Column Packing Material for Liquid Chromatography and Method for Producing the Same

Abstract

Disclosed are: a novel column filler which is for a liquid chromatography, is alkali resistant, has the separability and pressure resistance required as a column filler for a liquid chromatography, and can be mass-produced on a commercial basis; and a method for producing same. The column filler for a liquid chromatography is composed of eggshells or seashells that have been crushed, and subjected to a treatment using a calcium removing agent and to a biopolymer removal treatment. The method for producing a column filler for a liquid chromatography includes subjecting crushed eggshells or seashells to a treatment using a calcium removing agent and to a biopolymer removal treatment.

Description

Technical Field

[0001] The present invention relates to a column packing material for liquid chromatography and a method for producing the same, and more particularly, to an alkali-resistant column packing material for liquid chromatography that can use an alkaline mobile phase and a method for producing the same.

Background Art

[0002] Conventionally, chemically modified silica gels such as octadecyl silica (ODS silica) have been widely used as column packing materials for liquid chromatography. However, silica gel-based packing materials have the drawback that dissolution of silica gel and detachment of chemical modification occur under an alkaline aqueous solution, and a mobile phase with a high pH cannot be used. On the other hand, since various drugs and the like can be separated well in an alkaline condition, it is desired to use an alkaline mobile phase, and thus an alkali-resistant packing material is required.

[0003] The inventors of the present application have previously developed a porous spherical calcium carbonate-based high-performance liquid chromatography (HPLC) packing material as an alkali-resistant packing material (Non-Patent Document 1). This packing material is a highly alkali-resistant reversed-phase HPLC packing material in which the surface of artificially synthesized faujasite-type porous calcium carbonate crystal particles is coated with a hydrophobic group-containing polymer to impart a function of hydrophobic interaction.

[0004] It is also known to use crushed shells as a column packing material for liquid chromatography (Non-Patent Document 2). However, the separation performance is not always satisfactory.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] The above calcium carbonate-based HPLC packing material has mesopores with a surface of 20 nm as voids, can be stably used even under alkaline conditions, has sufficient separation ability and pressure resistance, and has excellent performance. However, it is difficult to mass-produce the barium titanate-type calcium carbonate crystal, and the current amount that can be synthesized while maintaining a uniform particle size and shape is about 7 g. That is, the known barium titanate-type calcium carbonate crystal is produced by a method of rapidly adding a calcium chloride solution to an aqueous solution containing sodium carbonate and PSS while stirring. However, if scaled up, it becomes difficult to control a uniform solution, the solution becomes non-uniform, and the particle size varies. For this reason, it is difficult to manufacture on a commercial scale.

[0007] Therefore, an object of the present invention is to provide a novel liquid chromatography column packing material and a method for producing the same, which are alkali-resistant, have the separation ability and pressure resistance required for a liquid chromatography column packing material, and can be mass-produced on a commercial basis.

Means for Solving the Problems

[0008] The inventors of the present application considered using eggshells as calcium carbonate particles. Eggshells are waste materials that are generated in large quantities in the food manufacturing industry, households, restaurants, etc., and can be obtained in large quantities at low cost. Since it was considered necessary to remove the remaining biopolymers from the crushed eggshells, the biopolymers were removed by treatment with sodium hypochlorite, and a test was conducted to coat them with a hydrophobic group-containing polymer and use them as a column packing agent as in Non-Patent Document 1, but the desired separation performance could not be achieved. As a result of further intensive studies to solve this problem, it was found that the desired separation performance can be achieved by further adding a step of washing the eggshells with EDTA, which is a calcium chelating agent, and the present invention was completed.

[0009] That is, the present invention provides the following. (1) A column packing agent for liquid chromatography comprising crushed eggshells or seashells that have been treated with a calcium removing agent and a biopolymer removing treatment. (2) The packing agent according to (1) comprising eggshells. (3) The packing agent according to (1) or (2), wherein the calcium removing agent is a calcium chelating agent or an acid. (4) The packing agent according to (3), wherein the calcium removing agent is a calcium chelating agent, and the calcium chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid or a salt thereof, glycol ether diamine tetraacetic acid or a salt thereof, and (1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid) or a salt thereof. (5) The packing agent according to (4), wherein the calcium chelating agent is ethylenediaminetetraacetic acid or a salt thereof. (6) The packing agent according to any one of (1) to (5), wherein the biopolymer removing treatment is an oxidizing agent treatment or a heat treatment. (7) The packing agent according to (6), wherein the biopolymer removing treatment is an oxidizing agent treatment, and the oxidizing agent is at least one selected from the group consisting of hypochlorous acid or a salt thereof, oxygen, ozone, and hydrogen peroxide. (8) The packing agent according to (7), wherein the oxidizing agent is hypochlorous acid or a salt thereof. (9) The filler according to any one of (1) to (8), wherein the number average particle diameter of the eggshell or shell is 1 μm to 1000 μm. (10) The filler according to any one of (1) to (9), having an organic group on the surface. (11) The filler according to (10), wherein the organic group is a hydrophobic group and the chromatography is reverse phase chromatography. (12) The filler according to (11), wherein the hydrophobic group is an alkyl group having 1 to 30 carbon atoms. (13) The filler according to any one of (1) to (12), wherein the 2θ peak value in X-ray diffraction coincides with the 2θ peak of calcite X-ray diffraction except for the 2θ peak value of 26.5°. (14) The filler according to (1), wherein the calcium remover is a weak acid or a weak acid buffer solution containing a weak acid and a salt. (15) The filler according to (14), wherein the weak acid is acetic acid. (16) In X-ray diffraction, the 2θ peak value exists at 26.5°, and except for the 2θ peak value of 26.5°, the 2θ peak value coincides with the 2θ peak value of calcite X-ray diffraction. When heated at 5 °C / min, the weight loss from 200 °C to 585 °C is 1 wt% or less, and the cumulative pore volume of pores with a diameter of less than 500 nm measured by mercury intrusion porosimetry is 0.05 mL / g or more. A column filler for liquid chromatography. (17) A method for producing a column filler for liquid chromatography according to (1), comprising treating the crushed eggshell or shell with a calcium remover and a biopolymer removal treatment. (18) The method according to (17), comprising treating the crushed eggshell or shell with a calcium remover and then treating it with an oxidizing agent. (19) The method according to (17) or (18), further comprising bonding an organic group to the surface of the eggshell or shell. (20) The method according to (19), comprising bonding an organic group to the surface of the eggshell or shell by bonding a polymer having a plurality of the organic groups to the surface of the eggshell or shell. (21) The method according to (19) or (20), wherein the organic group is a hydrophobic group and the chromatography is reverse-phase chromatography. (22) The method according to (21), wherein the hydrophobic group is an alkyl group having 1 to 30 carbon atoms. (23) Use of the eggshell or shell according to any one of (1) to (16) as a column packing material for liquid chromatography. (24) Use of the eggshell or shell described in any one of (1) to (16) for the production of a column packing material for liquid chromatography. (25) Liquid chromatography comprising applying a sample to a column packed with the column packing material for liquid chromatography according to any one of (1) to (16).

Advantages of the Invention

[0010] According to the present invention, there are provided a novel column packing material for liquid chromatography and a method for producing the same, which are alkali-resistant, have the separation ability and pressure resistance required as a column packing material for liquid chromatography, and can be mass-produced on a commercial basis.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] The eggshell used in the present invention is not particularly limited as long as it is the shell of a bird's egg, but a hen's eggshell with a large production volume and available inexpensively in large quantities is preferred. It is preferable to remove the eggshell membrane from the eggshell, dry it, and then grind it. The grinding of the eggshell can be mechanically performed using a mill or the like. The particle size of the ground eggshell can be appropriately set according to the purpose of column chromatography, but usually, the number average particle size (diameter) of about 5 μm to 20 μm is preferred. Note that commercially available ground eggshells are available after removing the eggshell membrane, drying, and grinding (for example, "Calhope" (registered trademark) manufactured by Kyupi Tama Co., Ltd. (diameter of about 10 μm)), so commercially available ground eggshells can be preferably used.

[0013] Next, the ground eggshell is treated with a calcium remover. The calcium remover may be any one that can dissolve fine particles of calcium carbonate, and preferable examples include calcium chelating agents and acids. By treating with a calcium remover and then washing, the minute calcium carbonate crystals contained in the pores of the ground eggshell can be removed.

[0014] The calcium chelating agent is not particularly limited, but preferable examples include at least one selected from the group consisting of ethylenediaminetetraacetic acid or its salts, glycol ether diamine tetraacetic acid or its salts, and (1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid) or its salts. Among these, EDTA or its salts are preferable. The calcium chelating agent is preferably used as an aqueous solution (which may be an aqueous buffer). The concentration of the calcium chelating agent in the aqueous solution can be set as appropriate, but it is about 10 mM to 1000 mM, preferably about 200 mM to 300 mM. For the treatment with the calcium chelating agent, it is preferable to adopt a method that allows sufficient contact between the calcium chelating agent and the ground eggshells. For example, it can be carried out by adding an aqueous solution of the calcium chelating agent to the ground eggshells, performing ultrasonic treatment, and then stirring. At this time, the amount of the aqueous solution of the calcium chelating agent to be added is usually about 2 to 100 times, preferably about 10 to 20 times, the weight of the ground eggshells. The treatment temperature is not particularly limited and can be above 0 °C and below 100 °C, but since it can be carried out at room temperature, it is preferably carried out at room temperature. The treatment time is usually about 1 hour to 5 hours. After the treatment with the calcium chelating agent, it is preferable to collect the ground eggshells by suction filtration or the like and wash them thoroughly with water.

[0015] Instead of the above-mentioned calcium chelating agent, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, water (H3O +)Acids such as acetic acid can also be used. As the conditions for the acid treatment, conditions are selected such that the calcium carbonate fine particles in the pores are dissolved, but the calcium carbonate forming the porous structure is not dissolved so much and the porous structure is maintained. Such conditions vary depending on whether the acid used is a strong acid or a weak acid, but it is preferable to use a weak acid such as acetic acid or phosphoric acid because it is easy to control. In the case of a strong acid, it is preferable to use a dilute acid. As the conditions for the acid treatment, it is preferable to adopt a method in which the acid and the ground eggshell can be sufficiently contacted. For example, it can be carried out by adding an acidic aqueous solution to the ground eggshell, performing ultrasonic treatment, and then stirring. The pH of the acid in the aqueous solution can be set as appropriate, but it is preferably about pH 1 to 7, more preferably about pH 3 to 6, and even more preferably pH 3 to 5. Also, a weak base can be added to the acid to form an acidic buffer solution. As a preferred example, an acidic buffer solution containing acetic acid and ammonium acetate can be mentioned. By adding a salt of a weak acid such as ammonium acetate salt to a weak acid such as acetic acid to make a buffer, the pH of the reaction system can be maintained by the buffering ability, and reproducibility can be easily obtained. There are advantages such as the pH can be controlled by changing the mixing ratio of the weak acid and the salt. In this case, the concentration of the salt of the weak acid in the buffer solution is preferably about 700 mM to 8 M. At this time, the amount of the acidic aqueous solution to be added is usually about 2 to 100 times, preferably about 10 to 20 times the weight of the ground eggshell. The treatment temperature is not particularly limited and can be above 0 °C and below 100 °C, but since it can be carried out at room temperature, it is preferably carried out at room temperature. The treatment time is usually about 30 minutes to 5 hours. After the acid treatment, it is preferable to collect the ground eggshell by suction filtration or the like and wash it sufficiently with water.

[0016] Next, after drying the pulverized eggshells after washing with water, a treatment for removing biopolymers is performed. The removal of biopolymers is carried out to remove the biopolymers remaining in the porous structure of the pulverized eggshells. The treatment for removing biopolymers can be performed by treatment with an oxidizing agent. Examples of the oxidizing agent include, but are not limited to, at least one selected from the group consisting of hypochlorous acid or its salts, oxygen, ozone, and hydrogen peroxide. Among these, hypochlorous acid or its salts are preferred. When using oxygen as the oxidizing agent, it is preferably heated to 400°C to 500°C. Also, when using hydrogen peroxide, ultraviolet irradiation can be used in combination.

[0017] The oxidizing agent is preferably used as an aqueous solution (which may be an aqueous buffer solution). The concentration of the oxidizing agent in the aqueous solution can be set as appropriate, but is usually about 1% to 20% by mass, preferably about 2% to 10% by mass. For the treatment with the oxidizing agent, it is preferable to adopt a method that allows the oxidizing agent and the pulverized eggshells to come into sufficient contact. For example, it can be carried out by adding an aqueous oxidizing agent solution to the pulverized eggshells, performing ultrasonic treatment, and then stirring. At this time, the amount of the aqueous oxidizing agent solution added is usually about 2 to 100 times the weight of the pulverized eggshells, preferably about 10 to 40 times. The treatment temperature is not particularly limited and can be carried out at a temperature above 0°C and below 100°C, but since it can be carried out at room temperature, it is preferably carried out at room temperature. The treatment time is usually about 12 to 96 hours, preferably about 24 to 72 hours. After the calcium chelating agent treatment, it is preferable to collect the pulverized eggshells by suction filtration or the like and wash them thoroughly with water.

[0018] Also, the treatment for removing biopolymers can also be carried out by heating. Heating can be carried out under vacuum or in an inert gas, but since it can be carried out in the atmosphere, it is simple and low-cost and preferable to carry out in the atmosphere. In this case, since oxygen is present, it can also be considered as an oxidizing agent treatment with oxygen. The temperature during heating is the temperature at which the biopolymer decomposes and is lower than the temperature at which calcium carbonate thermally decomposes, and is preferably about 400°C to 500°C as described above.

[0019] In the above description, the biopolymer removal treatment is performed after the calcium removal agent treatment, but the order may be reversed or it may be possible to perform them simultaneously. However, as described above, it is preferable to perform the biopolymer removal treatment after the calcium removal agent treatment.

[0020] By washing the powder after the oxidizing agent treatment or heat treatment, eggshell-derived calcium carbonate particles having a porous structure with a pore diameter of about 100 nm to 500 nm and no chemical modification can be obtained. As specifically shown in the following examples, the 2θ peak value of the X-ray diffraction of these eggshell-derived calcium carbonate particles coincides with the 2θ peak value of the X-ray diffraction of calcite except for the 2θ peak value of 26.5°. The 2θ peak value of 26.5° is not seen in the X-ray diffraction of calcite and is considered to be a peak value due to the eggshell. In addition, although there is a 2θ peak at about 32° in the eggshell-derived calcium carbonate particles, this peak is difficult to see in FIG. 29 etc. and is a peak that also exists in calcite. Further, the weight loss from 200 °C to 585 °C when the temperature is raised at 5 °C / min is 1 wt% or less, preferably 0.5 wt% or less. Also, the cumulative pore volume of pores with a diameter of less than 500 nm measured by the mercury intrusion method is 0.05 mL / g or more, and usually 0.05 mL / g to 0.10 mL / g. When treated with a calcium chelating agent such as EDTA and an oxidizing agent such as sodium hypochlorite, it is usually 0.06 mL / g or more and less than 0.07 mL / g. When treated with an acid or acidic buffer such as acetic acid + ammonium acetate buffer and an oxidizing agent such as sodium hypochlorite, it is usually 0.07 mL / g or more and 0.08 mL / g or less. However, if it is 0.05 mL / g or more, it is not limited to these ranges.

[0021] These calcium carbonate particles can be used as a packing material for a normal phase chromatography column as they are, but usually, a desired organic group, for example, a functional group, an ionic group, an organic group having an affinity for some substance, etc. is bonded to the surface of the particles, and they can be used as a packing material for a liquid chromatography column. That is, a hydrophobic group is bonded to be used as a packing material for a reverse phase or hydrophobic chromatography column, an ionic group is bonded to be used as a packing material for an ion exchange chromatography column, a hydrophilic group is bonded to be used as a packing material for a hydrophilic interaction chromatography column, a chiral selector is bonded to be used as a packing material for a chiral chromatography column, and an organic group having a specific binding ability with some ligand is bonded to be used as a packing material for an affinity chromatography column. More specifically, in the case of a packing material for a reverse phase chromatography column, as the hydrophobic group, an alkyl group having 1 to 30 carbon atoms is preferable, and particularly an alkyl group having 4 to 18 carbon atoms is preferable. In the case of a packing material for a hydrophobic chromatography column, examples of the hydrophobic group include a phenyl group. In the case of a packing material for an ion exchange chromatography column, examples of the ionic organic group include sulfonic acid, carboxylic acid, phosphoric acid, amino, amine group, etc. In the case of hydrophilic interaction chromatography, examples of the hydrophilic group include diol, amide, amino, cyano group, etc. In the case of chiral chromatography, examples include polysaccharide derivatives, proteins, kinin derivatives, helical vinyl polymers, etc. Further, examples of the organic group having a specific binding ability with some ligand include various antigens, haptens, antibodies, receptors, enzymes, protein A, protein G, etc.

[0022] The method of bonding a desired organic group to the surface of calcium carbonate particles is itself known as described in the above Non-Patent Document 1. For example, a desired group can be bonded to a polymer of a compound having a pair of carboxyl groups capable of chelating calcium, and by treating calcium carbonate particles with this polymer, a desired group can be bonded to the surface of the calcium carbonate particles. For example, in the following Examples, calcium carbonate particles are treated with poly(maleic acid-alt-1-octadecene) (PMAcO), which is a polymaleic acid derivative obtained by polymerizing a maleic acid derivative having an octadecyl group. Two carboxylic acid ions of maleic acid chelate calcium ions to bond the polymer to the surface of the calcium carbonate particles, thereby bonding the octadecyl group to the surface of the calcium carbonate particles to obtain a column packing material for reverse phase chromatography. The molecular weight of the polymer is not limited in any way, and it may be produced using easily available raw materials. For example, since poly(maleic anhydride-alt-1-octadecene) (PMAO) with a number average molecular weight of 30,000 to 50,000 is commercially available, it can be hydrolyzed and used.

[0023] As a method for binding a calcium chelating polymer such as PMAcO to the surface of calcium carbonate particles derived from eggshells, a method of treating the calcium carbonate particles derived from eggshells with a polymer solution can be preferably employed. The solvent of the polymer solution is not particularly limited as long as it can dissolve the polymer. For example, a water-soluble organic solvent such as acetone is preferable. The concentration of the polymer in the solution can be set appropriately, but usually, it is about 0.02 w / v% to 20 w / v%, preferably about 0.5 w / v% to 5 w / v%. For the treatment with the polymer, it is preferable to adopt a method that allows sufficient contact between the polymer and the calcium carbonate particles derived from eggshells. For example, it can be carried out by adding a polymer solution to the calcium carbonate particles derived from eggshells, performing ultrasonic treatment, and then stirring. At this time, the amount of the polymer solution to be added is usually about 2 to 80 times, preferably about 8 to 30 times the weight of the calcium carbonate particles derived from eggshells. The treatment temperature is not particularly limited and is possible within a temperature range higher than the melting point and lower than the boiling point of the solvent. However, since it can be carried out at room temperature, it is preferably carried out at room temperature. The treatment time is usually about 6 to 96 hours, preferably about 12 to 48 hours. After the polymer treatment, it is preferable to collect and dry the calcium carbonate particles derived from eggshells by suction filtration while washing with the solvent of the polymer solution.

[0024] In the above description, ground eggshells were used as the raw material, but ground shells can also be used instead of ground eggshells. As the shell, the shells of edible shellfish that are discarded as waste are preferable. Examples of edible shellfish include scallops, clams, clams, oysters, abalones, and ark shellfish, but are not limited thereto. Even when using shells, the above-described various treatments for eggshells can be carried out in the same manner. Note that eggshells are easier to grind than shells, and it is easier to obtain a pulverized product with a uniform particle size. Therefore, it is preferable to use eggshells as the raw material.

[0025] The column packing material for liquid chromatography of the present invention is a waste product, and since it uses eggshells or seashells that are discarded and supplied in large quantities at low cost as raw materials, mass production is possible. Therefore, it can be preferably used not only as a column packing material for analytical chromatography but also as a packing material for preparative columns. Furthermore, as specifically shown in the following examples, it can be stably used under alkaline conditions and has sufficient separation ability and pressure resistance.

[0026] Hereinafter, the present invention will be specifically described based on examples. However, the present invention is not limited to the following examples.

Examples

[0027] Example 1 1. Preparation of Ground Eggshells As ground eggshells, "Calhope" (registered trademark) manufactured by Cupi Tama Co., Ltd. was purchased. A part was taken, osmium was vacuum-evaporated for 10 seconds, and when observed with a scanning electron microscope (SEM), it was confirmed that a porous structure with a diameter of about 100 nm to 500 nm existed on the particle surface. Also, when the particle size distribution was measured with a particle size distribution meter, it was about 5 μm to 30 μm, and the number average particle diameter was 10.2 μm. The image taken with the above scanning electron microscope (SEM) is shown in Fig. 6.

[0028] 2. EDTA Treatment 30 g of ground eggshells were placed in a 1 L flask, and 500 mL of a 250 mM EDTA aqueous solution was added. Ultrasonic treatment was performed for 1 minute to sufficiently disperse the ground eggshells, and in this state, using a shaker, the mixture was stirred at 125 rpm at room temperature for 2 hours so that the ground eggshells did not precipitate. Then, suction filtration was performed while washing with a sufficient amount of water, and it was dried at 60 °C overnight.

[0029] 3. Sodium Hypochlorite (NaClO) Treatment To the ground eggshells after the above treatment, 500 mL of a 5 mass% NaClO aqueous solution was added, and ultrasonic treatment was performed for 3 minutes to sufficiently disperse the ground eggshells. In this state, using a shaker, the mixture was stirred at 125 rpm at room temperature for 48 hours so that the ground eggshells did not precipitate. Then, suction filtration was performed while washing with a sufficient amount of water, and it was dried overnight at 60 °C. The SEM image of the eggshells after the above treatment is shown in Fig. 7.

[0030] 4. Synthesis of PMAcO In a 100 mL eggplant flask, 3.0 g of commercially available PMAO (molecular weight 30,000 - 50,000) (manufactured by Sigma - Aldrich) was completely dissolved in 27 ml of acetone. 3 ml of water was added thereto, and a hydrolysis reaction was carried out by vigorously stirring at room temperature overnight. After evaporating acetone and water using an evaporator, it was dissolved in 10 ml of acetone, and reprecipitation purification by dropping while ice - cooling into 500 ml of water was carried out in three portions. At this time, white transparent polymer crystals were obtained. After suction - filtering the obtained polymer, the solvent was evaporated under vacuum conditions to obtain the target PMAcO.

[0031] 5. Modification of calcium carbonate particles derived from eggshells with PMAcO 500 mg of PMAcO was weighed into a 1 L eggplant flask and completely dissolved in 500 ml of acetone. 25.0 g of the calcium carbonate particles derived from eggshells obtained in 3 was added, and after shaking the eggplant flask, the slurry was subjected to ultrasonic treatment for 3 minutes so that the particles were completely wetted with the solvent. This slurry was stirred at 125 rpm at room temperature for 24 hours using a shaker so that the particles did not precipitate. Then, suction filtration was performed while washing with acetone, and it was dried overnight at 60 °C to obtain calcium carbonate particles derived from eggshells modified with PMAcO.

[0032] 6. Physical property evaluation (1) Measurement by X - ray diffractometer (XRD) The analysis results by X-ray diffraction are shown in Fig. 1. In Fig. 1, "Calcite" represents the results for calcite, "Eggshell" represents the results for crushed eggshells, "Bare (NaClO)" represents the results for the particles before PMAcO treatment where the EDTA treatment in 2 above was omitted and the NaClO treatment in 3 was performed, "Bare (EDTA NaClO)" represents the results for the particles before PMAcO treatment obtained in 3 above, and "Eggshell-PMAcO" represents the results for the particles obtained in 5 above.

[0033] As shown in Fig. 1, it was clarified that in the finally obtained PMAcO-modified particles, each peak also coincides with the calcite peak, indicating that the calcite structure is maintained.

[0034] (2) Dispersion test The dispersion test of the particles obtained in 5 above was carried out. The particles were put into water and methanol, sonicated, and then the dispersion was confirmed. The particles did not disperse in water but dispersed in methanol, which is an organic solvent. Therefore, it was confirmed that the particle surface became hydrophobic due to the modification of PMAcO.

[0035] (3) Analysis by Fourier transform infrared spectroscopy (FT-IR) The modification of PMAcO was evaluated by FT-IR. The results are shown in Fig. 2. In Fig. 2, "PMAcO's CH2" represents the results for PMAcO, "Eggshell-PMAcO" represents the results for the particles obtained in 5 above, "Bare (EDTA+NaClO)" represents the results for the particles before PMAcO treatment obtained in 3 above, and "Eggshell" represents the results for crushed eggshells.

[0036] After the modification of PMAcO, since the alkyl peak at 2924 cm -1 derived from PMAcO appears, the modification of PMAcO could be confirmed.

[0037] 7. Preparation of the column The particles obtained in the above 5 were packed into a semi-preparative column (inner diameter 10 mm x length 150 mm) using the wet packing method. This was specifically carried out as follows. 25 g of the particles obtained in the above 5 were suspended in 125 mL of chloroform and sonicated to form a slurry. The slurry was injected into a separatory column packer, and methanol was pumped at a constant pressure of 22 MPa for 60 minutes. Then, water / methanol (=50 / 50, v / v) was flowed for 30 minutes to replace the solvent in the column. After packing, excess particles were scraped off with a microspatula, and then the column was capped and stored at room temperature.

[0038] 8. Backpressure Evaluation of the Column The backpressure of the column prepared in the above 7 was measured. The backpressure when the mobile phase of water / acetonitrile (=50 / 50, v / v) was pumped at different flow rates is shown in Figure 3, and the backpressure when the mobile phase mixing ratio was changed with a mobile phase flow rate of 3.0 ml / min is shown in Figure 4.

[0039] As shown in Figure 3, the backpressure is proportional to the flow rate of the mobile phase, and this backpressure is not significantly different from that of a commercially available semi-preparative ODS column of the same size. It was confirmed that it has sufficient pressure resistance for practical use. Also, as shown in Figure 4, in the water / acetonitrile mobile phase, it becomes a convex curve with the maximum when the mixing ratio of water is 80%, which is consistent with the ratio of viscosities when water / acetonitrile is simply mixed. Since such a stable backpressure was obtained, it was clarified that the preparative column prepared in 7 is practical.

[0040] 9. Analysis of Basic Pharmaceuticals Using an Alkaline Mobile Phase Using the column prepared in [7] above, separation analysis of a mixed sample of two types of basic psychotropic drugs, imipramine and clomipramine, was performed. As the mobile phase, a 0.1 M sodium hydroxide aqueous solution / acetonitrile mixture was used. The proportion of the 0.1 M sodium hydroxide aqueous solution in the mobile phase was changed in 10% increments from 40% (v / v) to 70% (v / v). When the proportion of the 0.1 M sodium hydroxide aqueous solution was 70%, the pH was approximately 13. The log k (retention coefficient representing the distribution ratio between the hydrophobic field of the stationary phase and the mobile phase) at various mobile phase mixing ratios is shown in the left figure of Fig. 5, and the chromatogram is shown in the right figure of Fig. 5.

[0041] As shown in Fig. 5, it was confirmed that the preparative column prepared in [5] above also showed reverse-phase retention behavior in the retention of basic psychotropic drugs under alkaline conditions of pH 13. As shown in the right figure of Fig. 5, in the case of an injection volume of 10 μg each, when the mixing ratio of the sodium hydroxide aqueous solution in the sodium hydroxide aqueous solution / acetonitrile mobile phase was 60% or more, the resolution Rs was 1.5 or more, and the two types of basic psychotropic drugs could be completely separated.

[0042] Example 2 1. Method 1-1. Surface treatment of eggshells Acetic acid and ammonium acetate salt were mixed at a ratio of 1:1 and diluted with ultrapure water to prepare 500 mL each of 3 M and 6 M ammonium acetate buffers (pH = 4.7). Since the pK of acetic acid a is 4.7, the condition of pH = 4.7 is the condition with the largest buffering capacity. 50 g of eggshell powder was weighed into a 5 L plastic bucket, 500 mL of the prepared ammonium acetate buffer was added, and it was stirred at 600 rpm at room temperature using a stirrer and a magnetic stir bar. After 2 hours, 4 hours, or 24 hours had passed, the reaction solution was transferred to a 1 L beaker and decanted for about 3 minutes, the solution part was discarded, and the precipitated eggshell powder was suction-filtered while washing with a sufficient amount of water, dried overnight at 60 °C, and then the surface shape of the particles was observed with a scanning electron microscope (SEM).

[0043] Next, by changing the mixing ratio of acetic acid and ammonium acetate salt from 1:1 to 10:1, the pH was adjusted to 3.7, and then diluted with ultrapure water to prepare 500 mL each of 300 mM, 600 mM, and 900 mM ammonium acetate buffers (pH = 3.7). Similar to the case of pH = 4.7, 50 g of eggshell powder was weighed into a 5 L poly bucket, 500 mL of the prepared ammonium acetate buffer was added, and it was stirred at 600 rpm at room temperature using a stirrer and a magnetic stir bar. After 2 hours, 4 hours, or 24 hours, the reaction solution was transferred to a 1 L beaker and decanted for about 3 minutes, the solution part was discarded, and the precipitated eggshell powder was suction filtered while washing with a sufficient amount of water, dried overnight at 60 °C, and then the surface shape of the particles was observed by SEM.

[0044] In addition, to confirm the reproducibility of the surface treatment method with ammonium acetate buffer, the surface treatment of eggshells was carried out in three batches under the same conditions of pH, concentration, and reaction time, and the surface structure, particle size, and recovery rate of the eggshell powder were compared.

[0045] 1-2 Removal of organic matter by NaClO 27 g of eggshell powder that had been acid-treated with 900 mM ammonium acetate buffer (pH = 3.7) for 2 hours was added to a 1 L Erlenmeyer flask containing 500 mL of 5 wt% aqueous NaClO solution, and it was shaken at 100 rpm at room temperature for 48 hours using a shaker. After suction filtration and drying overnight at 60 °C, the surface shape of the particles was observed by SEM, and the organic matter content was measured by thermogravimetric analysis.

[0046] 1-3 Modification with PMAcO PMAO was hydrolyzed to synthesize PMAcO. 3 g of PMAO was weighed into a 100 mL eggplant-shaped flask, completely dissolved in 27 mL of acetone, and then 3 mL of pure water was added. After carrying out the hydrolysis reaction with vigorous stirring overnight at room temperature, the solvent was evaporated using an evaporator. It was dissolved in 10 mL of acetone, and reprecipitation purification was carried out by dropwise adding it to 500 mL of ice-cooled pure water, and white transparent polymer crystals were obtained. After the obtained polymer was suction filtered, the solvent was evaporated under vacuum conditions to obtain the target product, PMAcO.

[0047] Next, 0.5 g of the obtained PMAcO was weighed into a 1 L Erlenmeyer flask and dissolved in 500 mL of acetone. 26 g of eggshells after NaClO treatment were added, and ultrasonic treatment was applied for 3 minutes to make the particles completely wet with the solvent, and then shaken at 100 rpm for 24 hours at room temperature using a shaker. Then, suction filtration was carried out while washing with acetone, and it was dried overnight at 60 °C to obtain eggshells modified with PMAcO, Eggshell-PMAcO particles. Regarding the particles before and after modification with PMAcO, evaluation of the dispersibility in water and methanol, and measurement of the amount of PMAcO modification by thermogravimetric analysis were carried out.

[0048] 1-4 Basic evaluation of Eggshell-PMAcO 1-4-1 Preparation of semi-preparative column The eggshell-PMAcO prepared by acid treatment with ammonium acetate buffer, followed by NaClO treatment and PMAcO modification, was packed into a semi-preparative column (inner diameter 10 mm × 150 mm) by the wet packing method. 25 g of Eggshell-PMAcO particles were suspended in 180 mL of chloroform, injected into a column packer for separation, the flow rate was kept constant at 20 mL / min, and methanol was pumped for 5 minutes. Subsequently, the pressure was kept constant at 30 MPa, and methanol was pumped for 60 minutes. Then, water / methanol (50 / 50, v / v) was pumped for 30 minutes to replace the solvent in the column. After packing, the column was capped and stored at room temperature. Also, as a comparison, particles without acid treatment, particles without PMAcO modification, and commercially available crushed ODS particles with an average particle diameter of 10 μm were packed into columns of the same size in the same manner. Note that the names of the columns prepared using EDTA in Example 1 and each column prepared in this example were determined as shown in Table 1, and the names shown in Table 1 were used in subsequent studies.

[0049]

Table 1

[0050] 1-4-2. Comparison of retention behaviors The retention behaviors of the five types of columns shown in Table 1 were confirmed. The hydrophobic compound tert-butylbenzene was used as the sample. First, tert-butylbenzene was weighed into a screw tube, and methanol was added and dissolved to prepare a 10 mg / mL sample. Then, a plot was created with the ratio of water in the mobile phase on the horizontal axis and the retention factor obtained from the resulting chromatogram on the vertical axis to confirm the magnitude of the column's retention force and whether the column exhibits reverse-phase retention behavior. Note that in subsequent studies, the mobile phase was a mixed solvent of water and methanol, the column temperature was 25°C, and the detection wavelength of the chromatogram was 254 nm.

[0051] 1-4-3. Comparison of van Deemter plots For the column that showed reverse-phase retention behavior in the examination of 1-4-2, samples were injected at eight flow rates of 0.5 mL / min, 1 mL / min, 1.5 mL / min, 2 mL / min, 2.5 mL / min, 3 mL / min, 4 mL / min, and 5 mL / min, and the theoretical plate height was obtained from each of the resulting chromatograms. Then, a van Deemter plot with the mobile phase linear velocity on the horizontal axis and the theoretical plate height on the vertical axis was created to compare the separation ability. As the sample, 10 mg / mL of tert-butylbenzene prepared in 4.3.2 was used.

[0052] 1-4-4. Quantitative Analysis For the column prepared by acid treatment with ammonium acetate buffer, the retention capacity, that is, the maximum injection amount of the sample that the column can hold at one time, was investigated. As the sample, 20 mg / mL, 30 mg / mL, 40 mg / mL, and 50 mg / mL of tert-butylbenzene dissolved in methanol were used, and by comparing the shapes of the chromatograms obtained by increasing the sample injection amount into the column, the maximum injection amount at which the peak shape does not collapse was taken as the retention capacity.

[0053] Next, in order to confirm the quantitativeness of the analysis results obtained when a sample within the retention capacity was injected into the column, a calibration curve was created with the injection amount of tert-butylbenzene on the horizontal axis and the peak area of the chromatogram on the vertical axis.

[0054] Also, in order to confirm the reproducibility of the analysis results obtained when a sample not exceeding the retention capacity was injected into the column, the same amount of tert-butylbenzene was continuously injected 6 times, and the relative standard deviations of the retention time and the theoretical plate height were calculated from the resulting chromatograms.

[0055] 1-5. Separation of Basic Compounds 1-5-1. Retention Behavior of Basic Compounds The separation of two basic compounds was carried out under alkaline conditions. Samples were prepared by dissolving Imipramine and Clomipramine in pure water at a concentration of 100 mg / mL each. In addition, when the mobile phase contains non-volatile compounds such as sodium hydroxide, complicated operations such as desalting are required when removing the mobile phase after fractionating the sample, so it is considered undesirable as fractionation conditions. By adding 1 mL of volatile triethylamine (TEA) to 1 L of water as the mobile phase, an alkaline mobile phase with a pH of 11.5 was prepared. Then, the separation of the two basic compounds was performed by changing the mixing ratio of the TEA aqueous solution and methanol in the mobile phase, and the resolution of the chromatograms obtained under each condition was determined. In addition, a plot was created with the ratio of the TEA aqueous solution in the mobile phase on the horizontal axis and the retention factor obtained from the chromatogram on the vertical axis to confirm the retention behavior of the column.

[0056] 1-5-2. Retention Capacity of Basic Compounds The retention capacity of Eggshell-PMAcO was investigated under the conditions where the ratio of the TEA aqueous solution in the mobile phase was 50% and 60%. As the sample, a mixed sample of Imipramine and Clomipramine at 100 mg / mL each prepared in 4.4.1 was used. From the chromatograms obtained by increasing the sample injection volume into the column, the resolution of the peaks of the two components was calculated, and the maximum injection volume of the sample with complete separation, that is, a resolution of 1.5 or more, was taken as the retention capacity.

[0057] 2. Results 2-1. Surface Treatment of Eggshell The SEM image of the eggshell before acid treatment is shown in Fig. 8. Also, the SEM image of the eggshell after acid treatment with two concentrations of ammonium acetate buffer (pH = 4.7) for 2 hours is shown in Fig. 9.

[0058] As shown in Fig. 9, when the concentration of ammonium acetate salt is 3 M or 6 M, a uniform fine porous structure appears on the entire surface of the eggshell, and it is clearly confirmed that the surface area has expanded compared to before the acid treatment. From the above, the surface area expansion using 3 M and 6 M ammonium acetate buffers (pH = 4.7) was successful.

[0059] 2-1-2. Change in pH It was predicted that by lowering the pH of the ammonium acetate buffer, that is, making the ammonium acetate buffer a stronger acid, the eggshell surface could be sufficiently dissolved even at a lower concentration. Fig. 10 shows the SEM image of the eggshell after acid treatment with a 900 mM ammonium acetate buffer (pH = 3.7) for 2 hours.

[0060] Compared with Fig. 8, in the case of 900 mM shown in Fig. 10, a uniform fine porous structure appears on the entire surface of the eggshell, and it is clearly confirmed that the surface area has expanded. From the above, the surface area expansion using a 900 mM ammonium acetate buffer (pH = 3.7) was successful at reaction times of 2 hours, 4 hours, and 24 hours. However, since no significant change was observed in the surface structure even when the reaction time was extended, it was determined that a reaction time of 2 hours was sufficient.

[0061] Fig. 11 shows the results of measuring the crystal structure of the particles reacted with a 900 mM ammonium acetate buffer (pH = 3.7) for 2 hours by X-ray diffraction (XRD).

[0062] From Fig. 11, it was confirmed that the particles acid-treated with the ammonium acetate buffer had a calcite crystal structure of calcium carbonate, and no crystals derived from acetic acid such as ammonium acetate and calcium acetate were precipitated.

[0063] 2-1-3. Removal of biopolymers by NaClO After acid treatment with 900 mM ammonium acetate buffer at pH = 3.7, the SEM image of the eggshell after decomposing organic matter with 500 mL of 5 wt% aqueous NaClO solution is shown in Fig. 12, and the results of measuring the organic matter content by thermogravimetric analysis are shown in Fig. 13. Note that the thermogravimetric analysis shown in Fig. 13 shows the results of heating at a rate of 5 °C / min.

[0064] From Fig. 12, it was confirmed that the uniform porous structure obtained by acid treatment was maintained without being lost even after NaClO treatment. Regarding Fig. 13, it was determined that up to 200 °C was the moisture contained in the particles, 200 °C to 585 °C was the organic matter, and the weight loss after 585 °C was due to the combustion of CaCO3. The raw eggshell powder contained 2.13% organic matter, but it increased to 2.75% after acid treatment. This is presumably due to the ammonium acetate salt attached to the eggshell surface. On the other hand, when the acid-treated eggshell was treated with NaClO, a large weight loss near 300 °C was not observed, and the organic matter content decreased to 0.67%, confirming the successful removal of organic matter by NaClO.

[0065] 2-1-4. Modification of PMAcO Regarding the Eggshell-PMAcO particles prepared by modifying eggshells with PMAcO obtained by hydrolysis of PMAO, the dispersibility in water and methanol was observed. Also, the TG measurement results before and after modification of PMAcO are shown in Fig. 14.

[0066] The particles before PMAcO modification were dispersed in both water and methanol, but after PMAcO modification, they did not disperse in water, confirming that the particle surface became hydrophobic. Also, from Fig. 14, a significant weight loss was observed near 300 °C after PMAcO modification. The weight loss from 200 °C to 350 °C after PMAcO modification was 0.53%, while the weight loss from 200 °C to 350 °C before PMAcO modification was 0.13%. Therefore, the modification amount of PMAcO was determined to be 0.40%, which is the difference between 0.53% and 0.13%.

[0067] 2-2. Basic Evaluation of Eggshell-PMAcO 2-2-1. Comparison of Retention Behaviors To confirm the retention behaviors of the five types of columns shown in Table 1 above, the results of plotting with the ratio of water in the mobile phase on the horizontal axis and the retention coefficient obtained from the resulting chromatogram on the vertical axis are shown in Figure 15.

[0068] From Figure 15, for the EDTA column, Acetic acid column, and No acid column, a linear relationship was found between the water ratio in the mobile phase and the retention coefficient, indicating reverse-phase retention behavior. Also, since the retention coefficients of these three types of Eggshell-PMAcO columns are almost the same, it was shown that the presence or absence of acid treatment and the reagent used for acid treatment do not affect the retention power of the Eggshell-PMAcO column.

[0069] On the other hand, for the No PMAcO column, the retention coefficient hardly increased even when the water ratio in the mobile phase was increased, and no linear relationship was found between the water ratio in the mobile phase and the retention coefficient, so reverse-phase retention behavior was not observed. Therefore, it was shown that the reverse-phase retention behavior exhibited by the three types of Eggshell-PMAcO columns described above is due to the modification with PMAcO. When the water ratio in the mobile phase was 20%, a larger retention coefficient was obtained for the No PMAcO column than for the reverse-phase Eggshell-PMAcO column, which is considered to be due to the adsorption effect between the filler surface and the sample. While reverse-phase columns retain samples by the partition equilibrium between the stationary phase and the mobile phase, it is considered that adsorption equilibrium, rather than partition equilibrium, occurred on the surface of the stationary phase of the No PMAcO column.

[0070] In addition, for the ODS column, since a linear relationship holds between the water ratio in the mobile phase and the retention factor, the reversed-phase retention behavior was confirmed. When comparing under the condition that the water ratio in the mobile phase is the same, a larger retention factor was obtained for the ODS column than for the reversed-phase Eggshell-PMAcO column. However, by increasing the water ratio in the mobile phase compared to the ODS column, for example, when the retention factor of the ODS column at a water ratio of 15% in the mobile phase is similar to that of the Eggshell-PMAcO column at 50%, it can be said that Eggshell-PMAcO can retain the sample for a similar time as the ODS column.

[0071] 2-2-2. Comparison of van Deemter plots 2-2-2-1. Presence or absence of acid treatment Fig. 16 shows the results of comparing the separation ability of the Acetic acid column and the No acid column using van Deemter plots.

[0072] From Fig. 16, at all measured mobile phase linear velocities, the value of the theoretical plate height of the Acetic acid column was smaller than that of the No acid column. Thus, it was confirmed that the Acetic acid column has a higher separation ability.

[0073] From these results, it was shown that acid treatment has the effect of significantly improving the separation ability of the column. It is speculated that when acid treatment is performed, the particle surface becomes a uniform porous structure, and as a result of uniform surface modification of PMAcO, the dispersibility in the chloroform slurry during packing is improved, and the packing state is improved. Also, it is considered that the term A due to multi-channel diffusion among the elements constituting the value of the theoretical plate height became smaller because the particle size became more uniform by acid treatment.

[0074] 2-2-3. Quantitative analysis 2-2-3-1. Retention capacity To investigate the retention volume of the Acetic acid column prepared in this example, that is, the maximum injection volume of the sample that the column can hold at one time, the sample concentration was fixed at 20 mg / mL, and the injection volume was increased up to 100 μL, which is the maximum injection volume of the injector. The obtained chromatogram is shown in Figure 17.

[0075] From Figure 17, it was confirmed that the sample was retained without the peak shape collapsing when the sample injection volume was up to 2 mg. Therefore, it was determined that a larger amount of sample needed to be injected to determine the retention volume. Thus, the sample injection volume was fixed at 100 μL, which is the maximum injection volume of the injector, and the sample concentration was increased from 20 mg / mL to 50 mg / mL. The obtained chromatogram is shown in Figure 18. In Figure 18, the lower the sample concentration, the lower the peak.

[0076] In the chromatogram up to an injection volume of 2.0 mg shown in Figure 17, no peak tailing was observed, and all the samples injected within about 6 minutes from the start of measurement were eluted. On the other hand, in the chromatogram shown in Figure 18, significant peak tailing was observed when the injection volume was 3.0 mg or more, and some of the injected samples remained in the column even after 8 minutes from the start of measurement. Therefore, it was confirmed that the partition equilibrium due to hydrophobic interaction was disrupted. Thus, it was determined that 2.0 mg, which retained the sample while maintaining the peak shape without disrupting the partition equilibrium, was the retention volume of the Acetic acid column.

[0077] 2-2-3-2. Quantitativeness The quantitativeness of the analysis results obtained when injecting a sample in an amount within the retention volume of 2.0 mg into the Acetic acid column was confirmed. Figure 19 shows a calibration curve created with the sample injection volume on the horizontal axis and the peak area of the chromatogram on the vertical axis.

[0078] From Figure 19, since a linear relationship was established between the sample injection volume and the peak area, it was confirmed that the analysis results within an injection volume of 2.0 mg were quantitative.

[0079] 2-2-3-3. Reproducibility The reproducibility of the analysis results obtained when injecting a sample in an amount not exceeding the retention volume into the Acetic acid column was confirmed. The same amount of the sample was continuously injected 6 times, and the obtained chromatograms are shown in Fig. 20.

[0080] From Fig. 20, no significant differences were observed in the retention times and shapes of the chromatograms obtained by continuously injecting the sample 6 times. Since the RSD of the retention times obtained from the 6 chromatograms was 0.118% and the RSD of the theoretical plate height was 0.489%, the reproducibility of the analysis results of the Acetic-Acid column was shown.

[0081] As described above, since the analysis results of the Acetic acid column have quantitativeness and reproducibility, it was confirmed that the Acetic acid column is a column that can be used for quantitative analysis.

[0082] 2-3. Separation of Basic Compounds 2-3-1. Retention Behavior of Basic Compounds The mixing ratio of the aqueous TEA solution and methanol in the mobile phase was changed, and the chromatograms obtained by separating two basic compounds, Imipramine and Clomipramine, are shown in Fig. 21. Also, the resolution of each peak obtained from the chromatogram of Fig. 21 is shown in Table 2. Furthermore, the relationship between the ratio of the aqueous TEA solution and the retention coefficient obtained from the chromatogram of Fig. 21 is shown in Fig. 22.

[0083] [Table 2]

[0084] As shown in Table 24, the resolution determined from the chromatogram shown in Fig. 21 was 1.5 or more when the ratio of the TEA aqueous solution was 50% or more, and complete separation was achieved. Further, from Fig. 22, since a linear relationship holds between the ratio of the TEA aqueous solution in the mobile phase and the retention factor, in the retention of basic compounds under alkaline conditions as well, the reverse-phase retention behavior was shown, similar to the retention of hydrophobic compounds under neutral conditions.

[0085] 2-3-2. Retention Capacity of Basic Compounds From the examination of the retention behavior in 2-3-1, it was confirmed that when the ratio of the TEA aqueous solution in the mobile phase was 50% or more, complete separation of the two basic compounds was achieved, and that the resolution of the two peaks increased by increasing the ratio of the TEA aqueous solution. Therefore, the retention capacity was examined for the case where the ratio of the TEA aqueous solution was 50% where the sharpest peak was obtained and separation in a short time was possible, and for the case where the ratio of the TEA aqueous solution was 60% where, although the elution time was long, the elution times of the two peaks were further apart. The chromatograms obtained when the ratio of the TEA aqueous solution was 50% and 60% are shown in Fig. 23 and Fig. 24, respectively. Also, the resolution determined from the chromatograms of Fig. 23 and Fig. 24 is shown in Table 3. In Fig. 23 and Fig. 24, the peaks become higher in the order of higher concentration.

[0086]

Table 3

[0087] From Table 3, when the ratio of the TEA aqueous solution was 50%, although the resolution was 1.5 or more and complete separation was achieved for injections up to 2.5 mg each shown by the thick green line in Fig. 4-27, when it was 5.0 mg or more for each, the two peaks overlapped and the resolution was less than 1.5. On the other hand, when the ratio of the TEA aqueous solution was 60%, although the elution took 60 minutes, a long time, the resolution was 1.5 or more and complete separation was achieved for injections up to 5.0 mg each shown by the thick line in Fig. 24.

[0088] As described above, it was shown that Eggshell-PMAcO can separate basic compounds up to 5.0 mg each, achieving complete separation of basic compounds with a weight 500 times that of Example 1, which only achieved separation of 0.01 mg each.

[0089] In particular, the peak of clomipramine when the ratio of the TEA aqueous solution is 60% is significantly tailing and it takes 60 minutes for elution. However, by performing gradient elution to increase the methanol ratio in the mobile phase during the analysis, it may be possible to improve the peak shape and achieve high-speed separation in a shorter time.

[0090] Example 3 Various physical properties As a result of measuring the particle size distribution by a particle size distribution analyzer using EDTA and the acid treatment method, for untreated eggshells, it was 6.0 ± 3.0 μm (RSD: 51%), for those treated with EDTA, it was 8.4 ± 3.5 μm (RSD: 41%), and for those treated with ammonium acetate, it was 8.7 ± 3.5 μm (RSD: 40%). The treatment conditions with EDTA were the same as the method of "2. EDTA treatment" in Example 1, and the treatment conditions with acetic acid buffer were the same as the method of "2-1-2. Change in pH" in Example 2. The results are shown in FIGS. 25 and 26.

[0091] As shown in FIGS. 25 and 26, by performing EDTA treatment or acid treatment, the particle size increased and the distribution became narrower. This result is considered to be due to the dissolution of eggshells with a very small particle size during the treatment, resulting in an increase in the average particle size and a narrower distribution due to a decrease in small particles. Since a more uniform particle size leads to higher column separation ability, an improvement in the performance as a filler can be expected by EDTA and acid treatment.

[0092] Furthermore, each SEM image is shown in Fig. 27. The particle size distribution was evaluated from the SEM images in Fig. 27 using ImageJ. For the untreated sample, it was 3.2 ± 3.5 μm (RSD: 109%); for the EDTA-treated sample, it was 8.8 ± 5.2 μm (RSD: 59%); and for the ammonium acetate-treated sample, it was 9.2 ± 5.2 μm (RSD: 56%) (Table 4). For the untreated sample, the particle size of around 1 - 2 μm was the most abundant, and there were also many particles with a size of less than 1 μm. In contrast, by treating with EDTA or ammonium acetate, similar to the measurement results with the particle size analyzer, the very small eggshells dissolved, resulting in an increase in particle size and a narrower distribution.

[0093] As described above, it was confirmed that both from the particle size analyzer and the SEM images, treating with EDTA and acid treatment increased the particle size and narrowed the distribution.

[0094] [Table 4]

[0095] The surface area, pore volume, and pore diameter by EDTA and acid treatment methods were measured by mercury intrusion porosimetry. The results are shown in Table 5 below and Fig. 28. With mercury intrusion porosimetry, larger pores can be measured compared to the nitrogen adsorption method. The pores of about 1 μm in size are the most abundant, which is due to the gaps between the eggshell particles. Those with a size of 500 nm or more are due to the gaps and have nothing to do with the macropores of the eggshell. Therefore, when only focusing on the size range of 1 nm - 500 nm, it can be confirmed that the volume is larger for those treated with EDTA and acid treatment compared to the untreated and the case of only using NaClO.

[0096] It can be said that acid treatment and EDTA treatment expanded the pores less than 500 nm. In chromatography, pores on the nm order affect the separation ability, so it is considered to affect the improvement of the performance as a packing material.

[0097] [Table 5]

[0098] As can be seen from Table 5 and Figure 28, in pores less than 500 nm, it can be seen that acid treatment results in more pore expansion than EDTA treatment. Also, treatment with NaClO causes a slight expansion. When only NaClO is treated, a slight expansion can be confirmed at 100 nm or less. This is an expansion at a pore size different from that of EDTA and acid treatment, and is considered to be due to the removal of organic substances. That is, EDTA and acid expand macropores by dissolving calcium carbonate, while NaClO expands mesopores by removing organic substances.

[0099] From these results, it can be expected that treatment with EDTA and acid can expand pores less than 500 nm and dissolve fine eggshell powder, thereby improving the performance as a filler.

[0100] XRD The crystal structure of the eggshell powder before and after treatment was evaluated by an X-ray diffractometer (XRD). The results are shown in Figure 29. It was confirmed that the eggshell powder has a calcite crystal structure and that there is no change in the calcite crystal structure even after NaClO treatment, EDTA treatment, acid treatment, and PMAcO modification reaction. Therefore, it was confirmed that all treatments do not affect the crystal structure. Also, peaks were obtained at the following numerical values. 23.1, 26.5, 29.4, 31.5, 36.0, 39.4, 43.2, 47.3, 47.5, 48.6, 56.8, 57.4, 58.4 2θ / degree

[0101] The modification of PMAcO was evaluated by FT-IR. The results are shown in Figures 30 and 31.

[0102] As shown in Figures 30 and 31, since a peak derived from the alkyl of PMAcO at 2924 cm -1 was confirmed after the modification of PMAcO, the modification of PMAcO was confirmed.

Claims

1. A column packing material for liquid chromatography, which is composed of crushed eggshells or eggshells that have been treated with a calcium remover and a biopolymer remover, and has a porous structure formed from calcium carbonate.

2. The packing material according to claim 1, which is composed of eggshells.

3. The packing material according to claim 1 or 2, wherein the calcium remover is a calcium chelating agent or an acid.

4. The packing material according to claim 3, wherein the calcium remover is a calcium chelating agent, and the calcium chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid or a salt thereof, glycol ether diamine tetraacetic acid or a salt thereof, and (1,2-bis(o-aminophenoxide)ethane-N,N,N',N'-tetraacetic acid) or a salt thereof.

5. The packing material according to claim 4, wherein the calcium chelating agent is ethylenediaminetetraacetic acid or a salt thereof.

6. The packing material according to any one of claims 1 to 5, wherein the biopolymer removal treatment is an oxidizing agent treatment or a heat treatment.

7. The packing material according to claim 6, wherein the biopolymer removal treatment is an oxidizing agent treatment, and the oxidizing agent is at least one selected from the group consisting of hypochlorous acid or a salt thereof, oxygen, ozone, and hydrogen peroxide.

8. The packing material according to claim 7, wherein the oxidizing agent is hypochlorous acid or a salt thereof.

9. The packing material according to any one of claims 1 to 8, wherein the number average particle diameter of the eggshells or eggshells is 1 μm to 200 μm.

10. The packing material according to any one of claims 1 to 9, which has an organic group on the surface.

11. The packing material according to claim 10, wherein the organic group is a hydrophobic group, and the chromatography is reverse phase chromatography.

12. The packing material according to claim 11, wherein the hydrophobic group is an alkyl group having 1 to 30 carbon atoms.

13. The packing material according to any one of claims 1 to 12, wherein the 2θ peak value of X-ray diffraction coincides with the 2θ peak of calcite X-ray diffraction except for the 2θ peak value of 26.5°.

14. The packing material according to claim 1, wherein the calcium remover is a weak acid or a weak acid buffer solution containing a weak acid and a salt.

15. The packing material according to claim 14, wherein the weak acid is acetic acid.

16. In X-ray diffraction analysis, the 2θ peak value is present at 26.5°, and outside this 26.5° 2θ peak value, the 2θ peak value coincides with the 2θ peak value of the X-ray diffraction of calcite. When the temperature is raised at 5 °C / min, the weight loss from 200 °C to 585 °C is 1 wt% or less, and the cumulative pore volume of pores with a diameter of less than 500 nm measured by mercury intrusion porosimetry is 0.05 mL / g or more. A column packing material for liquid chromatography.

17. A method for producing the column packing material for liquid chromatography according to claim 1, comprising treating the crushed eggshell or shell with a calcium removing agent and a biopolymer removing treatment.

18. The method according to claim 17, comprising treating the crushed eggshell or shell with a calcium removing agent and then treating it with an oxidizing agent.

19. The method according to claim 17 or 18, further comprising bonding an organic group to the surface of the eggshell or shell.

20. The method according to claim 19, comprising bonding an organic group to the surface of the eggshell or shell by bonding a polymer having a plurality of the organic groups to the surface of the eggshell or shell.

21. The method according to claim 19 or 20, wherein the organic group is a hydrophobic group and the chromatography is reverse phase chromatography.

22. The method according to claim 21, wherein the hydrophobic group is an alkyl group having 1 to 30 carbon atoms.

23. Use of the eggshell or shell according to any one of claims 1 to 16 as a column packing material for liquid chromatography.

24. Use of the eggshell or shell described in any one of claims 1 to 16 for the production of a column packing material for liquid chromatography.

25. Liquid chromatography, comprising applying a sample to a column filled with the column packing material for liquid chromatography according to any one of claims 1 to 16.

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