Hydrochloric acid

Example 12

There has been a growing interest in the fabrication of nanofibers derived from natural polymers due to their ability to mimic the structure and function of extracellular matrix. Electrospinning is a simple technique to obtain nano-micro fibers with customized fiber topology and composition (FIGS. 33A and 33B). The chitosan electrospun nanofibers have recently been extensively studied due to the favorable properties of chitosan such as controllable biodegradation, good biocompatibility and high mechanical strength. Currently, chitosan can be electrospun from a solution of chitosan dissolved in either trifluoroacetic acid (TFA) or acetic acid (HAc). However, processes to remove residual acid and acid salts from the electrospun material generally resulted in a swelling of fibers and deterioration of the nano-fibrous structure. Crosslinking in combination with neutralization methods also had not been effective at preventing loss of nano-fibrous structure.

The current study aimed to improve and maintain nano-fibrous and porous structure of the electrospun membranes by introducing a new post electrospinning chemical treatment. Membrane thickness was tripled in this research in order to increase the general tearing strength. Scanning electron micrograph (SEM) examination (FIG. 33C) and transmission electron micrograph (TEM) examination (FIG. 33D) showed Fiber diameters of the triethanolamine/N-tert-butoxycarbonyl (TEA/t-BoC) treated membranes ranged from 40 nm to 130 nm while fiber diameters were not able to be determined for the Na₂CO₃ group. Membranes treated by TEA/tboc (FIG. 34A) exhibited more nano-scale fibrous structure than membranes treated by saturated Na₂CO₃ (FIGS. 35B-35D, as seen demonstrated in scanning electron micrographs. After immersion in PBS for 24 hours, membranes treated by TEA/tboc exhibited less than 30% swelling (FIG. 34B) and retained their nanofibrous structure, compared with membranes treated by Na₂CO₃ (FIGS. 35B-35D) or compared with the non-treated chitosan membrane (FIG. 35A). After soaking the TEA/tBoc treated membranes in water overnight, membranes still kept the porous structure. In both, the before and after water status, fibers kept diameters in the nanometer range (FIG. 35C). TEA/tBoC modified nanofiber membranes also well preserved their fibrous structure over 4 weeks in physiological solution compared with Na₂CO₃ treated membranes (FIG. 35D).

Chitosan membranes treated by TEA/tboc showed better nano-fiber morphology characteristics than membranes neutralized by saturated Na₂CO₃ solution before and after being soaked in PBS. Retention of the nanofibrous structure for guided tissue regeneration applications may be of benefit for enabling nutrient exchange between soft gingival tissue and bone compartments and for mimicking the natural nanofibrillar components of the extracellular matrix during regeneration.

Example 41

[Figure (not displayed)]
1) Synthesis of Compound 42-1

[Figure (not displayed)]

Potassium carbonate (110 mg) was added to a solution of Compound 39 (200 mg) and ethyl 2-bromoacetate (100 mg) in DMF (5 mL), and the resulting mixture heated to 80° C. and stirred for 1 h under nitrogen protection. The reaction mixture was cooled to room temperature, and filtered. The filter cake was washed with ethyl acetate (2 mL). The filtrate was concentrated to obtain Compound 42-1. LCMS (ESI) m/z: 606 (M+1).

2) Synthesis of Compound 42-2

[Figure (not displayed)]

An aqueous solution of lithium hydroxide monohydrate (1M, 0.7 mL) was added to a solution of Compound 42-1 (200 mg) in tetrahydrofuran (5 mL), and the resulting mixture was stirred at 26° C. for 1 h under nitrogen protection. The reaction mixture was acidified to pH=5-6 with an aqueous solution of dilute hydrochloric acid (1M), and extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Compound 42-2. LCMS (ESI) m/z: 578 (M+1).

3) Synthesis of Compound 42

[Figure (not displayed)]

Methylamine hydrochloride (18 mg) was added to a solution of Compound 42-2 (100 mg), HATU (80 mg), and triethylamine (50 mg, 494.12 μmol) in dichloromethane (5 mL), and the resulting mixture was stirred at 26° C. for 1 h. The reaction mixture was acidified to pH=5-6 with an aqueous solution of dilute hydrochloric acid (1M), and extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue obtained from the concentration was purified by preparative TLC and preparative HPLC to obtain Compound 42. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.68 (s, 1H), 7.95 (d, J=8.3 Hz, 1H), 7.88 (d, J=1.5 Hz, 1H), 7.76 (dd, J=8.3, 1.8 Hz, 1H), 7.31-7.36 (m, 1H), 7.29 (dd, J=8.8, 2.0 Hz, 1H), 4.51 (s, 2H), 2.90 (d, J=5.0 Hz, 3H), 2.84 (q, J=7.7 Hz, 2H), 1.62 (s, 6H), 1.29 ppm (t, J=7.5 Hz, 3H); LCMS (ESI) m/z: 591 (M+1).

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.