JPS59195153A - Production of porous carrier - Google Patents

Abstract

PURPOSE:To obtain a carrier having low adsorptivity and excellent strength, reproducibility, column efficiency, etc. by bringing a porous carrier having a silanol group on the surface such as silica gel, etc. and an org. silane compd. into reaction in the presence of a surface active agent to cover the silanol group roughly thoroughly. CONSTITUTION:A porous carrier having a silanol group on the surface such as a silica gel, porous glass, diatomaceous earth or the like is treated at 20-100 deg.C in a soln. mixture composed a water-soluble silane coupling agent, for example, gamma-glycidyl oxypropyl trimethoxysilane or the like and water of 3-10pH or water and <=30wt% water-soluble org. solvent in the presence of a surface active agent, more particularly an anionic surface active agent so that roughly the whole silanol group is chemically bound with the org. silane and is covered on said carrier. The carrier for a chromatography which has a high resolving power and permits an easy increase in speed is thus obtd. without decreasing the hole diameter and decreasing the adsoption of solute such as protein.

Description

DETAILED DESCRIPTION OF THE INVENTION 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 optical fillers 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 1 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 divinylbenzene gels have been developed and used, but they are inferior in the most important aspects of adsorption (hydrophobic N absorption) 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 are still insufficient.

Recently, it has been proposed to chemically bond a substrate and a stationary phase by esterifying a silicon-based chromatographic column with alcohol (Japanese Patent Laid-Open No. 4'7-7222
issue). However, the resulting S i -0''-C bond is easily hydrolyzed and not only adsorbs solute but also 5i-0-8
A method was proposed for coating a porous carrier with pores by first chemically bonding with an i-bond and then secondarily reacting with a chemical substance that can react with this epoxy group (Japanese Patent Application Laid-Open No. 1989-1993). !-!ri≠7, JP-A-13-6t7jt). This is to remove the surface silanol groups that did not participate in the reaction in the secondary chemical bonding reaction, and to prevent the adsorptive silanol from being exposed on the surface. but,"
J, Amer, Chem, Soc,”72 77 A
-7r, zFc4), ('rizo), 1.5HAP
IROAND 1. When surface silanol groups were measured with reference to the methyl red adsorption method reported by M. KoLTHOFF et al., a considerable amount of silanol groups (23 μmail/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
The performance of porous glass with glycerolpropylsilane bonds has been reported, such as Fj/l, -320 (77/l), etc., and the adsorptivity, operability, and recovery rate are considerably improved compared to conventional carriers. However, 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 silyl groups on the carrier surface and has low adsorption properties.

A third object of the present invention is to provide an excellent porous carrier without narrowing the fine pore diameter inherent in the porous carrier.

A third object of the present invention is to provide a chromatography carrier with excellent mechanical strength, reproducibility, column efficiency (performance), and sample recovery rate.

That is, the above object of the present invention is to reduce the silanol groups on the pore surface of a porous carrier such as silica gel or porous glass,
This is achieved by reacting an organic silane compound with 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 the surfactant include cationic surfactants (e.g., laurylamine acetate, lauryltrimethylammonium chloride, stearyltrimethylammonium chloride, distearyldimethylammonium chloride, polyoxyethyl alkylamine, etc.), anionic surfactants (e.g., Sodium lauryl sulfate, triethanolamine lauryl sulfate, ammonium lauryl sulfate, sodium polyoxyethylene alkyl ether sulfate, triethanolamine polyoxyethylene alkyl ether sulfate, etc.), nonionic surfactants (e.g., polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene stearyl ether, rubitan 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 group and organic silane is 20 to 1000
C, but in terms of reaction efficiency (rate), what is the reaction temperature?
- Tata 0C is preferred.

The porous carrier of the present invention is a porous carrier having pores such as silica gel, porous glass, diatomaceous material, etc., and having 7 lanol groups on the surface.

The porous carrier used in the present invention has silanol groups on the pore surface of O0S/mm2 or more, preferably j silanol groups/mm2.
Those having the above are optimal. Further, the porous carrier can have any shape, and the particle size of the porous glass is 1~! 00 ttm, preferably 30
-20θμm, pore size is average pore diameter ~too
A carrier is applicable.

The organic silane compound in the present invention is used as a silane coupling agent, and has 7 to 3 lower alkoxy group atoms in 7 molecules, and is preferably water or an aqueous solution with a pH of ~10, or an aqueous solution containing 30 wt% or less of a water-soluble organic solvent. Preferably, those soluble in For example, there are compounds represented by general formulas (I) and (If).

(In the formula, R1 and R2 are not particularly limited, and X is the number of carbon atoms/~
The alkoxy group of λ, Y%2 represents a methyl group, an ethyl group, or an alkoxy group having 7 to λ carbon atoms. ) General formula (I), (■
), such as r-glycidyloxypropyltrimethoxysilane, γ-, glycidoxypropyldimethylethoxysilane, r-glycidoxypropylmethyljethoxysilane, r-aminopropyltriethoxysilane, r- Mercaptopropyltrimethoxysilane, N-β(aminoethyl)-doaminopropylmethyldimethoxysilane, vinyltriethoxysilane/, bis(
Water-soluble silane coupling agents such as 2-hydroxyethyl)aminopropyltriethoxysilane (p prepared with water or hydrochloric acid, potassium hydroxide, phosphate, etc.) (J to I
An aqueous solution of O or 3C)H (one that is soluble in water containing a water-soluble organic solvent of % or more by weight) may be used.

In the present invention, the method of reacting an organic silane compound with a porous carrier is to impregnate a porous carrier having a silanonyl group in an organic silane diluted with a water solvent or a water-organic solvent,
Furthermore, a surfactant is added to this solution, and normal reflux is carried out at a predetermined temperature for a predetermined time.Next, the produced silanized product is separated and washed several times with a water solvent, a methanol solvent, or an acetone solvent. , 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, hydrochloric acid, potassium hydroxide, phosphate, etc. (aqueous solutions of J to IO), and water-soluble organic solvents (e.g., methanol,
Examples include aqueous solutions containing 3 (1) wt % or less of ethanol, isopropanol, acetone, dioxane).

Preferably p I-(
A water solvent at t-4 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 the "J. Amer. , ChemSoc″ 72 7
74-7r2Fed,, (/9!0), 1.5HAP
IROAND 1. The methyl red adsorption method reported by M. KoLTHOFF 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 to be used can be 1-10 times the total amount of silanol groups in the porous carrier, but for the purpose of completely covering the surface, it is preferably 7 to 5 times the amount by mole. 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° to 7° relative to the total pore surface area (expressed in m2) of the porous carrier used in the reaction. gμmorph rn2. On the other hand, J, Chromatog mentioned above.

According to Sci″/44 3/l~320 (/97A), it is 22°3 μmol/m2 for the porous carrier, and JP-A-11-49≠1 and JP-A-11-6T7-6 According to the issue, /≠./μmol/m2.

Tokukai Sho≠6-72ri No. 6 too! Otsu. An amount of organosilane compound of 3 μmol/m2 is used. From this comparison, the detailed explanation of the present invention is clear.

The carrier obtained according to the present invention as described above has extremely superior performance as a carrier for chromatography, especially as a carrier for gel filtration, compared to conventional carriers. 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 onto the surface is sufficient, it does not damage the fine pore diameter of the porous carrier, and furthermore, the alkyl group of the introduced chemical substance,
This is because there is very little hydrophobic interaction (hydrophobic adsorption) between the sample and the carrier surface, which is presumed to be caused by the aryl group.

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 (and the degree of swelling varies even with different M media). It provides a hard gel that does not change.Therefore, it is possible to produce a chromatography carrier with very high separation ability, easy speedup, and 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 gel filtration carrier, an organic solvent-based carrier, etc. It can be used as a base material for separation carriers such as gel filtration carriers, ion exchange chromatography carriers, affinity chromatography carriers, and enzyme immobilization carriers.

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 porous glass 209 with the following physical properties at /2 o 0 c for ≠ hours, put it in a third flask equipped with a reflux condenser, add sodium lauryl sulfate Q and geta g to distilled water! It was immersed in a solution of 0rnl and stirred for 75 minutes at room temperature.

Shape: Crushed Particle form: 3 talf μm Specific surface area weight ♂ Am2/g Average pore diameter: 2110 The solution diluted with Qrnl is 3
The pH of this solution was adjusted to about 7°θ with an IN KOH aqueous solution, and the solution was heated for 30 minutes.

Subsequently, the mixture was allowed to react for 000% on an oil bath. After cooling,
The above silane-treated porous glass is separated into a furnace, and M distilled water/,
Washed twice with thornl. Furthermore, acetone ltom
The silanized porous glass was prepared by washing it twice with l and vacuum drying at 0C,,2CmmHF for r hours.
I got g.

This carrier was packed into a stainless steel column with an inner diameter of 7 s mm and a length of too 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/"GPc20 model" (product name: Waters ■) Detector: Ultraviolet absorption detector (2♂Omm) Eluent diphosphate buffer (1713 mol, pH 7.0) (l/lo mol) Contains Nace) Flow rate: / ml/min Sample injection volume 20, JG solution, 20 μl or s.

μl (protein), soμl (dextran) Mass, various dextran samples are used to compare the change in pore diameter of the carrier before (untreated porous glass) and after (silanized porous glass) reaction with the silane coupling agent. A 1-stop positive curve was created using the text run elution practice and is shown in Figure 1.

A calibration curve is a calibration line that represents the fractionation range of a porous carrier when it is used as a gel ξ-seaion (GPC).

That is, the degree and range of molecular weight of a substance that can be separated by chromatography6 is expressed by measuring with standard dextran.

Comparative Example 1 The same column as in Example 1 was packed with a silane-treated fl-type glass carrier obtained by reacting under completely opposite conditions except that the surfactant sodium lauryl sulfate was not added. Performance was examined under the same conditions.

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.

The reaction was carried out under rapid flow for a period of time without distilling off the methanol produced. 7-run treated porous glass door to door and toluene 100
It was washed three times with TLl, twice with 100 ml of acetone, and then dried. Next, the obtained silane-treated porous glass was
) (Impregnated with 2ooml of hydrochloric acid aqueous solution of 3, 1l-00C
The reaction was carried out for 172 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.

In addition, the dry compositions of all the silane-treated carriers and the carriers of Comparative Examples 1 and 2 are shown in this example, as well as the elution amount (elution position) and recovery rate of various proteins, and the carrier surface coverage by the methyl red method. was measured. The results are shown in Table 1.

The recovery rate was measured using the ultraviolet absorption log/
The value of /T (λ, 2fOmm) was expressed as ioo% and expressed as a percentage of the ultraviolet absorption log//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.

Examples 2 to 6 Using the same porous glass as in Example 1, and reacting it under the same conditions as in Example 1 using various organic silanes and various surfactants, measurement results of the characteristic values of the obtained carrier. are shown in Table 2.

Example 7 Shape: Crushed Particle size: 10-/2 μm Specific surface area: 3m27g Average pore size: 2≠OA Using porous glass with the above physical properties, the same organic silane as in Example 1 was used under the same conditions. After the run treatment, the carrier obtained by washing and drying was wet-filled and various proteins were measured under the same conditions. The obtained recovery rate and j8 output chart are shown in Table 3 and Figure 2.

Table 3 Regarding the ring-opening modification of epoxy groups when using an epoxy group-containing organic silane in the organic silane 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 at a reaction time of 'QJ time'.

To open the epoxy group, the silane-treated carrier must be fully 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 shape 10±λμm Specific surface area: ≠00m/g Average pore diameter: 10OA Silica gel carrier 209 having the above physical properties was prepared using sodium lauryl sulfate. C#, r-glycidyloxypropyltrimethoxysilane/≠. The results of compositional analysis of the obtained carrier in a dry state after the reaction was carried out under the same operating conditions as in Example 1 using distilled water λ75ml are shown below.

The obtained constant silane-treated support showed good performance similar to porous glass and Example 11.The physical properties of the porous support after seven runs of treatment (dextran calibration curve, composition analysis, protein recovery rate, methyl The method of the present invention has extremely low sample adsorption as a chromatography photoreparation carrier (in addition, it does not damage the fine pore diameter inherent in porous carriers, It can be seen that the surface is coated with the necessary minimum amount of organic silane treatment agent in a form close to a monomolecular layer.

[Brief explanation of drawings]

Figure 1 is a calibration curve based on dextran elution practice.
Untreated porous glass/and silanized porous glass 2 are shown. FIG. 2 is an elution chart 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. Patent Applicant Fuji Photo Film Co., Ltd. Figure 2 4 5 10 Sango Fang (mJ) Figure 3 and To: B Konya (hrs) Procedural Amendment 1, Indication of Case Showa SR Year Patent Application No. 70t02
No. 2, Title of the invention Process for producing porous carrier 3, Relationship to the case of the person making the amendment Name of the applicant for the pin (520
) Fuji Photo Film Co., Ltd. Contact Light 〒]06 2-26-3o' Nishi-Azabu, Minato-ku, Tokyo;. Fuji Photo Film Co., Ltd. Tokyo Head Office Telephone: (406) 2537 4° Comparison of Amendment Contents of 5° Amendment in the "Detailed Description of the Invention" column of the specification BA honorary will be amended as follows. /) Page/Hiroyukime's rl, 1vf, KoLTHOFFJ f "I, I
vi, K (correct as JLTHOFFj. 2) Correct "0.5 solution" in line l♂ of page 13 to "0.S salary bath standard". 3) Correct as "io ~/2μyrJ k 1-70~-i0μm" on page 20, line 3. Hiroshi] “Epoxy group” on page 22, line 13? Corrected to "generally epoxy group".

Claims (1)

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