
3. The method of claim 1, wherein the epoxysilylating agent is gamma- (2, 3-glycidoxy) propyltrimethoxysilane.
4. The preparation method according to claim 1, wherein the mass ratio of the silica gel to the epoxysilylation agent is 1:2 to 1:4, and the reaction time of the activated silica gel for epoxy modification is 0.5 to 2 hours; the reaction temperature is 70-100 ℃.
5. The method according to claim 1, wherein the pH value of the boric acid buffer is 7.0 to 9.0, and the feed-to-solution ratio of the activated silica gel to the boric acid buffer is 1:8 to 1:10 in terms of mass-to-volume ratio in g/mL.
6. The method of claim 1, wherein the hydrophilic polymer is soluble starch, cellulose, or dextran.
7. The preparation method according to claim 6, wherein the mass ratio of the silica gel to the soluble starch is 1:2 to 1:4, and the reaction time of modifying the epoxy-based modified silica gel with the hydrophilic polymer is 20 to 24 hours; the reaction temperature is 70-100 ℃.
8. The preparation method according to claim 1, wherein the pH value of the citrate buffer is 2.0-4.0, the feed-to-liquid ratio of the silica gel to the citrate buffer is 1:8-1:10 in terms of g/mL by mass-to-volume ratio, the reaction time for hydrolyzing the hydrophilic polymer modified silica gel is 1-3 hours, and the reaction temperature is 70-90 ℃.
9. A hydrophilic silica gel chromatographic packing for separating water-soluble polymer and protein, prepared by the preparation method as claimed in any one of claims 1 to 8.
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| CN104826618B (en) * | 2015-04-20 | 2017-05-24 | 华东理工大学 | Aminodiol hydrophilic chromatography stationary phase and preparation method thereof |
| CN108404893A (en) * | 2018-03-29 | 2018-08-17 | 浙江月旭材料科技有限公司 | A kind of silica gel chromatographic column filling material, preparation method and applications |
| CN109174010A (en) * | 2018-08-31 | 2019-01-11 | 深圳市迪莫斯环保科技有限公司 | One kind is except modified formaldehyde silica gel material and preparation method thereof |
| CN113786817B (en) * | 2021-08-31 | 2022-06-14 | 浙江月旭材料科技有限公司 | Modified silica gel material and preparation method and application thereof |
| CN113952944B (en) * | 2021-12-23 | 2022-03-29 | 浙江湃肽生物有限公司深圳分公司 | Method for purifying nonapeptide-1 |
| CN114601805A (en) * | 2022-01-13 | 2022-06-10 | 南昌立健药业有限公司 | Cefminox sodium powder for injection and preparation method thereof |
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