Sodium Hydroxide

Example 19

The above silk solutions were transformed to a silk powder through lyophilization to remove bulk water and chopping to small pieces with a blender. pH was adjusted with sodium hydroxide. Low molecular weight silk (−25 kDa) was soluble while high molecular weight silk (−60 kDa) was not.

The lyophilized silk powder can be advantageous for enhanced storage control ranging from 10 days to 10 years depending on storage and shipment conditions. The lyophilized silk powder can also be used as a raw ingredient in the pharmaceutical, medical, consumer, and electronic markets. Additionally, lyophilized silk powder can be re-suspended in water, HFIP, or an organic solution following storage to create silk solutions of varying concentrations, including higher concentration solutions than those produced initially.

In an embodiment, aqueous pure silk fibroin-based protein fragment solutions of the present disclosure comprising 1%, 3%, and 5% silk by weight were each dispensed into a 1.8 L Lyoguard trays, respectively. All 3 trays were placed in a 12 ft² lyophilizer and a single run performed. The product was frozen with a shelf temperature of ≤−40° C. and held for 2 hours. The compositions were then lyophilized at a shelf temperature of −20° C., with a 3 hour ramp and held for 20 hours, and subsequently dried at a temperature of 30° C., with a 5 hour ramp and held for about 34 hours. Trays were removed and stored at ambient conditions until further processing. Each of the resultant lyophilized silk fragment compositions were able to dissolve in aqueous solvent and organic solvent to reconstitute silk fragment solutions between 0.1 wt % and 8 wt %. Heating and mixing were not required but were used to accelerate the dissolving rate. All solutions were shelf-stable at ambient conditions.

In an embodiment, an aqueous pure silk fibroin-based protein fragment solution of the present disclosure, fabricated using a method of the present disclosure with a 30 minute boil, has a molecular weight of about 57 kDa, a polydispersity of about 1.6, inorganic and organic residuals of less than 500 ppm, and a light amber color.

In an embodiment, an aqueous pure silk fibroin-based protein fragment solution of the present disclosure, fabricated using a method of the present disclosure with a 60 minute boil, has a molecular weight of about 25 kDa, a polydispersity of about 2.4, inorganic and organic residuals of less than 500 ppm, and a light amber color.

Example 1

<Step (A): Synthesis of porous particle having glycidyl group>

27.8 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 11.3 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were dissolved in 58.7 g of diethyl succinate as a diluent, and nitrogen gas was bubbled for 30 minutes to provide an oil phase.

Next, separately from the oil phase, 10.0 g of PVA-224 (manufactured by Kuraray Co., Ltd., polyvinyl alcohol having a degree of saponification of 87.0% to 89.0%) as a dispersion stabilizer and 10.0 g of sodium chloride as a salting-out agent were dissolved in 480 g of ion exchanged water to provide an aqueous phase.

The aqueous phase and the oil phase were placed in a separable flask and dispersed at a rotation speed of 430 rpm for 20 minutes using a stirring rod equipped with a half-moon stirring blade, then the inside of the reactor was purged with nitrogen, and the reaction was carried out at 60° C. for 16 hours.

After that, the resulting polymer was transferred onto a glass filter and thoroughly washed with hot water at about 50 to 80° C., denatured alcohol, and water in the order presented to obtain 100.4 g of a porous particle (carrier al).

The amount of glycidyl methacrylate used was 79.8 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 20.2 mol % based on the total amount of the monomers.

<Step (B): Introduction reaction of alkylene group>

98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier α1 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (920 mol % based on glycidyl methacrylate) of 1,4-butanediol were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 ml of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, then the porous particle (carrier β1) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 152 g of a carrier β1.

The progress of the reaction was confirmed by the following procedure.

A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of an absorbance peak at 908 cm⁻¹ due to the glycidyl group in the infrared absorption spectrum.

As a result, no absorbance peak at 908 cm⁻¹ was observed by FT-IR.

<Step (C): Introduction Reaction of Glycidyl Group>

150 g of the carrier β1 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide.

After cleaning, the carrier β1 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours.

After completion of the reaction, the obtained product was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 172 g of a porous particle into which a glycidyl group had been introduced (carrier γ1).

The introduction density of the glycidyl group in the obtained carrier γ1 was measured by the following procedure.

5.0 g of the carrier γ1 was sampled, and the dry mass thereof was measured and as a result, found to be 1.47 g. Next, the same amount of the carrier γ1 was weighed into a separable flask and dispersed in 40 g of water, 16 mL of diethylamine was added while stirring at room temperature, and the resulting mixture was heated to 50° C. and stirred for 4 hours. After completion of the reaction, the reaction product was transferred onto a glass filter and thoroughly washed with water to obtain a porous particle A into which diethylamine had been introduced.

The obtained porous particle A was transferred into a beaker and dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L hydrochloric acid with the point at which the pH reached 4.0 as the neutralization point.

From this, the amount of diethylamine introduced into the porous particle A into which diethylamine had been introduced was calculated, and the density of the glycidyl group of the carrier γ1 was calculated from the following expression.

As a result, the density of the glycidyl group was 880 μmol/g.
Density(μmol/g) of glycidyl group={0.1×volume(μL) of hydrochloric acid at neutralization point/dry mass(g) of porous particle into which glycidyl group has been introduced}<Step (D): Introduction Reaction of Polyol>

150 g of the carrier γ1, 600 mL of water, and 1000 g (13000 mol % based on glycidyl group) of D-sorbitol (log P=−2.20, manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.

After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which polyol had been introduced (carrier 61).

The obtained carrier 61 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 1.

<Evaluation of Alkali Resistance>

The alkali resistance was evaluated by calculating the amount of a carboxy group produced by hydrolysis of sodium hydroxide according to the following procedure.

First, 4 g of the packing material was dispersed in 150 mL of a 0.5 mol/L potassium chloride aqueous solution, and titration was carried out using 0.1 mol/L sodium hydroxide aqueous solution with the point at which the pH reached 7.0 as the neutralization point. From this, the amount of a carboxy group before hydrolysis included in the packing material was calculated from the following expression.
Amount(μmol/mL) of carboxy group=0.1×volume(μL) of sodium hydroxide aqueous solution at the time of neutralization/apparent volume (mL) of packing material

Here, the apparent volume of the packing material is the volume of the packing material phase measured after preparing a slurry liquid by dispersing 4 g of the packing material in water, transferring the slurry liquid to a graduated cylinder, and then allowing the same to stand for a sufficient time.

Subsequently, 4 g of the packing material was weighed into a separable flask, 20 mL of a 5 mol/L sodium hydroxide aqueous solution was added, and the resulting mixture was treated at 50° C. for 20 hours while stirring at 200 rpm. The mixture was cooled, then the packing material was collected by filtration, then washed with a 0.1 mol/L HCl aqueous solution and water in the order presented, and the amount of a carboxy group contained in the obtained packing material was calculated by the same method as above. From the difference between the amount of a carboxy group before and that after the reaction with the 5 mol/L sodium hydroxide aqueous solution, the amount of a carboxy group produced by the reaction with the 5 mol/L sodium hydroxide aqueous solution was calculated. As a result, the amount of a carboxy group produced was 21 μmol/mL.

If the amount of a carboxy group produced is 40 μmol/mL or less, the alkali resistance is considered to be high.

<Evaluation of Non-Specific Adsorption>

The obtained packing material was packed into a stainless steel column (manufactured by Sugiyama Shoji Co., Ltd.) having an inner diameter of 8 mm and a length of 300 mm by a balanced slurry method. Using the obtained column, a non-specific adsorption test was carried out by the method shown below.

The column packed with the packing material was connected to a Shimadzu Corporation HPLC system (liquid feed pump (trade name: LC-10AT, manufactured by Shimadzu Corporation), autosampler (trade name: SIL-10AF, manufactured by Shimadzu Corporation), and photodiode array detector (trade name: SPD-M10A, manufactured by Shimadzu Corporation)), and a 50 mmol/L sodium phosphate buffer aqueous solution as a mobile phase was passed at a flow rate of 0.6 mL/min.

Using the same sodium phosphate aqueous solution as the mobile phase as a solvent, their respective sample solutions of 0.7 mg/mL thyroglobulin (Mw of 6.7×105), 0.6 mg/mL γ-globulin (Mw of 1.6×105), 0.96 mg/mL BSA (Mw of 6.65×104), 0.7 mg/mL ribonuclease (Mw of 1.3×104), 0.4 mg/mL aprotinin (Mw of 6.5×103), and 0.02 mg/mL uridine (Mw of 244) (all manufactured by Merck Sigma-Aldrich) are prepared, and 10 μL of each is injected from the autosampler.

The elution time of each observed using the photodiode array detector at a wavelength of 280 nm was compared to confirm that there was no contradiction between the order of elution volume and the order of molecular weight size.

As a result, the elution volumes of the samples from the column packed with the packing material 1 were 8.713 mL, 9.691 mL, 9.743 mL, 10.396 mL, 11.053 mL, and 11.645 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced. When there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof, there was no non-specific adsorption, which is indicated as 0 in Table 1, and when there was a contradiction therebetween, non-specific adsorption was induced, which is thus indicated as X.

The porous particle (carrier al) obtained in the same manner as in Example 1 was subjected to the step D of Example 1.

<Step (D): Introduction Reaction of Polyol>

98 g of carrier al, 600 mL of water, and 1000 g (3050 mol % based on glycidyl group) of D-sorbitol (manufactured by KANTO CHEMICAL CO., INC.) were placed in a 3 L separable flask and stirred to form a dispersion.

After that, 10 g of potassium hydroxide was added, the temperature was raised to 60° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 15 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 130 g of a porous particle into which a polyol had been introduced (carrier δ7).

The carrier δ7 was classified into 16 to 37 μm using a sieve to obtain 115 g of a packing material 7.

The alkali resistance of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 7 was 120.3 μmol/mL, resulting in poor alkali resistance.

Further, the non-specific adsorption of the obtained packing material 7 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.606 mL, 9.769 mL, 9.9567 mL, 10.703 mL, 11.470 mL, and 12.112 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

Example 2

A porous particle (carrier al) was obtained in the same manner as in Example 1, and then a packing material 2 was obtained as follows.

98 g of the carrier α1 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.

After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (580 mol % based on the glycidyl group) of 1,4-cyclohexanedimethanol were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 ml of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, then the resulting porous particle (carrier $2) bonded to a diol compound including an alkylene group in the structure thereof was collected by filtration and then washed with 1 L of ion exchanged water to obtain 165 g of a carrier 32.

The progress of the reaction was confirmed by the following procedure.

A part of the dry porous particle into which an alkylene group had been introduced was mixed with potassium bromide, and the resulting mixture was pelletized by applying a pressure and then measured using FT-IR (trade name: Nicolet (registered trademark) iS10, manufactured by Thermo Fisher Scientific Inc.) to check the height of a absorbance peak at 908 cm⁻¹ due to the glycidyl group in the infrared absorption spectrum.

As a result, no absorbance peak at 908 cm⁻¹ was observed by FT-IR.

<Step (C): Introduction Reaction of Glycidyl Group>

150 g of the carrier $2 was weighed onto a glass filter and thoroughly cleaned with dimethylsulfoxide. After cleaning, the carrier $2 was placed in a separable flask, 262.5 g of dimethyl sulfoxide and 150 g of epichlorohydrin were added, the resulting mixture was stirred at room temperature, 37.5 ml of a 30% sodium hydroxide aqueous solution (manufactured by KANTO CHEMICAL CO., INC.) was further added, and the resulting mixture was heated to 30° C. and stirred for 6 hours. After completion of the reaction, the porous particle was transferred onto a glass filter and thoroughly washed with water, acetone, and water in the order presented to obtain 180 g of a porous particle into which a glycidyl group had been introduced (carrier γ2).

The introduction density of the glycidyl group in the obtained carrier γ2 was measured in the same manner as in Example 1. As a result, the density of the glycidyl group was 900 μmol/g.

<Step (D): Introduction Reaction of Polyol>

150 g of the carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether. After cleaning, the carrier γ2 was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g (5760 mol % based on the glycidyl group) of ethylene glycol (log P=−1.36) were placed in the separable flask, and stirring and dispersion were carried out. After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours. The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a polyol-introduced porous particle (carrier δ2). The carrier δ2 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 2.

The alkali resistance of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 15.2 μmol/mL, and it was confirmed that the packing material 2 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 2 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.814 mL, 9.635 mL, 9.778 mL, 10.37 mL, 10.898 mL, and 12.347 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 8 was obtained in the same manner as in Example 1 except that 150 g of ethylene glycol was used instead of 1,4-butanediol as an alkylene group-introducing agent.

The alkali resistance of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced in the packing material 8 was 108.4 μmol/mL, resulting in poor alkali resistance.

Further, the non-specific adsorption of the obtained packing material 8 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.708 mL, 9.8946 mL, 10.6452 mL, 11.5374 mL, and 12.1656 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

Example 3

A carrier γ2 was obtained in the same manner as in Example 2.

150 g of the obtained carrier γ2 was weighed onto a glass filter and thoroughly cleaned with diethylene glycol dimethyl ether.

After cleaning, the porous particle was placed in a 1 L separable flask, 150 g of diethylene glycol dimethyl ether and 150 g of polyethylene glycol #200 (manufactured by KANTO CHEMICAL CO., INC., average molecular weight of 190 to 210, log P is unclear, but the close compound tetraethylene glycol (Mw of 194) has a log P of −2.02) (1790 mol % based on glycidyl group) were placed in the separable flask, and stirring and dispersion were carried out.

After that, 1.5 mL of a boron trifluoride diethyl ether complex was added, the temperature was raised to 80° C. while stirring at 200 rpm, and the resulting mixture was subjected to the reaction for 4 hours.

The mixture was cooled, and then the reaction product was collected by filtration and washed thoroughly with water to obtain 152 g of a porous particle into which a polyol had been introduced (carrier 63).

The carrier δ3 was classified into 16 to 37 μm using a sieve to obtain 140.5 g of a packing material 3.

The alkali resistance of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 16.1 μmol/mL, and it was confirmed that the packing material 3 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 3 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.517 mL, 9.241 mL, 9.47 mL, 10.034 mL, 10.484 mL, and 11.927 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 9 was obtained in the same manner as in Example 2 except that no glycidyl group was introduced and no polyol was introduced. That is, the carrier $2 obtained in the step (B) of Example 2 was used as the packing material 9.

The non-specific adsorption of the obtained packing material 9 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.590 mL, 10.316 mL, 9.603 mL, 10.484 mL, 13.863 mL, and 12.861 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

Example 4

A packing material 4 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 90.0 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 10.0 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 11.5 μmol/mL, and it was confirmed that the packing material 4 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 4 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 7.52 mL, 8.214 mL, 8.451 mL, 9.062 mL, 9.511 mL, and 11.915 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 10 was obtained in the same manner as in Example 1 except that 150 g (480 mol % based on glycidyl methacrylate) of 1,10-decanediol was used instead of 1,4-butanediol as an alkylene group-introducing agent.

The non-specific adsorption of the obtained packing material 10 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.991 mL, 10.15 mL, 10.063 mL, 10.691 mL, 12.172 mL, and 11.531 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

Example 5

A packing material 5 was obtained in the same manner as in Example 3 except that 21.5 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 17.6 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase.

The amount of glycidyl methacrylate used was 66.2 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 33.8 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 18.3 μmol/mL, and it was confirmed that the packing material 5 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 5 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.692 mL, 9.434 mL, 9.625 mL, 10.236 mL, 10.759 mL, and 12.457 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 11 was obtained in the same manner as in Example 3 except that 13.7 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 25.4 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 46.4 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 53.6 mol % based on the total amount of the monomers.

The non-specific adsorption of the obtained packing material 11 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 8.872 mL, 10.131 mL, 9.82 mL, 10.422 mL, 12.782 mL, and 12.553 mL, and it was confirmed that there was a contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that non-specific adsorption was induced. Because of this, the alkali resistance was not evaluated.

It was confirmed that the exclusion limit molecular weights of the packing materials obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were all 1,000,000 or more.

Example 6

A packing material 6 was obtained in the same manner as in Example 3 except that 33.2 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 5.9 g of ethylene glycol dimethacrylate (trade name: NK Ester 1G, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 29.3 g of butyl acetate, 29.3 g of chlorobenzene, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 88.7 mol % based on the total amount of the monomers, and the amount of ethylene glycol dimethacrylate used was 11.3 mol % based on the total amount of the monomers.

The alkali resistance of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the amount of a carboxy group produced was 12.5 μmol/mL, and it was confirmed that the packing material 6 had excellent alkali resistance.

Further, the non-specific adsorption of the obtained packing material 6 was evaluated in the same manner as in Example 1. As a result, the elution volumes of the samples were 9.613 mL, 10.427 mL, 10.444 mL, 11.066 mL, 11.582 mL, and 12.575 mL, and it was confirmed that there was no contradiction between the order of the molecular weights of the samples and the order of the elution volumes thereof and that no non-specific adsorption was induced.

A packing material 12 was obtained in the same manner as in Example 3 except that 37.1 g of glycidyl methacrylate (trade name: Blemmer G (registered trademark) manufactured by NOF Corporation), 2.0 g of glycerin-1,3-dimethacrylate (trade name: NK Ester 701, SHIN-NAKAMURA CHEMICAL Co., Ltd.), 58.7 g of diethyl succinate, and 1.9 g of 2,2′-azobis(2,4-dimethylvaleronitrile) were used to provide an oil phase. The amount of glycidyl methacrylate used was 96.7 mol % based on the total amount of the monomers, and the amount of glycerin-1,3-dimethacrylate used was 3.3 mol % based on the total amount of the monomers.

Packing into a stainless steel column using the obtained packing material 12 was attempted. However, the back pressure was high, making liquid feeding difficult, and this made it impossible to carry out the packing. Because of this, neither of the evaluations was able to be carried out.

Results of the above Examples and Comparative Examples are shown in Table 1.

From the above results, by adopting the configuration of the present invention, a packing material having suppressed non-specific adsorption and high alkali resistance can be obtained.

When no hydrophobic portion is provided or when the alkylene chain is short, the alkali resistance is low as shown in Comparative Examples 1 and 2. In addition, it was found that when the alkylene chain is too long or when no hydrophilic portion is provided, the hydrophobicity is strong, and non-specific adsorption is induced as shown in Comparative Examples 3 and 4. In addition, in Comparative Example 5 having many repeating units derived from a polyfunctional monomer, it was found that non-specific adsorption was induced, and in Comparative Example 6 having fewer repeating units derived from a polyfunctional monomer, it was found that the back pressure applied to the apparatus was high, making column packing difficult.

Example 1

In a 2 L stainless steel container, 730 g of aluminum hydroxide powder (commercially available from KANTO CHEMICAL CO., INC., Cica special grade) were added into 1110 mL of 48% sodium hydroxide solution (commercially available from KANTO CHEMICAL CO., INC., Cica special grade), and they were stirred at 124° C. for 1 hour to give a sodium aluminate solution (First Step).

After the sodium aluminate solution was cooled to 80° C., ion exchange water was added into the sodium aluminate solution to achieve a total amount of 1500 mL.

After 96 mL of the sodium aluminate solution were separated into a 1 L stainless steel container, pure water was added into the solution to achieve a total amount of 730 mL (concentration of the sodium aluminate solution: 0.8 mol/L). The solution was stirred with keeping a temperature thereof at 25° C., and the solution was aerated with carbon dioxide in an aeration amount of 0.7 L/min. for 60 minutes to give adjusted aluminum hydroxide slurry (low-crystallinity aluminum compound=pseudo-boehmite) (Second Step).

Separately, 49.5 g of magnesium oxide powder (commercially available from KANTO CHEMICAL CO., INC., special grade) were added to 327 mL of pure water, and they were stirred for 1 hour to give magnesium oxide slurry.

In a 1.5 L stainless steel container, the magnesium oxide slurry and the adjusted aluminum hydroxide slurry were added into 257 mL of pure water, and they were stirred at 55° C. for 90 minutes to cause a first-order reaction. As a result, a reactant containing hydrotalcite nuclear particles was prepared (Third Step).

Then, pure water was added to the reactant to give a solution in a total amount of 1 L. The solution was put into a 2 L autoclave, and a hydrothermal synthesis was performed at 160° C. for 7 hours. As a result, hydrotalcite particles slurry was synthesized (Fourth Step).

To the hydrotalcite particles slurry were added 4.3 g of stearic acid (3 parts by mass with respect to 100 parts by mass of hydrotalcite particles) with keeping a temperature of the hydrotalcite particles slurry at 95° C. to perform a surface treatment on particles (Fifth Step). After the hydrotalcite particles slurry of which particles were surface treated was filtered and washed, a drying treatment was performed at 100° C. to give solid products of hydrotalcite particles. The produced hydrotalcite particles were subjected to an elemental analysis, resulting in that Mg/Al (molar ratio)=2.1.

In accordance with a method of Example 1 described in Japanese Laid-Open Patent Publication No. 2003-048712, hydrotalcite particles were synthesized.

In 150 g/L of NaOH solution in an amount of 3 L were dissolved 90 g of metal aluminum to give a solution. After 399 g of MgO were added to the solution, 174 g of Na₂CO₃ were added thereto and they were reacted with each other for 6 hours with stirring at 95° C. As a result, hydrotalcite particles slurry was synthesized.

To the hydrotalcite particles slurry were added 30 g of stearic acid (3 parts by mass with respect to 100 parts by mass of hydrotalcite particles) with keeping a temperature of the hydrotalcite particles slurry at 95° C. to perform a surface treatment on particles. After the hydrotalcite particles slurry of which particles were surface treated was cooled, filtered and washed to give solid matters, a drying treatment was performed on the solid matters at 100° C. to give solid products of hydrotalcite particles.

Example 2

In a 2 L stainless steel container, 730 g of aluminum hydroxide powder (commercially available from KANTO CHEMICAL CO., INC., Cica special grade) were added into 1110 mL of 48% sodium hydroxide solution (commercially available from KANTO CHEMICAL CO., INC., Cica special grade), and they were stirred at 124° C. for 1 hour to give a sodium aluminate solution (First Step).

After the sodium aluminate solution was cooled to 80° C., ion exchange water was added into the sodium aluminate solution to achieve a total amount of 1500 mL.

After 96 mL of the sodium aluminate solution were separated into a 1 L stainless steel container, pure water was added into the solution to achieve a total amount of 730 mL (concentration of the sodium aluminate solution: 0.8 mol/L). The solution was stirred with keeping a temperature thereof at 30° C., and the solution was aerated with carbon dioxide in an aeration amount of 0.7 L/min. for 90 minutes to give adjusted aluminum hydroxide slurry (low-crystallinity aluminum compound=pseudo-boehmite) (Second Step).

Separately, 49.5 g of magnesium oxide powder (commercially available from KANTO CHEMICAL CO., INC., special grade) were added to 327 mL of pure water, and they were stirred for 1 hour to give magnesium oxide slurry.

In a 1.5 L stainless steel container, the magnesium oxide slurry and the adjusted aluminum hydroxide slurry were added into 257 mL of pure water, and they were stirred at 55° C. for 90 minutes to cause a first-order reaction. As a result, a reactant containing hydrotalcite nuclear particles was prepared (Third Step).

Then, pure water was added to the reactant to give a solution in a total amount of 1 L. The solution was put into a 2 L autoclave, and a hydrothermal synthesis was performed at 160° C. for 7 hours. As a result, hydrotalcite particles slurry was synthesized (Fourth Step).

To the hydrotalcite particles slurry were added 4.3 g of stearic acid (3 parts by mass with respect to 100 parts by mass of hydrotalcite particles) with keeping a temperature of the hydrotalcite particles slurry at 95° C. to perform a surface treatment on particles (Fifth Step). After the hydrotalcite particles slurry of which particles were surface treated was filtered and washed, a drying treatment was performed at 100° C. to give solid products of hydrotalcite particles.

Solid products of hydrotalcite particles were produced in a same manner as in Comparative Example 1 except that reaction conditions of 95° C. and 6 hours for synthesis of the hydrotalcite particles slurry in Comparative Example 1 were changed to hydrothermal reaction conditions of 170° C. and 6 hours.

Example 3

In a 2 L stainless steel container, 730 g of aluminum hydroxide powder (commercially available from KANTO CHEMICAL CO., INC., Cica special grade) were added into 1110 mL of 48% sodium hydroxide solution (commercially available from KANTO CHEMICAL CO., INC., Cica special grade), and they were stirred at 124° C. for 1 hour to give a sodium aluminate solution (First Step).

After the sodium aluminate solution was cooled to 80° C., ion exchange water was added into the sodium aluminate solution to achieve a total amount of 1500 mL.

After 96 mL of the sodium aluminate solution were separated into a 1 L stainless steel container, pure water was added into the solution to achieve a total amount of 730 mL (concentration of the sodium aluminate solution: 0.8 mol/L). The solution was stirred with keeping a temperature thereof at 60° C., and the solution was aerated with carbon dioxide in an aeration amount of 0.7 L/min. for 60 minutes to give adjusted aluminum hydroxide slurry (low-crystallinity aluminum compound=pseudo-boehmite) (Second Step).

Separately, 49.5 g of magnesium oxide powder (commercially available from KANTO CHEMICAL CO., INC., special grade) were added to 327 mL of pure water, and they were stirred for 1 hour to give magnesium oxide slurry.

In a 1.5 L stainless steel container, the magnesium oxide slurry and the adjusted aluminum hydroxide slurry were added into 257 mL of pure water, and they were stirred at 55° C. for 90 minutes to cause a first-order reaction. As a result, a reactant containing hydrotalcite nuclear particles was prepared (Third Step).

Then, pure water was added to the reactant to give a solution in a total amount of 1 L. The solution was put into a 2 L autoclave, and a hydrothermal synthesis was performed at 160° C. for 7 hours. As a result, hydrotalcite particles slurry was synthesized (Fourth Step).

To the hydrotalcite particles slurry were added 4.3 g of stearic acid (3 parts by mass with respect to 100 parts by mass of hydrotalcite particles) with keeping a temperature of the hydrotalcite particles slurry at 95° C. to perform a surface treatment on particles (Fifth Step). After the hydrotalcite particles slurry of which particles were surface treated was filtered and washed, a drying treatment was performed at 100° C. to give solid products of hydrotalcite particles.

In accordance with a method of Example 1 described in Japanese Laid-Open Patent Publication No. 2013-103854, hydrotalcite particles were synthesized.

Into a 5 L container were added 447.3 g of magnesium hydroxide (d50=4.0 μm) and 299.2 g of aluminum hydroxide (d50=8.0 μm), and water was added thereto to achieve a total amount of 3 L. They were stirred for 10 minutes to prepare slurry. The slurry had physical properties of d50=10 μm and d90=75 μm. Then, the slurry was subjected to wet grinding for 18 minutes (residence time) by using Dinomill MULTILAB (wet grinding apparatus) with controlling a slurry temperature during grinding by using a cooling unit so as not to exceed 40° C. As a result, ground slurry had physical properties of d50=1.0 μm, d90=3.5 μm, and slurry viscosity=5000 cP. Then, sodium hydrogen carbonate was added to 2 L of the ground slurry such that an amount of the sodium hydrogen carbonate was ½ mole with respect to 1 mole of the magnesium hydroxide. Water was added thereto to achieve a total amount of 8 L, and they were stirred for 10 minutes to give slurry. Into an autoclave was put 3 L of the slurry, and a hydrothermal reaction was caused at 170° C. for 2 hours. As a result, hydrotalcite particles slurry was synthesized.

To the hydrotalcite particles slum were added 6.8 g of stearic acid (3 parts by mass with respect to 100 parts by mass of hydrotalcite particles) with keeping a temperature of the hydrotalcite particles slurry at 95° C. to perform a surface treatment on particles. After solids were filtered by filtration, the filtrated cake was washed with 9 L of ion exchange water at 35° C. The filtrated cake was further washed with 100 mL of ion exchange water, and a conductance of water used for washing was measured. As a result, the conductance of this water was 50 μS/sm (25° C.). The water-washed cake was dried at 100° C. for 24 hours and was ground to give solid products of hydrotalcite particles.

Example 5

In a 2 L stainless steel container, 730 g of aluminum hydroxide powder (commercially available from KANTO CHEMICAL CO., INC., Cica special grade) were added into 1110 mL of 48% sodium hydroxide solution (commercially available from KANTO CHEMICAL CO., INC., Cica special grade), and they were stirred at 124° C. for 1 hour to give a sodium aluminate solution (First Step).

After the sodium aluminate solution was cooled to 80° C., ion exchange water was added into the sodium aluminate solution to achieve a total amount of 1500 mL.

After 192 mL of the sodium aluminate solution were separated into a 1 L stainless steel container, pure water was added into the solution to achieve a total amount of 730 mL (concentration of the sodium aluminate solution: 1.6 mol/L). The solution was stirred with keeping a temperature thereof at 30° C., and the solution was aerated with carbon dioxide in an aeration amount of 0.7 L/min. for 90 minutes to give adjusted aluminum hydroxide slurry (low-crystallinity aluminum compound=pseudo-boehmite) (Second Step).

Separately, 49.5 g of magnesium oxide powder (commercially available from KANTO CHEMICAL CO., INC., special grade) were added to 327 mL of pure water, and they were stirred for 1 hour to give magnesium oxide slurry.

In a 1.5 L stainless steel container, the magnesium oxide slurry and the adjusted aluminum hydroxide slurry were added into 257 mL of pure water, and they were stirred at 55° C. for 90 minutes to cause a first-order reaction. As a result, a reactant containing hydrotalcite nuclear particles was prepared (Third Step).

Then, pure water was added to the reactant to give a solution in a total amount of 1 L. The solution was put into a 2 L autoclave, and a hydrothermal synthesis was performed at 160° C. for 7 hours. As a result, hydrotalcite particles slurry was synthesized (Fourth Step).

To the hydrotalcite particles slurry were added 4.3 g of stearic acid (3 parts by mass with respect to 100 parts by mass of hydrotalcite particles) with keeping a temperature of the hydrotalcite particles slurry at 95° C. to perform a surface treatment on particles (Fifth Step). After the hydrotalcite particles slurry of which particles were surface treated was filtered and washed, a drying treatment was performed at 100° C. to give solid products of hydrotalcite particles.

In accordance with a method of Example 1 described in Japanese Laid-Open Patent Publication No. H06-136179, hydrotalcite particles were synthesized.

To 1 L of water were added 39.17 g of sodium hydroxide and 11.16 g of sodium carbonate with stirring, and they were heated to 40° C. Then, to 500 mL of distilled water were added 61.28 g of magnesium chloride (19.7% as MgO), 37.33 g of aluminum chloride (20.5% as Al₂O₃), and 2.84 g of ammonium chloride (31.5% as NH₃) such that a molar ratio of Mg to Al, Mg/Al, was 2.0 and a molar ratio of NH₃ to Al, NH₃/Al, was 0.35. As a result, an aqueous solution A was prepared. The aqueous solution A was gradually poured into a reaction system of the sodium hydroxide and the sodium carbonate. The reaction system after pouring had pH of 10.2. Moreover, a reaction of the reaction system was caused at 90° C. for about 20 hours with stirring to give hydrotalcite particles slurry.

To the hydrotalcite particles slurry were added 1.1 g of stearic acid, and a surface treatment was performed on particles with stirring to give a reacted suspension. The reacted suspension was subjected to filtration and water washing, and then the reacted suspension was subjected to drying at 70° C. The dried suspension was ground by a compact sample mill to give solid products of hydrotalcite particles.