JPH0410587B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a porous carrier. More specifically, the present invention relates to a method for producing a porous carrier suitable for chromatography. Conventionally, crosslinked dextran, polyacrylamide, and the like have been known as carriers for chromatography of biochemical substances such as proteins. Since these carriers have low adsorption to water-soluble substances, they are used as packing materials for chromatography for analysis and purification. However, these gels have low mechanical strength and can only be used at low flow rates. Furthermore, it is known that changes in pH, ionic strength, and eluent composition change the degree of swelling of the carrier, reducing column efficiency, and a separation carrier with high column stability has been desired. Therefore, organic polymer gels such as methacrylate gels, polyvinyl alcohol gels, and styrene vinylbenzene gels have been developed and used, but they are inferior in the most important aspects of adsorption (hydrophobic adsorption) and separation. Also, similar to the gel described above, improvements have been made in terms of mechanical strength, changes in eluent composition, etc., but these have been insufficient. Recently, it has been proposed to chemically bond a substrate and a stationary phase by including a step of esterifying a silicon-based chromatographic support with alcohol (Japanese Patent Application Laid-Open No. 7296/1983). However, the obtained Si−
O-C bonds are susceptible to hydrolysis, which not only adsorbs solutes but also reduces chromatographic reproducibility and column efficiency. Furthermore, by firstly chemically bonding a silane coupling agent having an epoxy group through Si-O-Si bonds, and then secondarily reacting with a chemical substance that can react with this epoxy group, porous particles having pores are formed. A method of coating a sexual carrier was proposed (Japanese Patent Application Laid-open No. 5941/1983,
No. 66756). In this method, the surface silanol groups that have not participated in the reaction in the primary chemical bonding reaction are covered by the secondary reaction, and the adsorbable silanol is not exposed on the surface. However, “J.Amer.Chem, Soc.”
72 776-782Feb., (1950). I. SHAPIRO AND
When we measured silanol groups on the surface using the methylred adsorption method reported by IMKoLTHOFF et al., we found that a considerable amount of silanol groups (23 μmol/g) were exposed on the surface, and they adsorbed samples such as proteins and enzymes. The recovery rate also decreases. In addition, the polymer layer formed on the surface of the carrier due to the secondary reaction narrows the fine pore diameter originally possessed by the porous carrier that is the substrate. Furthermore, since the reacting chemical substances do not react uniformly, the pore size distribution widens, resulting in a decrease in performance as a gel chromatography carrier. Furthermore, J.
Chromatog, Sci. 14 316-320 (1976) and others have reported the performance of porous glass with glycerolpropylsilane bonds, and although it has considerably improved adsorption, operability, and recovery rate compared to conventional carriers. ,
Since the reaction between the silanol groups on the surface of the porous glass and the silane coupling agent is insufficient, unreacted silanol groups remain, and the recovery rate of proteins, enzymes, etc. is low due to adsorption by the silanol groups. Therefore, the present inventors conducted intensive studies on a method of almost completely reacting the silanol groups on the surface of the pores of a porous carrier with an organic silane compound to coat the carrier, and as a result, the present invention was achieved. The first object of the present invention is to provide a porous carrier that almost completely covers the silanol groups on the carrier surface and has low adsorption properties. A second object of the present invention is to provide an excellent porous carrier without narrowing the fine pore diameter inherent in the porous carrier. The third object of the present invention is to improve mechanical strength, reproducibility,
Our objective is to provide a chromatography carrier with excellent column efficiency (performance) and sample recovery rate. That is, the above object of the present invention is achieved by reacting the silanol groups on the pore surface of a porous carrier such as silica gel or porous glass with an organic silane compound using a surfactant. The most important point in the present invention is to add a surfactant as a catalyst into the reaction system when chemically bonding the silanol groups on the surface of the porous carrier and the organic silane. Examples of surfactants include cationic surfactants (for example, laurylamine acetate, lauryltrimethylammonium chloride, stearyltrimethylammonium chloride, distearyldimethylammonium chloride, polyoxyethylene alkylamine, etc.);
Anionic surfactants (e.g., sodium laurate, triethanolamine lauryl sulfate, ammonium lauryl sulfate, sodium polyoxyethylene alkyl ether sulfate, triethanolamine polyoxyethylene alkyl ether sulfate, etc.), nonionic surfactants (e.g., polyoxyethylene alkyl ether triethanolamine sulfate, etc.), (oxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether sorbitan monolaurate, etc.). In the present invention, the surfactant has the effect of significantly promoting the chemical bonding reaction between the silanol groups on the surface of the porous carrier and the organic silane, and almost completely covering the surface silanol groups. In particular, anionic surfactants are most suitable. The reaction temperature between silanol groups and organic silane is 20 to 100℃,
From the viewpoint of reaction efficiency (speed), the reaction temperature is preferably 85 to 95°C. The porous carrier of the present invention has pores made of silica gel, porous glass, or diatomaceous earth, and has silanol groups on the surface. The porous carrier used in the present invention has 0.5 or more silanol groups/mm ² , preferably 5 silanol groups on the pore surface.
It is best to have at least 100% of the number of particles/ ^mm2 . Further, the porous carrier may have any shape, and the particle size of the porous carrier may be 1 to 500 μm,
Preferably, a carrier having a pore size of 30 to 200 μm and an average pore size of 5 to 5000 Å is applicable. The organic silane compound in the present invention is used as a silane coupling agent, has 1 to 3 lower alkoxy group atoms in one molecule, and has water or PH
30wt of 3-10 aqueous solution or water-soluble organic solvent
% or less is preferable. For example, there are compounds represented by the general formulas () and (). (In the formula, R ₁ and R ₂ are not particularly limited, X is an alkoxy group having 1 to 2 carbon atoms, Y and Z are methyl groups,
It represents an ethyl group and an alkoxy group having 1 to 2 carbon atoms. ) Compounds represented by the general formulas () and (), such as γ-glycidyloxypropyltrimethoxysilane, γ-glycidoxypropyldimethylethoxysilane, γ-glycidoxypropylmethyldiethoxysilane, and γ-aminopropyltrimethoxysilane. Water-soluble silanes such as ethoxysilane, γ-mercaptopropyltrimethoxysilane, N-β(aminoethyl)-γ-aminopropylmethyldimethoxysilane, vinyltriethoxysilane, bis(2-hydroxyethyl)aminopropyltriethoxysilane, etc. Coupling agents (water or hydrochloric acid, potassium hydroxide,
Add 1wt to an aqueous solution with a pH of 3 to 10 adjusted with phosphate, etc., or water containing 30wt% or less of a water-soluble organic solvent.
% or more). In the present invention, the method of reacting a porous carrier with an organic silane compound involves impregnating a porous carrier having silanol groups in an organic silane diluted with a water solvent or a water-organic solvent, and then adding a surfactant to this solution. and normal reflux is carried out at a predetermined temperature and for a predetermined time. Next, the generated silanized product is separated,
After washing several times with a water solvent, methanol solvent, or acetone solvent, it is dried under reduced pressure. In the present invention, it is preferable to carry out the reaction in the presence of a solvent from the viewpoint of stability and operability with respect to uniformity of surface treatment when chemically bonding the organic silane to the silanol groups of the porous carrier. Examples of solvents include water or an aqueous solution with a pH of 3 to 10 adjusted with hydrochloric acid, potassium hydroxide, phosphate, etc., and water-soluble organic solvents (e.g., methanol, ethanol, isopropanol, acetone, dioxane).
Examples include aqueous solutions containing 30 wt% or less.
PH adjusted preferably with hydrochloric acid, potassium hydroxide
6-8 water solvent is optimal. In addition, since the amount of organic silane used for a porous carrier depends on the amount of surface silanol groups present in the carrier, the present inventors used "J.Amer. .
Chem, Soc” 72 776-782Fed., (1950).I.
The methyl red adsorption method reported by SHAPIRO AND IMKOLTHOFF et al. was used. The amount of organic silane to be used was determined based on the amount of surface silanol groups obtained from this measurement method. The amount of organic silane used can be 1 to 10 times the total amount of silanol groups in the porous carrier, but is preferably 4 to 5 times the amount to completely cover the surface. If the amount is too large, disadvantages such as narrowing of the pore size will occur. That is, the amount of the organosilane compound used in the present invention is 7.9 to 9.8 μmol/m ² based on the total pore surface area (expressed in m ² ) of the porous carrier used in the reaction. In contrast, the aforementioned “J.Chromatog, Sci” 14
316-320 (1976) for porous supports.
According to JP-A ^No. 55-5941 and JP-A-55-66756, it is 14.1 μmol/m ² .
JP-A-46-7296 also uses an amount of organosilane compound of 56.3 μmol/m ² . From this comparison, the advantages of the present invention are clear. The carrier obtained according to the present invention as described above is
Compared to conventional carriers, it has extremely superior performance as a carrier for chromatography, especially as a carrier for gel filtration. Its first feature is low adsorption. This is because the silanol groups on the surface of the carrier are almost completely covered with organic silane, so that the adsorption of solutes (proteins, biochemical related substances, etc.) by the remaining silanol groups is extremely small. Second, since a small amount of the chemical substance (organosilane) introduced to the surface is sufficient, it does not damage the fine pore size of the porous carrier, and it is also assumed that this is due to the alkyl group and aryl group of the introduced chemical substance. This is because there is very little hydrophobic interaction (hydrophobic adsorption) between the sample and the carrier surface. Third, inorganic carriers such as silica gel and porous glass, which can be easily produced by conventional methods, have a much narrower pore size distribution than organic polymer carriers.
To provide a hard gel whose degree of swelling does not change even if different solvents are used. Therefore, it is possible to produce a carrier for chromatography that has very high separation ability, can be easily increased in speed, and has good column stability. Therefore, the carrier obtained in the present invention has extremely excellent performance, and by selecting the organic silane (silane coupling agent) described above and using it in the reaction, it can be used as a hydrophilic gelling carrier or an organic solvent-based carrier. Carrier for gel filtration, carrier for ion exchange chromatography,
It can be used as a base material for affinity chromatography carriers and separation carriers such as enzyme immobilization carriers. EXAMPLES Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto. Example 1 After drying 20 g of porous glass with the following physical properties at 120°C for 4 hours, it was placed in a three-necked flask equipped with a reflux condenser, and 0.49 g of sodium lauryl sulfate was added to 50 ml of distilled water.
and stirred at room temperature for 15 minutes. Shape: Crushed Particle shape: 35-77 μm Specific surface area: 86 m ² /g Average pore diameter: 240 Å Furthermore, a solution of 3.53 g of γ-glycidyloxypropyltrimethoxysilane diluted with 64 ml of distilled water was poured into a three-necked flask. , the PH of this solution is
The pH was adjusted to about 7.0 with a KOH aqueous solution and stirred for 30 minutes. Subsequently, the mixture was reacted on an oil bath at 90°C for 6 hours. After cooling, the silane-treated porous glass was separated and washed five times with 150 ml of distilled water. The glass was further washed twice with 150 ml of acetone and dried under reduced pressure at 50° C. and 20 mmHg for 8 hours to obtain about 21 g of silane-treated porous glass. This carrier was packed into a stainless steel column with an inner diameter of 7.5 mm and a length of 600 mm, and its performance as a packing material for gel permeation chromatography in an aqueous solvent system was investigated under the following conditions. Measurement conditions Equipment: ALC/"GPC204 type" (product name: Waters Co., Ltd.) Detector: Ultraviolet absorption detector (280 mm) Eluent: Phosphate buffer (1/15 mol, PH7.0) (1/
(Contains 10 mol NaCl) Flow rate: 1 ml/min Sample injection amount: 0.5% solution, 20 μ or 50 μ (protein), 50 μ (dextran) In order to compare the change in the pore diameter of the carrier after the reaction (silanized porous glass) and after the reaction (silanized porous glass), a calibration curve was created using a practical dextran elution technique and is shown in FIG. A calibration curve is a calibration curve that represents the fractionation range of a porous carrier when the carrier is used as a gel perfusion (GPC). That is, the degree and range of molecular weight of a substance that can be separated and chromatographically measured using standard dextran is expressed. Comparative Example 1 A silane-treated porous glass carrier obtained by reacting under exactly the same conditions as in Example 1 except that the surfactant sodium lauryl sulfate was not added was packed into the same column as in Example 1, and the reaction was carried out under the same conditions. We investigated the performance. Comparative Example 2 The same porous glass as in Example 1 was used and impregnated with the same organic silane in a solution diluted with 100 ml of toluene. 8 under reflux while distilling off the methanol produced.
Allowed time to react. Separate the silane-treated porous glass, wash it three times with 100 ml of toluene, and wash it twice with 100 ml of acetone.
After washing twice, it was dried. Next, the obtained silane-treated porous glass was impregnated with 200 ml of aqueous hydrochloric acid solution of PH3, and
C. for 12 hours to remove the alkoxy group bonded to the silicon atom and to hydrolyze and ring-open the epoxy group. The carrier obtained by thorough washing with water and drying under reduced pressure was packed into the same column as in Example 1, and its performance was examined under the same conditions. Further, the results of compositional analysis in the dry state of the silanized carrier in this example and the carriers in Comparative Examples 1 and 2 are shown below. [Table] Furthermore, the elution amount (elution position) and recovery rate of various proteins and the carrier surface coverage by the methyl red method were measured. The results are shown in Table 1. The recovery rate is measured by measuring the ultraviolet absorption of each sample solution.
The value of log1/T (λ280 mm) was taken as 100%, and it was expressed as a percentage of the ultraviolet absorption log1/T value (sample concentration was adjusted to be the same) of each sample that passed through the packed column. In this case, the measurements were performed three times and the average was determined. However, it was assumed that each protein was not inactivated. [Table] [Table] Examples 2 to 6 Using the same porous glass as in Example 1, the carriers obtained were reacted under the same conditions as in Example 1 using various organic silanes and various surfactants. The measurement results of the characteristic values are shown in Table 2. [Table] [Table] Example 7 Shape: crushed particle size: 10 to 20 μm Specific surface area: 93 m ² /g Average pore size: 240 Å The same organic silane as in Example 1 using porous glass of the above material. After silane treatment under the following conditions, the carrier obtained by washing and drying is wet-filled,
Various proteins were measured under the same conditions. The determined recovery rate and elution chart are shown in Table 3 and Figure 2. [Table] For ring-opening modification of epoxy groups when using an epoxy group-containing organosilane in the organosilane treatment of a porous carrier according to the method of the present invention, dry gel is mixed with dry DMF in which hydrogen chloride is dissolved. A certain amount of the gel was added to open the epoxy groups of the gel to form a chlorohydrin type, and residual hydrogen chloride was back determined using sodium methylate to quantify the amount of epoxy groups present in the gel. As a result, as shown in FIG. 3, it was found that in the method of the present invention, 100% of the epoxy groups in the gel could be ring-opened and modified with a reaction time of 6 hours. In general, when opening the epoxy group, Comparative Example 2
The silane-treated carrier must be sufficiently impregnated with an acidic aqueous solution and thoroughly washed with water to open the epoxy group. Furthermore, the reaction time for treatment with organic silanes other than epoxy group-containing organic silanes was one hour, and the reaction proceeded sufficiently, and the performance as a chromatography carrier was also satisfactory. Example 8 Shape: Crushed Particle size: 10±2μm Specific surface area: 400m ² /g Average pore size: 100Å 20g of silica gel carrier with the above physical properties was mixed with 2.0g of sodium lauryl sulfate, 14.5g of γ-glycidyloxypropyltrimethoxysilane, and distilled. After reacting with 275 ml of water under the same operating conditions as in Example 1, the results of compositional analysis of the obtained carrier in a dry state are shown below. [Table] The obtained silane-treated carrier showed good performance similar to that of porous glass and Example 1. The physical properties of the porous carrier after silane treatment (dextran calibration curve, composition analysis, protein recovery rate,
From the results of the surface coverage (based on the methylred method),
The method of the present invention has extremely low sample adsorption as a chromatography separation carrier, and furthermore, it does not damage the fine pore diameter inherent in the porous carrier, and the carrier surface is treated with almost monomolecules using the minimum necessary organic silane treatment agent. It can be seen that the coating treatment is applied in a form similar to that of a layer.

[Brief explanation of drawings]

FIG. 1 is a calibration curve from a dextran elution experiment, showing untreated porous glass 1 and silane-treated porous glass 2. FIG. 2 is a chart of elution of various proteins using the porous carrier of the present invention. FIG. 3 is a graph showing the change in the amount of epoxy groups when the epoxy groups of the dried gel are subjected to ring-opening modification.

Claims (1)

[Claims] 1. A method for producing a porous carrier, which comprises reacting a silanol group of the porous carrier with an organic silane compound in the presence of a surfactant.