CN101234340A - Methylpropenoic acid-2-hydroxy propyl ester silica--based polymer-bonded phase, preparation and application thereof - Google Patents
Methylpropenoic acid-2-hydroxy propyl ester silica--based polymer-bonded phase, preparation and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of chromatographic column filling and the procedures of the preparation method are as follows: (1) silica gel activation; (2) methyl vinyl bonding; (3) silica gel deactivation; (4) initiated polymerization. The application is as following: acetonitrile-water as moving phase, gradient condition is of 0 to 20min, acetonitrile is of 5 to 40 percent; gradient condition is of 20 to 25min, acetonitrile is of 40 to 5 percent. The standard protein and crude protein are separated from each other and three components can be obtained: ovalbumin, trypsin inhibitor and chymotrypsin inhibitor chromatogram map. The column filling preparation method has the advantages of high column efficiency, short separation time, good effect of protein separation and wide range of suitable pH value.
Description
Technical field
The present invention relates to the preparation of chromatographic column filler, relate in particular to the preparation of the polymer chromatographic column filler that is coated on the silica matrix.
Background technology
Splitter is the core of chromatographic technique, and fixedly the research of phase then is the basis that various high performance liquid chromatographies are rely and set up and develop.In the face of in the research and production to the demand of high purity substance, new packing and the Separation of Proteins filler of developing more effective preparation isolation technics, preparative chromatography are vital.Fixing that research and development are good with separative efficiency height, selectivity that large biological molecule adapts, activity reclaims and mass recovery is good, the life-span is long is the target that people pursue all the time mutually.Use the fixing widely silica matrix bonding that is mainly mutually to fix two classes mutually with polymer substrate mutually in the current high performance liquid chromatography.The two each has its unique advantage and range of application, and silica matrix bonding phase mechanical strength is big, but anti-pH narrow range 2-7.5.And the fixing mutually anti-pH wide ranges 0-14 of polymer substrate, but in organic solvent, be easy to swelling.If the strong point of the two is combined, learn from other's strong points to offset one's weaknesses, promptly make supporting mass with silica matrix, outside coated polymer layer, thus obtain a kind of new packing, more help quick separation and the active recovery of large biological molecule.This kind new packing can tolerate pH2-10 than ordinary silicon gel matrix filler tolerance pH range widens, also swelling can not take place in solvent.The permeance property of this kind filler is good in addition, and post forces down, at 100% methyl alcohol, and flow velocity 1ml/min, chromatographic column: under 150 * 4.6mm i.d condition, it only is 5.3MPa that post is pressed, the quick wash-out that therefore can adapt under the high solvent flow velocity separates.
Summary of the invention
The object of the invention provides a kind of methacrylic acid-2-hydroxy propyl ester silica polymer-bound phase chromatographic column filler and preparation method thereof.This project is finished bullets under Beijing's natural science fund assistance: 2073028.
Methacrylic acid-2-hydroxy propyl ester silica polymer-bound phase, its structural formula is:
N is the degree of polymerization, and the scope of n value is 2-7.
Preparing above-mentioned methacrylic acid-2-hydroxy propyl ester silica polymer-bound can realize mutually by the following method:
(1) silica gel activating: press hydrochloric acid, silica gel volume, mass ratio 2-6ml/g, with percent by volume is that 20% the hydrochloric acid and the silica gel in 30nm-300nm aperture are mixed with mixture, heat 90-100 ℃ of reaction backflow 2-6 hour, suction filtration, spend deionised water to neutral, 105 ℃ were dried by the fire 4-8 hour, and 50 ℃ vacuum drying 4-6 hour, activated silica gel standby.
(2) bonding methyl ethylene: be mixed with mixture by methyl ethylene dichlorosilane, silica gel volume, mass ratio 1-3ml/g methyl ethylene dichlorosilane and above-mentioned activated silica gel, the dry toluene that adds 5-7 times of methyl ethylene dichlorosilane volume again, logical N under 100-120 ℃, mechanical agitation, liquid level
2Reaction condition under backflow 18-24 hour, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel.
Reaction equation:
(3) passivation: press bonding methyl ethylene silica gel, trim,ethylchlorosilane quality, volume ratio 1-4g/ml, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, the dry toluene that adds 8-10 times of trim,ethylchlorosilane volume again is in the down logical N of 100-120 ℃, mechanical agitation, liquid level
2Under the reaction condition backflow 18-24 hour, reaction was finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets passivation silica gel.
Reaction equation:
(4) initiated polymerization: take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the initator of the bonding ethylene base silica gel quality 5/1000ths-2 percent of passivation, 10-15 times of dry toluene is at 100-120 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition, methyl-prop diluted acid-2-the hydroxy propyl ester of the bonding ethylene base silica gel quality of one above-mentioned passivation of 1ml/min dropping ten/First Five-Year Plan branch, the crosslinking agent divinylbenzene of the bonding ethylene base silica gel quality of 5/1000ths-2 percent above-mentioned passivation, the initator of the bonding ethylene base silica gel quality of 5/1000ths-2 percent above-mentioned passivation and the mixed liquor of 2-4 times of dry toluene, after treating all to add, reacted 18-24 hour, after finishing, reaction uses earlier the dry toluene suction filtration, washing, use the methyl alcohol suction filtration again, washing gets methacrylic acid-2-hydroxy propyl ester silica chromatographic column filler.
Reaction equation:
N is the degree of polymerization, and the scope of n value is 2-7.
Above-mentioned initator is benzoyl peroxide or azodiisobutyronitrile.
Use above-mentioned methacrylic acid-2-hydroxy propyl ester silica polymer-bound protein that is separated, as mobile phase, gradient condition is 0-20min, acetonitrile 5%-40% with acetonitrile-water; 20-25min, acetonitrile 40%-5% separates standard protein or egg albumin crude product, obtains three components, is respectively ovalbumin, trypsin inhibitor, chymotrypsin protein inhibitor chromatogram.Liquid chromatograph: island Tianjin-20A; Detect wavelength: 280nm and 254nm.
The present invention compared with prior art has post and imitates high, disengaging time weak point, effective to Separation of Proteins, advantages such as while applicable pH range broad.
Description of drawings
Fig. 1,13,14,15,16 are followed successively by the liquid chromatogram of four standard protein mixing sample introductions of 5 embodiment
1-papain (papain); 2-insulin (insulin); 3-hemoglobin (BHb); 4-lysozyme (lysozyme)
Phase flows: acetonitrile-water; Gradient condition: 0-20min, 5% acetonitrile-40% acetonitrile; 20-25min, 40% acetonitrile-5% acetonitrile.
Fig. 2 is a crude protein sample liquid chromatogram
Sample: egg albumin crude product
Phase flows: acetonitrile-water; Gradient condition: 0-20min, 5% acetonitrile-40% acetonitrile; 20-25min, 40% acetonitrile-5% acetonitrile.
Fig. 3 protein keeps and the mobile elution volume graph of a relation mutually of alkali
Chromatographic column: 150 * 4.6mm i.d. silica gel polymer filler, phase flows: the A:0.1%TFA aqueous solution, B:0.1%TFA acetonitrile solution, 20-min from 5%B to the 100%B linear gradient.Flow velocity: 1ml/min, UV-280nm.
Peaks:1.Yeast ribonuclease (yeast ribonuclease), 2.Glucose oxidase (catalase), (3.Lysozyme lysozyme), 4.Cytochrome C (cromoci), (5.Ribonnuclease ribalgilase), 6.Bovineserum albumin (bovine serum albumin(BSA)), 7.Chick egg albumin (egg albumin).
Fig. 4 methacrylic acid-2-hydroxy propyl ester silica polymer chromatographic column filler infrared spectrogram
Fig. 5 silica gel body infrared spectrogram
Fig. 6 silica gel particle amplifies 20,000 times of sem photographs
Fig. 7 silica gel particle amplifies 1,000 times of sem photographs
Fig. 8 methacrylic acid-2-hydroxy propyl ester silica polymer chromatographic column filler amplifies 20,000 times of sem photographs
Fig. 9 base acrylic acid-2-hydroxy propyl ester silica polymer chromatographic column filler amplifies 1,000 times of sem photographs
Figure 10 drip water meter surface tension angular measurement is fixed, photo before the globule drippage
Figure 11 drip water meter surface tension angular measurement is fixed, globule drippage back photo
The logarithm of Figure 12 molecular weight and corresponding retention volume curve map
Use high performance liquid chromatograph Waters 600, the siliceous polymer-bound phase of self-control macropore chromatographic column, EISD, mobile phase: oxolane, flow velocity: 0.6mL/min.Adopt the standard P s standard specimen of known different molecular weight: from 666 to 2,000,000g.mol
-1, measure retention volume separately, make curve map with the logarithm of molecular weight with corresponding retention volume then.Can find out adequate proteins matter separating ranges 1000-3000000 from curve map.
Wei1: silica gel particle
Wei2: bonding ethylene base silica gel
Wei3: methacrylic acid-2-hydroxy propyl ester silica polymer filler
The specific embodiment
Embodiment 1:(annotates: " bonding phase " and " chromatographic column filler " are general in this area, write as the bonding phase because of title is no more than 25 words, but generally all be accustomed to being write as chromatographic column filler, so hereinafter usefulness be chromatographic column filler.)
(1) silica gel activating
By hydrochloric acid, silica gel volume, mass ratio is 2ml/g, is that 20% hydrochloric acid and 30nm pore size silica gel are mixed with mixture with percent by volume, heats 90 ℃ of reactions and refluxes 2 hours, suction filtration, spend deionised water to neutral, 105 ℃ of bakings 4 hours, and 50 ℃ of vacuum drying 4 hours, activated silica gel is standby.
(2) bonding methyl ethylene
Press methyl ethylene dichlorosilane, silica gel, volume/mass is 1ml/g, methyl ethylene dichlorosilane and above-mentioned activated silica gel are mixed with mixture, add the dry toluene of 6 times of methyl ethylene dichlorosilane volumes again, logical N under 100 ℃, mechanical agitation, liquid level
2Reaction condition refluxed 20 hours down, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel.
(3) passivation
Press bonding methyl ethylene silica gel, trim,ethylchlorosilane, mass/volume is 2g/ml, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, add the dry toluene of 8 times of trim,ethylchlorosilane volumes again, in the down logical N of 110 ℃, mechanical agitation, liquid level
2Refluxed 24 hours under the reaction condition, earlier with dry toluene suction filtration, washing, use methyl alcohol suction filtration, washing again after reaction is finished, get passivation silica gel.
(4) initiated polymerization
Take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the initator of the bonding ethylene base silica gel quality 5/1000ths of passivation, 12 times of dry toluenes are at 100 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition, the initator of the bonding ethylene base silica gel quality of one of the crosslinking agent divinylbenzene of the bonding ethylene base silica gel quality of one of the methyl-prop diluted acid-2-hydroxy propyl ester of the bonding ethylene base silica gel quality of 1ml/min dropping 1/10th above-mentioned passivation, percentage above-mentioned passivation, percentage above-mentioned passivation and the mixed liquor of 2 times of dry toluenes, after treating all to add, reacted 18 hours, after finishing, reaction uses dry toluene suction filtration, washing earlier, use methyl alcohol suction filtration, washing again, get methacrylic acid-2-hydroxy propyl ester silica chromatographic column filler.
The sign of chromatograph packing material (can be the sign of the obtained filler of arbitrary preparation embodiment)
Infrared: get methacrylic acid-2-hydroxy propyl ester silica polymer that macro porous silica gel and the foregoing description make, carry out infrared spectrum analysis, the infrared spectrogram of macro porous silica gel and polymer filler is respectively as Fig. 4 and shown in Figure 5, as can be seen from Figure:
(1) benzene ring structure: wave number 1630.4 and 1519.4, polymerization silica gel itself does not have 1519.4 absworption peak;
(2) methyl structural: wave number 2948.3 and 2881.2;
(3) Si-CH3 structure: wave number 1145.5 belongs to the wherein symmetrical deformation vibration of methyl, and characteristics are strong, wide.
(4) hydroxyl structure: wave number 3700-3200 wherein belongs to the free silica hydroxyl near 3690, and 3500-3200 belongs to alcoholic extract hydroxyl group and forms the broad peak that produces behind the hydrogen bond.Wave number 1411.7-1200 belongs to hydroxyl in-plane bending vibration peak.Hydroxyl peak obviously broadens after relatively can obviously finding out bonding, illustrates that silicon hydroxyl major part is reacted away.
ESEM
Get methacrylic acid-2-hydroxy propyl ester silica polymer that macro porous silica gel and the foregoing description make, adopt ESEM, carry out morphology analysis.Fig. 6 and Fig. 7 are the silica gel particles of not participating in the chemical bonding reaction, and Fig. 8 and Fig. 9 are the silica gel polymer chromatograph packing materials that makes by the foregoing description.Fig. 6 and Fig. 8 according to silica gel as can be seen in that to participate in the structural change of reaction front and rear surfaces little, the very thin pore structure that does not influence silica gel of polymer layer be described, meet experiment and expect.Fig. 7 and Fig. 9 disperse in order before and after reaction according to silica gel particle as can be seen, and intergranular serious adhesion does not take place because of polymerisation.
The surface tension angle experiment
Get methacrylic acid-2-hydroxy propyl ester silica polymer that macro porous silica gel and the foregoing description make, the hydrophilicity that adopts drip water meter surface tension angle measuring instrument to carry out synthetic filling characterizes.Figure 10 is the photo before the globule does not drop onto the filler compressing tablet, and dark plane, figure below both had been the compressing tablet of prepared filler.The photo that Figure 11 in time absorbs for globule drippage back.Two figure are mutually according to not changing at its hydrophilicity of filler that silica gel is carried out obtain after the polymerization as can be seen, also belong to the total hydrophilic filler, surface tension is 0 degree, meet the experiment expection, this kind hydrophilic filler and water miscible large biological molecule have good mass transfer ability, are suitable for the separation of large biological molecule.
Thermogravimetric analysis
Methacrylic acid-2-hydroxy propyl ester silica the polymer-bound of the present invention preparation is reached body silica gel mutually carry out thermogravimetric analysis, amount of samples is about 10mg, and the intensification scope is 20 ℃-850 ℃, and programming rate is 10 ℃/min, carries out thermogravimetric analysis under the air ambient.Methacrylic acid-2-hydroxy propyl ester silica polymer-bound has an obvious weightless peak between 320.97 ℃-360.72 ℃, the weightlessness of calculating after the deduction silica gel body blank is 2.73%.Learn that by theory calculating the phosphorus content of this kind bonding phase is about 2.11%.
The evaluation of chromatograph packing material
(1) chromatographic column is loaded
Get the filler 2.3g that the foregoing description makes, as suspending agent, the absolute ethyl alcohol after the ultrasonic expeling gas is as dress post displacement fluid with the acetone after the ultrasonic expeling gas, and filling restriction pressure is 55MP, one of filling 25 * 4.6mm chromatographic column is carried out the Separation of Proteins experiment.The pneumatic reciprocating pump formula of packing column machine: Haskel packing column machine, the high pure nitrogen steel cylinder is as pneumatic source.
(2) chromatograph packing material Protein Separation experiment
Use above-mentioned methacrylic acid-2-hydroxy propyl ester silica polymer chromatographic column filler, with acetonitrile-water as mobile phase, flow velocity 1ml/min, gradient condition is 0-20min, acetonitrile 5%-40%; 20-25min, acetonitrile 40%-5% separates standard protein, obtains the chromatogram shown in the figure one.As can be seen from the figure, four standard proteins have all been realized baseline separation, the peak shape symmetry, and the response height does not have hangover, and analysis speed is fast, and all protein all went out the peak in seven minutes, and the papain retention time is 2.54 minutes; The insulin retention time is 4.85 minutes; The BHb retention time is 5.7 minutes; The lysozyme retention time is 6.1 minutes.Liquid chromatograph: island Tianjin-20A; Detect wavelength: 280nm and 254nm, be 25 minutes detection time.
Use above-mentioned methacrylic acid-2-hydroxy propyl ester silica polymer chromatographic column, with acetonitrile-water as mobile phase, flow velocity 1ml/min, gradient condition is 0-20min, acetonitrile 5%-40%; 20-25min, acetonitrile 40%-5% separates the egg albumin crude product, and obtaining three components is respectively ovalbumin, trypsin inhibitor, chymotrypsin protein inhibitor, and chromatogram is shown in Figure 2.As can be seen from the figure, three component peaks have realized separating preferably, and analysis speed is fast, went out the peak and finish separation in ten minutes, liquid chromatograph: island Tianjin-20A; Detect wavelength: 280nm and 254nm, be 20 minutes detection time.
After adopting the sodium hydrate aqueous solution of pH10 to wash methacrylic acid-2-hydroxy propyl ester silica polymer chromatographic column 10000ml that the foregoing description makes continuously, the isolated protein effect is constant, and filler can use in the pH2-10 scope, as shown in Figure 3.
Chromatographic column: 150 * 4.6mm i.d. silica gel polymer filler, phase flows: the A:0.1%TFA aqueous solution, B:0.1%TFA acetonitrile solution, 20-min from 5%B to the 100%B linear gradient.Flow velocity: 1ml/min, UV-280nm.
Peaks:1.Yeast ribonuclease (yeast ribonuclease), 2.Glucose oxidase (catalase), (3.Lysozyme lysozyme), 4.Cytochrome C (cromoci), (5.Ribonnuclease ribalgilase), 6.Bovineserum albumin (bovine serum albumin(BSA)), 7.Chick egg albumin (egg albumin).
Adopt PS-gel filtration chromatography method to measure full infiltration and the full exclusion limit that the foregoing description makes methacrylic acid-2-hydroxy propyl ester silica polymer filler, determine the molecular weight ranges of the separable protein of this filler molecular exclusion chromatography.
Use high performance liquid chromatograph Waters 600, the siliceous polymer-bound phase of self-control macropore chromatographic column, EISD, mobile phase: oxolane, flow velocity: 0.6mL/min.Adopt the standard P s standard specimen of known different molecular weight: from 666 to 2,000,000g.mol-1 measures retention volume separately, makes curve map with the logarithm of molecular weight with corresponding retention volume then, as shown in Figure 7.Can find out adequate proteins matter separating ranges 1000-3000000 from curve map.
Embodiment 2:
(1) silica gel activating
By hydrochloric acid, silica gel volume/mass is 2ml/g, is that 20% hydrochloric acid and 100nm pore size silica gel are mixed with mixture with percent by volume, heats 90 ℃ of reactions and refluxes 2 hours, suction filtration, spend deionised water to neutral, 105 ℃ of bakings 4 hours, and 50 ℃ of vacuum drying 4 hours, activated silica gel is standby;
(2) bonding methyl ethylene
By methyl ethylene dichlorosilane, silica gel volume/mass is 1ml/g, methyl ethylene dichlorosilane and above-mentioned activated silica gel are mixed with mixture, the dry toluene that adds 6 times of methyl ethylene dichlorosilane volumes again, logical N under 100 ℃, mechanical agitation, liquid level
2Reaction condition refluxed 20 hours down, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel;
(3) passivation
By bonding methyl ethylene silica gel, trim,ethylchlorosilane mass/volume is 2g/ml, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, the dry toluene that adds 8 times of trim,ethylchlorosilane volumes again is in the down logical N of 110 ℃, mechanical agitation, liquid level
2Refluxed 24 hours under the reaction condition, earlier with dry toluene suction filtration, washing, use methyl alcohol suction filtration, washing again after reaction is finished, get passivation silica gel;
(4) initiated polymerization
Take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the initator of the bonding ethylene base silica gel quality 5/1000ths of passivation, 12 times of dry toluenes are at 100 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition, the initator of the bonding ethylene base silica gel quality of one of the crosslinking agent divinylbenzene of the bonding ethylene base silica gel quality of one of the methyl-prop diluted acid-2-hydroxy propyl ester of the bonding ethylene base silica gel quality of 1ml/min dropping 1/10th above-mentioned passivation, percentage above-mentioned passivation, percentage above-mentioned passivation and the mixed liquor of 2 times of dry toluenes, after treating all to add, reacted 18 hours, after finishing, reaction uses dry toluene suction filtration, washing earlier, use methyl alcohol suction filtration, washing again, get methacrylic acid-2-hydroxy propyl ester silica chromatographic column filler.
Embodiment 3:
(1) silica gel activating
By hydrochloric acid, silica gel volume/mass is 4ml/g, is that 20% hydrochloric acid and 200nm pore size silica gel are mixed with mixture with percent by volume, heats 90 ℃ of reactions and refluxes 3 hours, suction filtration, spend deionised water to neutral, 105 ℃ of bakings 5 hours, and 50 ℃ of vacuum drying 4 hours, activated silica gel is standby;
(2) bonding methyl ethylene
Press methyl ethylene dichlorosilane, silica gel, volume/mass is 3ml/g, and methyl ethylene dichlorosilane and activated silica gel are mixed with mixture, adds the dry toluene of 5 times of methyl ethylene dichlorosilane volumes again, logical N under 110 ℃, mechanical agitation, liquid level
2Reaction condition refluxed 18 hours down, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel;
(3) passivation
Press bonding methyl ethylene silica gel, trim,ethylchlorosilane, mass/volume is 3g/ml, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, add the dry toluene of 10 times of trim,ethylchlorosilane volumes again, in the down logical N of 120 ℃, mechanical agitation, liquid level
2Refluxed 20 hours under the reaction condition, earlier with dry toluene suction filtration, washing, use methyl alcohol suction filtration, washing again after reaction is finished, get passivation silica gel;
(4) initiated polymerization
Take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the centesimal initator of bonding ethylene base silica gel quality of passivation, 10 times of dry toluenes are at 110 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition, the initator of the bonding ethylene base silica gel quality of one of the crosslinking agent divinylbenzene of the methyl-prop diluted acid-2-hydroxy propyl ester of the bonding ethylene base silica gel quality of 1ml/min dropping 1/10th above-mentioned passivation, the bonding ethylene base silica gel quality of 5/1000ths above-mentioned passivation, percentage above-mentioned passivation and the mixed liquor of 3 times of dry toluenes, after treating all to add, reacted 20 hours, after finishing, reaction uses dry toluene suction filtration, washing earlier, use methyl alcohol suction filtration, washing again, get methacrylic acid-2-hydroxy propyl ester silica chromatographic column filler.
Embodiment 4:
(1) silica gel activating
By hydrochloric acid, silica gel volume/mass is 6ml/g, is that 20% hydrochloric acid and 300nm pore size silica gel are mixed with mixture with percent by volume, heats 100 ℃ of reactions and refluxes 6 hours, suction filtration, spend deionised water to neutral, 105 ℃ of bakings 8 hours, and 50 ℃ of vacuum drying 6 hours, activated silica gel is standby;
(2) bonding methyl ethylene
Press methyl ethylene dichlorosilane, silica gel, volume/mass is 2ml/g, and methyl ethylene dichlorosilane and activated silica gel are mixed with mixture, adds the dry toluene of 7 times of methyl ethylene dichlorosilane volumes again, logical N under 120 ℃, mechanical agitation, liquid level
2Reaction condition refluxed 24 hours down, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel;
(3) passivation
Press bonding methyl ethylene silica gel, trim,ethylchlorosilane, mass/volume is 4g/ml, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, add the dry toluene of 9 times of trim,ethylchlorosilane volumes again, in the down logical N of 110 ℃, mechanical agitation, liquid level
2Refluxed 24 hours under the reaction condition, earlier with dry toluene suction filtration, washing, use methyl alcohol suction filtration, washing again after reaction is finished, get passivation silica gel;
(4) initiated polymerization
Take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the initator of the bonding ethylene base silica gel quality 2 percent of passivation, 10 times of dry toluenes are at 120 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition, the initator of the crosslinking agent divinylbenzene of the methyl-prop diluted acid-2-hydroxy propyl ester of the bonding ethylene base silica gel quality of 1ml/min dropping 1/5th above-mentioned passivation, the bonding ethylene base silica gel quality of 2 percent above-mentioned passivation, the bonding ethylene base silica gel quality of 2 percent above-mentioned passivation and the mixed liquor of 4 times of dry toluenes, after treating all to add, reacted 24 hours, after finishing, reaction uses dry toluene suction filtration, washing earlier, use methyl alcohol suction filtration, washing again, get methacrylic acid-2-hydroxy propyl ester silica chromatographic column filler.
Embodiment 5:
(1) silica gel activating
Press hydrochloric acid, silica gel, volume/mass is 4ml/g, is that 20% hydrochloric acid and 100nm pore size silica gel are mixed with mixture with percent by volume, heat 95 ℃ of reactions and refluxed 5 hours, suction filtration, spend deionised water, 105 ℃ of bakings 7 hours to neutral, and 50 ℃ of vacuum drying 5 hours, activated silica gel is standby;
(2) bonding methyl ethylene
Press methyl ethylene dichlorosilane, silica gel, volume/mass is 1ml/g, and methyl ethylene dichlorosilane and activated silica gel are mixed with mixture, adds the dry toluene of 6 times of methyl ethylene dichlorosilane volumes again, logical N under 100 ℃, mechanical agitation, liquid level
2Reaction condition refluxed 22 hours down, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel;
(3) passivation
Press bonding methyl ethylene silica gel, trim,ethylchlorosilane, mass/volume is 1g/ml, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, add the dry toluene of 8 times of trim,ethylchlorosilane volumes again, in the down logical N of 115 ℃, mechanical agitation, liquid level
2Refluxed 18 hours under the reaction condition, earlier with dry toluene suction filtration, washing, use methyl alcohol suction filtration, washing again after reaction is finished, get passivation silica gel;
(4) initiated polymerization
Take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the initator of the bonding ethylene base silica gel quality 2 percent of passivation, 10 times of dry toluenes are at 120 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition, the initator of the crosslinking agent divinylbenzene of the methyl-prop diluted acid-2-hydroxy propyl ester of the bonding ethylene base silica gel quality of 1ml/min dropping 1/5th above-mentioned passivation, the bonding ethylene base silica gel quality of 2 percent above-mentioned passivation, the bonding ethylene base silica gel quality of 2 percent above-mentioned passivation and the mixed liquor of 2 times of dry toluenes, after treating all to add, reacted 24 hours, after finishing, reaction uses dry toluene suction filtration, washing earlier, use methyl alcohol suction filtration, washing again, get methacrylic acid-2-hydroxy propyl ester silica chromatographic column filler.
Claims (3)
1, a kind of methacrylic acid-2-hydroxy propyl ester silica polymer-bound phase, its structural formula is:
N is the degree of polymerization, and the scope of n value is 2-7.
2, prepare the method for the described methacrylic acid of claim 1-2-hydroxy propyl ester silica polymer-bound phase, it is characterized in that may further comprise the steps:
(1) silica gel activating: press hydrochloric acid, silica gel volume mass ratio 2-6ml/g, with percent by volume is that 20% the hydrochloric acid and the silica gel in 30nm-300nm aperture are mixed with mixture, heat 90-100 ℃ of reaction backflow 2-6 hour, suction filtration, washing are to neutral, 105 ℃ were dried by the fire 4-8 hour, and 50 ℃ vacuum drying 4-6 hour, activated silica gel standby;
(2) bonding methyl ethylene: press methyl ethylene dichlorosilane, silica gel volume mass ratio 1-3ml/g, methyl ethylene dichlorosilane and 30nm-300nm aperture activated silica gel are mixed with mixture, the dry toluene that adds 5-7 times of methyl ethylene dichlorosilane volume again, logical N under 100-120 ℃, mechanical agitation, liquid level
2Reaction condition under backflow 18-24 hour, reaction is finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets bonding methyl ethylene silica gel;
(3) passivation: compare 1-4ml/g by bonding methyl ethylene silica gel, trim,ethylchlorosilane volume mass, above-mentioned bonding methyl ethylene silica gel and trim,ethylchlorosilane are mixed with mixture, the dry toluene that adds 8-10 times of trim,ethylchlorosilane volume again is in the down logical N of 100-120 ℃, mechanical agitation, liquid level
2Under the reaction condition backflow 18-24 hour, reaction was finished the back earlier with dry toluene suction filtration, washing, uses methyl alcohol suction filtration, washing again, gets passivation silica gel;
(4) initiated polymerization: take by weighing above-mentioned bonding ethylene base silica gel through passivation, after 50 ℃ of vacuum drying, add the initator of the bonding ethylene base silica gel quality 5/1000ths-2 percent of passivation, 10-15 times of dry toluene is at 100-120 ℃, mechanical agitation, logical N
2After refluxing 3 hours under the condition; 1ml/min drips the methyl-prop diluted acid-2-hydroxy propyl ester of the bonding ethylene base silica gel quality of 1/1/10th-five above-mentioned passivation; the crosslinking agent divinylbenzene of the bonding ethylene base silica gel quality of 5/1000ths-2 percent above-mentioned passivation; the initator of the bonding ethylene base silica gel quality of 5/1000ths-2 percent above-mentioned passivation and the mixed liquor of 2-4 times of dry toluene; initator is benzoyl peroxide or azodiisobutyronitrile; after treating all to add; reacted 18-24 hour; after finishing, reaction uses earlier the dry toluene suction filtration; washing; use the methyl alcohol suction filtration again; washing gets methacrylic acid-2-hydroxy propyl ester silica bonding phase.
3. the application of a kind of methacrylic acid according to claim 1-2-hydroxy propyl ester silica polymer chromatographic column filler is characterized in that, acetonitrile-water is as the phase that flows, and gradient condition is 0-20min, acetonitrile 5%-40%; 20-25min, acetonitrile 40%-5% separates standard protein or thick protein, obtains three components: ovalbumin, trypsin inhibitor, chymotrypsin protein inhibitor chromatogram.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101774599A CN101234340A (en) | 2007-11-16 | 2007-11-16 | Methylpropenoic acid-2-hydroxy propyl ester silica--based polymer-bonded phase, preparation and application thereof |
| SK5016-2008A SK288165B6 (en) | 2007-11-16 | 2008-02-12 | Method of preparation and use poly(2-hydroxy propyl methacrylate) of chromatic phase bounded on silicate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101774599A CN101234340A (en) | 2007-11-16 | 2007-11-16 | Methylpropenoic acid-2-hydroxy propyl ester silica--based polymer-bonded phase, preparation and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN101234340A true CN101234340A (en) | 2008-08-06 |
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|---|---|---|---|
| CNA2007101774599A Pending CN101234340A (en) | 2007-11-16 | 2007-11-16 | Methylpropenoic acid-2-hydroxy propyl ester silica--based polymer-bonded phase, preparation and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101234340A (en) |
| SK (1) | SK288165B6 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102085477A (en) * | 2010-11-26 | 2011-06-08 | 江南大学 | Polymer coated silica gel high performance liquid chromatography filler as well as preparation method and application thereof |
| CN108772042A (en) * | 2018-07-04 | 2018-11-09 | 林思思 | A kind of anthraquinone derivative bonded silica gel stationary phase, preparation method and ginsenoside detection application |
| CN108889276A (en) * | 2018-07-04 | 2018-11-27 | 林思思 | A kind of exclusive separation silica gel solid phase of ginsenoside and preparation method |
-
2007
- 2007-11-16 CN CNA2007101774599A patent/CN101234340A/en active Pending
-
2008
- 2008-02-12 SK SK5016-2008A patent/SK288165B6/en not_active IP Right Cessation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102085477A (en) * | 2010-11-26 | 2011-06-08 | 江南大学 | Polymer coated silica gel high performance liquid chromatography filler as well as preparation method and application thereof |
| CN108772042A (en) * | 2018-07-04 | 2018-11-09 | 林思思 | A kind of anthraquinone derivative bonded silica gel stationary phase, preparation method and ginsenoside detection application |
| CN108889276A (en) * | 2018-07-04 | 2018-11-27 | 林思思 | A kind of exclusive separation silica gel solid phase of ginsenoside and preparation method |
Also Published As
| Publication number | Publication date |
|---|---|
| SK288165B6 (en) | 2014-03-04 |
| SK50162008A3 (en) | 2009-06-05 |
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