JP5080413B2 - Method for producing coated particles - Google Patents

Description

  The present invention relates to a method for producing coated particles.

  For example, a calcium phosphate compound has a structure in which calcium ions and phosphate groups are regularly arranged at high density, and has an adsorption ability based on electrostatic interaction as an amphoteric ion exchanger. For this reason, particles composed of this calcium phosphate compound (calcium phosphate compound particles) are widely used as adsorbents for separation columns that separate biologically related substances such as proteins, nucleotides, nucleic acids, and cells (for example, (See Patent Document 1).

  In such calcium phosphate compound particles, for example, when separating proteins, if the protein is an acidic protein, the carboxyl group contained in the structure is coordinated and adsorbed to the calcium ions of the calcium phosphate compound particles, Alternatively, when the protein is a basic protein, the amino group contained in the structure is ion-bonded and adsorbed to the phosphate group of the calcium phosphate compound particle. Therefore, proteins are separated based on the difference in the adsorption capacity of each protein with respect to the calcium phosphate compound particles.

  However, since the calcium phosphate compound has a smaller positive charge due to calcium ions than a negative charge due to a phosphate group, the adsorption ability to adsorb a substance such as an acidic protein having a negative charge is small.

  Therefore, in columns (adsorbers) equipped with calcium phosphate compound particles as adsorbents, I) the recovery rate of adsorbed materials with negative charges contained in the sample solution is low, and II) adsorption with negative charges in the sample solution. When a plurality of substances are contained, each adsorbate elutes almost simultaneously at a relatively early stage, resulting in a problem that a sufficient resolution cannot be obtained.

Japanese Patent Laid-Open No. 10-153588

  As a result of intensive studies by the present inventors for the purpose of solving such problems, the coated particles obtained by coating the surface of the calcium phosphate compound particles with a polyamine compound having a positive charge are positively charged. Therefore, it is found that the coated particles having excellent adsorption ability and separation ability can be obtained regardless of the charged state of the adsorbate, and the surface of the calcium phosphate compound particles is coated with a polyamine compound. The inventors have already filed for the particles.

  Such coated particles can be produced by preparing a dispersion containing calcium phosphate compound particles and a polyamine compound and heating the dispersion. There is a demand for a manufacturing method in which the bonding can be stably performed and manufacturing can be performed with a higher yield.

  Such a problem occurs not only in the case of calcium phosphate compound particles having at least the vicinity of the surface composed of calcium phosphate compound, but also in various particles.

  Therefore, an object of the present invention is to provide a method for producing coated particles, which can produce coated particles that can stably obtain excellent adsorption ability and separation ability regardless of the charged state of the adsorbate. is there.

Such an object is achieved by the present invention described in ( 1 ) below.
(1) A method for producing a coated particle in which the surface of the particle is coated with a polyamine compound,
After preparing a dispersion containing the particles and the polyamine compound, the dispersion is supplied as a droplet into a heated atmosphere, whereby the droplet is heated to form the polyamine compound on the surface of the particle. A method for producing coated particles, characterized in that:

  Thereby, irrespective of the charge state of the adsorbate, coated particles that can stably obtain excellent adsorption ability and separation ability can be produced with high yield.

  When producing coated particles in which the surface of the particles is coated with a polyamine compound, a dispersion containing these particles and the polyamine compound is supplied as a droplet in a heated atmosphere. If it is set as the structure which obtains the coating particle by which the surface of particle | grains was coat | covered with the polyamine type compound by heating, the coupling | bonding of the polyamine type compound with respect to the surface of particle | grain will be performed stably. Furthermore, since the obtained coated particles are produced without forming aggregates, the yield of the obtained coated particles can be improved.

  Hereinafter, the manufacturing method of the coated particle | grains of this invention is demonstrated in detail based on suitable embodiment shown to an accompanying drawing.

<Coated particles>
First, the coated particles produced by the method for producing coated particles of the present invention will be described.

  The coated particles produced by the method for producing coated particles of the present invention function as an adsorbent that adsorbs substances (adsorbents) such as proteins, nucleotides, nucleic acids, and biological substances such as cells on the surface thereof. It is. Such coated particles are used, for example, as a packing material (adsorbent) for a separation column that separates a plurality of types of adsorbate contained in a sample liquid in a form packed in a column of an adsorption device.

  In this case, each adsorbate contained in the sample liquid is adsorbed on the coated particles by allowing the sample liquid containing plural kinds of adsorbates to pass through the column. When the eluate is passed through the column, each adsorbate adsorbed on the coated particles is eluted with an elution time based on the difference in the adsorptivity to the coated particles and the difference in the affinity for the eluate. Therefore, each adsorbate is separated by fractionating the eluate that has passed through the column at predetermined time intervals.

  The coated particle used as such an adsorbent is a particle (carrier) whose surface is coated with a polyamine compound.

The particle is a part constituting the skeleton of the coated particle.
The constituent material of the particles is not particularly limited, and examples thereof include various ceramic materials such as calcium phosphate compounds, alumina, zirconia, and silica, carbon black, cellulose, diamond, and various resin materials.

  As the resin material, various thermosetting resins and various thermoplastic resins can be used. Specifically, as the thermoplastic resin, for example, polyamide (for example, nylon 6, nylon 6,6, nylon 6).・ 10, nylon 12), polyethylene, polypropylene, polystyrene, polyimide, acrylic resin, thermoplastic polyurethane, etc., as thermosetting resin, for example, epoxy resin, phenol resin, melamine resin, urea resin, unsaturated polyester, alkyd resin , Thermosetting polyurethane, and ebonide, and one or more of these can be used in combination.

  Among these, it is preferable that at least the vicinity of the surface of the particles is mainly composed of a ceramic material. Ceramic materials are suitable for use as an adsorbent skeleton for adsorbing various substances to the adsorbent because they have excellent adsorbability for various substances such as biological substances. When a positive charge is further imparted to the skeleton of such an adsorbent, the method for producing coated particles of the present invention is suitably applied.

  Further, as the ceramic material, a calcium phosphate compound is particularly preferable.

The calcium phosphate compound is represented by Ca ₁₀ (PO ₄ ) ₃ X ₂ and has a Ca / P ratio of 1.0 to 2.0, and calcium ions and phosphate groups are regularly arranged at high density. It has an arrayed structure and has an adsorption ability based on electrostatic interaction as an amphoteric ion exchanger. For this reason, particles composed mainly of calcium phosphate-based compounds at least near the surface have excellent adsorption ability for various substances having positive or negative charges. Therefore, various adsorbents such as biological substances on the surface. It is preferably used as a skeleton of an adsorbent that adsorbs benzene.

  As described above, when a positive charge is further imparted to the calcium phosphate compound having an adsorption ability based on electrostatic interaction as an amphoteric ion exchanger, the adsorption ability for various substances having a negative charge is further improved. The method for producing coated particles of the invention is preferably applied.

Examples of calcium phosphate compounds include hydroxyapatite (Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ), fluorapatite (Ca ₁₀ (PO ₄ ) ₆ F ₂ ), and chlorapatite (Ca ₁₀ (PO ₄ ) ₆ Cl _2. ), Fluorinated carbonate apatite (Ca ₁₀ (PO ₄ , CO ₃ ) ₆ F ₂ ), carbonated hydroxyapatite (Ca ₁₀ (PO ₄ , CO ₃ ) ₆ (OH) ₂ ), etc. Can be mixed and used.

Among them, the calcium phosphate-based compound, hydroxyapatite _{(Ca 10 (PO 4) 6} (OH) 2) as a main component is preferable. Hydroxyapatite is widely used as a biomaterial because it has an excellent affinity for biomaterials. Therefore, when a biomaterial is selected as the adsorbent, the adsorbate adsorbs on the coated particles very efficiently. It becomes like this. Further, since hydroxyapatite has a particularly low possibility of damaging the biological substance, the biological substance (adsorbing substance) can be adsorbed in a state where it is not altered or deteriorated.

  These calcium phosphate compounds can be synthesized by a known wet synthesis method, dry synthesis method, or the like. In this case, the calcium phosphate compound may contain a substance (raw material, etc.) remaining during the synthesis or a secondary reaction product generated during the synthesis.

  In the case where the particles are at least the surface of which is composed of a calcium phosphate compound, the particles may be composed entirely of a calcium phosphate compound. As shown in FIG. The surface of the particulate substrate 210 composed of, for example, a particle 200 in which the surface is coated with a calcium phosphate compound layer 220 composed mainly of a calcium phosphate compound may be used. If the particles are as shown in FIG. 1, the shape, size (average particle size, etc.), physical properties (density, etc.), etc. of the particles can be easily adjusted.

  Examples of the resin material constituting the substrate 210 include the same materials as described above, and one or more of them can be used in combination.

  Further, as shown in FIG. 1, the calcium phosphate compound layer 220 is one of fine particles 230 (hereinafter simply referred to as “microparticles 230”) mainly composed of a calcium phosphate compound near the surface of the substrate 210. It is preferable that the portion is formed by penetration. Thereby, the adhesiveness of the calcium-phosphate type compound layer 220 and the base material 210 can be made excellent. For this reason, peeling from the surface of the base material 210 of the calcium-phosphate-type compound layer 220 can be prevented suitably, ie, the intensity | strength of particle | grains and covering particle | grains can be made excellent.

  In this case, the calcium phosphate compound layer 220 can be formed, for example, by colliding porous particles mainly composed of the calcium phosphate compound with the surface of the substrate 210. According to this method, the calcium phosphate compound layer 220 can be formed easily and reliably.

  Moreover, it is preferable that the average particle diameter of particle | grains exceeds 1 micrometer, it is more preferable that it is about 2-200 micrometers, and it is further more preferable that it is about 2-100 micrometers.

  If the average particle size of the particles is more than 1 μm, clogging of the filter members 600 and 700 is surely prevented when the coated particles coated with the polyamine compound are applied as an adsorbent included in the adsorption device 400 described later. can do. Further, the liquid easily flows between the coated particles, and the liquid can be prevented from staying in the adsorption device 400. As a result, the coated particles exhibit excellent separation ability.

  In addition, in the case of particles composed of a calcium phosphate compound at least near the surface, generally, the calcium ion and phosphate groups exposed on the surface have a calcium ion exposure amount compared to the phosphate group exposure amount. Show a tendency to decrease. That is, the positive charge due to calcium ions tends to be smaller than the negative charge due to phosphate groups. And such a tendency is recognized by the case where the particle size of particle | grains is comparatively enlarged like more than 1 micrometer.

  Therefore, if the surface of the particle is coated with a polyamine compound and the coated particle has a positive charge due to an amino group in addition to a positive charge due to calcium ions, a negative charge and a positive charge are caused on the surface of the coated particle. Can be exposed in a balanced manner. As a result, the coated particles exhibit excellent adsorption ability for both a negatively charged substance and a positively charged substance.

The particles having such a structure are coated with a polyamine compound.
In the following, an example will be described in which particles whose surface is composed of a calcium phosphate compound at least near the surface are coated with a polyamine compound.

  When the particle surface is coated with a polyamine compound in this way, the amino group contained in the polyamine compound has a positive charge, and the phosphate group is exposed on the particle surface. Are bonded to phosphate groups, and those other than those bonded to phosphate groups are exposed on the surface of the coated particles. Therefore, at least the surface of the coated particle composed of a calcium phosphate compound has a positive charge due to an amino group in addition to a positive charge due to calcium ions, so a substance such as an acidic protein or nucleotide having a negative charge ( Adsorption ability to adsorbate is improved.

  In addition, since the charged state of the coated particles can be controlled by changing the coating amount of polyethyleneimine, the coating is performed so that the adsorptivity with respect to the charged state of the target adsorbate is optimal. The charge state of the particles can be adjusted. As a result, it is possible to obtain more excellent adsorption ability and separation ability for various adsorbates having different charge states.

  Examples of the polyamine compound include polyethyleneimine and polylysine. Among these, polyethyleneimine is particularly preferable.

  This polyethyleneimine is a polymer of ethyleneimine represented by the following chemical formula (1).

  Polyethyleneimines having such a structure include those in which ethyleneimine is linearly connected and those in which the ethyleneimine is connected in a branched form. For example, there are those represented by the following chemical formulas (2) and (3), respectively. However, in the present invention, it is preferable that they are connected in a branched manner.

［式中、ｍは２以上の整数を表す。］

[Wherein m represents an integer of 2 or more. ]

［式中、ｎは１以上の整数を表す。］

[Wherein n represents an integer of 1 or more. ]

  Since polyethyleneimine in which ethyleneimine is linked in a branched manner has a network structure as shown in chemical formula (3), when particles are coated, the particles are firmly coated with polyethyleneimine. be able to. Further, when an amino group is bonded to a phosphate group exposed on the surface of the particle, the calcium ion exposed on the surface of the particle can be exposed from the network structure without being covered. As a result, the coated particles can more reliably improve the adsorption ability for a negatively charged substance.

  In addition to the above-described compounds, the polyamine compound may be, for example, a polyethyleneimine derivative in which at least a part of hydrogen of polyethyleneimine is replaced with another element or atomic group.

  The average molecular weight of polyethyleneimine is preferably about 800 to 100,000, and more preferably about 5000 to 80,000. If the average molecular weight is too small, it is difficult to reliably cover the particles with polyethyleneimine, and it may be difficult to obtain a desired charged state of the coated particles. Moreover, when the average molecular weight exceeds the above upper limit, the particles may form a large agglomerate due to binding between polyethyleneimines, and it may be difficult to obtain coated particles having a large specific surface area.

  The coating amount of polyethyleneimine is preferably about 0.1 to 100 mg, more preferably about 0.5 to 50 mg, and further preferably about 0.5 to 15 mg with respect to 1 g of particles. When the coating amount is small, the effect of increasing the positive charge in the coated particles is small, and there is a possibility that the adsorbing ability with respect to an adsorbate having a particularly large negative charge cannot be sufficiently improved. Moreover, when there is much coating amount, there exists a possibility that it may become difficult for particles to make a big agglomerate by binding of polyethyleneimine and to obtain a covering particle with a large specific surface area.

  In addition, the coating amount of polyethyleneimine can be measured as follows, for example.

  First, 0.10 g of the coated particles and 20 mL of 1N sodium hydroxide solution are mixed, and shaken for 3 hours by vortexing for 15 seconds to obtain a suspension. Thereby, the polyethyleneimine which coat | covers a coating particle in a sodium hydroxide solution melt | dissolves.

  Next, the suspension is centrifuged, and the supernatant (stock solution) is collected. And a some dilution liquid is prepared by diluting a part of this stock solution in steps.

  Next, 20 μL of each of the stock solution and each diluted solution and 180 μL of CBB reagent (protein staining reagent) are mixed and allowed to stand for 20 minutes. Thereafter, the absorbance at 630 nm is measured for each sample, and a correlation diagram between the dilution factor and the absorbance is obtained.

  On the other hand, CBB staining and absorbance measurement are similarly performed on a polyethyleneimine solution (standard solution) having a known concentration and a plurality of dilutions obtained by serially diluting the polyethyleneimine solution, and the relationship between the dilution ratio and the absorbance is shown. Obtain a calibration curve.

  Then, the ratio between the slope of the calibration curve and the slope of the correlation diagram in the sample is obtained, and based on this, the amount of polyethyleneimine in the stock solution and the amount of polyethyleneimine per gram of the coated particles are calculated.

<Method for producing coated particles>
Next, the manufacturing method of the covering particle | grains of this invention which manufactures the covering particle | grain mentioned above is demonstrated.

  In the present embodiment, a case where coated particles are manufactured by coating the surface of particles (hydroxyapatite particles) entirely composed of hydroxyapatite with polyethyleneimine will be described as an example.

  Here, prior to describing the method for producing coated particles of the present invention, a spray dryer apparatus used in the method for producing coated particles of the present invention will be described.

  2 is a cross-sectional view showing the structure of a spray dryer apparatus used when the particle surface is coated with a polyamine-based compound, and FIG. 3 is a partial cross-section for illustrating the structure of a spray plate provided in the spray dryer apparatus shown in FIG. FIG. In the following, for convenience of explanation, the upper side in FIGS. 2 and 3 is referred to as “upper” and the lower side is referred to as “lower”.

<< Spray dryer device >>
The spray dryer apparatus 1 shown in FIG. 2 is installed in the heating chamber 2, the heating chamber 2, and rotates the spray disc 4 for supplying the dispersion liquid as droplets into the heating chamber 2. Rotating unit 9, storage tank 6 that stores the dispersion, liquid supply unit 3 that supplies the dispersion medium from the storage tank 6 to the spray board 4, and an air supply unit that supplies heated gas into the heating chamber 2 7 and an exhaust portion 8 for exhausting gas from the heating chamber 2.

  The heating chamber 2 has a cylindrical shape as a whole, and the inner diameter of the heating chamber 2 is almost constant toward the upper end or the lower end in the vertical direction, but gradually decreases from the middle toward the lower end. It is the structure opened by the through-hole 21 provided in. In the heating chamber 2 having such a configuration, when the dispersion is supplied as droplets from the spray disc 4, the surface of the hydroxyapatite particles contained in the dispersion is made of polyethyleneimine due to the dispersion being heated. The coated particles are formed by removing the dispersion medium, and the coated particles (powder) are collected from the through holes 21 by being stored under the heating chamber 2 by their own weight.

  As shown in FIG. 2, an air supply unit 7 that supplies heated gas into the heating chamber 2 is connected to the heating chamber 2.

  The air supply unit 7 includes a blower 71 that supplies gas (for example, air), a supply line 72 that guides the gas into the heating chamber 2, an opening 73 in which the supply line 72 opens in the vicinity of the spray board 4, and a gas It comprises a filter 74 for removing the dust inside, and the heated gas can pass through the vicinity of the spray platen 4 and be supplied into the heating chamber 2. Thereby, the droplet ejected from the spray disc 4 can be directly heated, and the coated particles can be obtained as a dry matter at an early stage.

  And the heating means 5 provided with the heater 51 is provided in the middle of the supply line 72 of this air supply part 7, The gas supplied by the air supply part 7 can be heated by supplying with electricity to the heater 51. It is like that. By setting it as this structure, since the gas supplied in the heating chamber 2 can be supplied in a high temperature state, the inside of the heating chamber 2 can be made into the state heated to high temperature.

  The heating chamber 2 is connected to an exhaust unit 8 including an exhaust line 81 and a storage chamber 82. Thereby, an excessive amount of gas supplied from the air supply unit 7 can be discharged to the outside through the exhaust unit 8. In the present embodiment, since the storage chamber 82 is provided in the middle of the exhaust line 81, fine coated particles mixed in the gas exhausted from the exhaust unit 8 are retained in the storage chamber 82 and collected. can do. Therefore, the discharge of the powder to the outside of the heating chamber 2 can be reliably prevented.

  In addition, a spray board 4 is installed on the upper side inside the heating chamber 2. A storage tank 6 is connected to the spray board 4 through the liquid feeding unit 3 so that the dispersion liquid can be supplied to the spray board 4. Yes.

  The liquid feeding unit 3 is provided in the middle of the liquid feeding line 32 and a liquid feeding line (pipe) 32 configured so that one end is connected to the storage tank 6 and the other end can supply the dispersion liquid to the spray board 4. And a high-pressure pump 31 for supplying the dispersion liquid to the spray disc 4. As shown in FIG. 3, the other end of the liquid feed line 32 (the end on the spray disc 4 side) supplies the dispersion liquid to the inside of the spray disc 4 through an opening 44 of the spray disc 4 described later. It is arranged in such a position that it can. With this configuration, the dispersion liquid stored in the storage tank 6 can be supplied to the spray board 4 via the liquid feeding line 32 by the operation of the high-pressure pump 31, and the dispersion liquid is sprayed as droplets. It can be supplied into the heating chamber 2 from the panel 4. At this time, since the inside of the heating chamber 2 is heated to a high temperature, the hydroxyapatite particles contained in the dispersion liquid are rapidly heated because the dispersion liquid supplied as droplets is rapidly heated. Are coated with polyethyleneimine, and the dispersion medium is removed to form coated particles.

  The rotating unit 9 is connected to a bearing 93 fixed to the heating chamber 2, a rotating shaft 92 extending upward from the center of the spraying plate 4 through the bearing 93, and an upper end of the rotating shaft 92. And a motor 91 that rotates the shaft 92 around its axis. By rotating the motor 91, the spray platen 4 connected to the lower end of the motor 91 is rotated.

  Further, in the present embodiment, as shown in FIG. 3, the liquid feeding line 32 is disposed so that the dispersion liquid can be injected into the opening 44 of the spray disc 4 through the inside of the bearing 93. Thereby, the dispersion liquid is supplied to the inside (gap) of the spray board 4, and the dispersion liquid is supplied (sprayed) into the heating chamber 2 as droplets by the rotation of the spray board 4.

  The spray platen 4 makes the dispersion liquid supplied from the storage tank 6 via the liquid feeding unit 3 centrifugal force is applied to the dispersion liquid by the rotation, and the dispersion solution is scattered from the outer peripheral direction of the spray platen 4 to form droplets. By doing so, it sprays in the heating chamber 2 as a droplet.

  As shown in FIG. 3, the spray disc 4 has a plurality of concentric upper discs 41 and lower discs 42 (a pair of discs) and a plurality of the discs 4 disposed on the outer periphery of the pair of discs 41, 42. Columnar body (spacer) 43.

  The upper disk 41 and the lower disk 42 are respectively fixed to the rotating shaft 92 at their central portions. In addition, the upper disk 41 is provided with a concentric opening 44, and through the opening 44, a gap formed between the upper disk 41 and the lower disk 42 is formed. A dispersion is supplied. In the present embodiment, an injection portion into which the dispersion liquid is injected is constituted by the gap.

  The columnar bodies 43 are arranged on the outer peripheral portions of the discs 41 and 42 at substantially equal intervals, thereby connecting the discs 41 and 42 and forming the ejection holes 45 between the columnar bodies 43. Thereby, when a centrifugal force is applied to the dispersion liquid supplied into the gap by the rotation of the disks 41 and 42, the dispersion liquid is subdivided by the columnar body 43 when pushed out from the spray disc 4. Thus, since the liquid is scattered from the outer peripheral direction of the discs 41 and 42 into droplets, they are ejected (sprayed) as droplets from the ejection holes 45.

  By using the spray dryer apparatus 1 configured as described above, the dispersion liquid can be supplied into the heating chamber 2 as droplets. By heating the droplets in the heating chamber 2, the hydroxyapatite particles Coated particles whose surfaces are coated with polyethyleneimine can be produced.

  Hereinafter, the manufacturing method which manufactures a covering particle using the spray dryer apparatus of this composition is explained.

  In the following, it is assumed that particles composed entirely of hydroxyapatite are produced using a wet synthesis method.

[S1: Step of Obtaining Slurry Containing Hydroxyapatite Aggregates]
In this step, calcium hydroxide (first raw material) and phosphoric acid (second raw material) are reacted with stirring to obtain a slurry containing hydroxyapatite aggregates.

  Specifically, in a container (not shown), an aqueous phosphoric acid solution is dropped and mixed while stirring a liquid containing calcium hydroxide.

  In this embodiment, a wet synthesis method using phosphoric acid as an aqueous solution is used. This makes it possible to synthesize hydroxyapatite (synthetic product) more easily and efficiently without requiring expensive production equipment.

  In the present invention, at least one of the first raw material and the second raw material may be used as a solution, or both may be used as a solution.

  Moreover, by carrying out this reaction with stirring, the reaction between calcium hydroxide and phosphoric acid can proceed efficiently, that is, the efficiency of those reactions can be improved.

[S2: Step of drying slurry to obtain hydroxyapatite powder]
In this step, the slurry obtained in the step [S1] is dried to obtain a powder.

  As the drying method, a spray drying method is preferably used. According to this method, a powder having a desired particle diameter can be obtained more reliably and in a short time.

  Through the steps as described above, hydroxyapatite (synthetic product) granules (hydroxyapatite particles) are obtained. The hydroxyapatite obtained by spray drying may be fired at about 150 to 1200 ° C, preferably about 200 to 800 ° C.

[S3: Step of coating the surface of hydroxyapatite particles with polyethyleneimine]
In this step, a particle / polyethyleneimine dispersion (preparation solution) is prepared by mixing the hydroxyapatite particles obtained in the step [S2], polyethyleneimine, and a dispersion medium (liquid). Thereafter, the dispersion is heated by supplying it as droplets into the heating atmosphere (heating chamber 2) to coat the surface of the hydroxyapatite particles with polyethyleneimine and remove the dispersion medium, thereby covering the particles. Is obtained in the heating chamber 2 as a dried product.

First, a polyethyleneimine dispersion is prepared.
Here, the method of adjusting the particle / polyethyleneimine dispersion, that is, the method of mixing the hydroxyapatite particles, polyethyleneimine, and the dispersion medium is not particularly limited. I) Hydroxyapatite particles and polyethyleneimine in the dispersion medium II) a method of adding hydroxyapatite particles to the dispersion medium and then adding polyethyleneimine to the dispersion medium; and III) a method of adding polyethyleneimine to the dispersion medium and then adding hydroxyapatite particles to the dispersion medium. Of these, the method III) is preferred. In the method III), after the polyethyleneimine is dissolved in the dispersion, the hydroxyapatite particles are dispersed in the dispersion medium, so that the polyethyleneimine can be uniformly contacted with the hydroxyapatite particles. Each of the resulting coated particles can be uniformly coated with polyethyleneimine.

Hereinafter, the method III) will be described in detail as a representative.
First, polyethyleneimine is dissolved in a dispersion medium to prepare a polyethyleneimine solution.

  Thereafter, hydroxyapatite particles are added and dispersed in this polyethyleneimine solution, and if necessary, a dispersion medium is added and mixed to obtain a particle / polyethyleneimine dispersion.

  As described above, if the dispersion medium is further added after adding the hydroxyapatite particles, the dispersibility of the hydroxyapatite particles in the hydroxyapatite particles / polyethyleneimine dispersion medium is further improved. The coated particles can be more uniformly coated with polyethyleneimine.

  Although it does not specifically limit as a dispersion medium, It is preferable that at least one of water and alcohol is included, and water, alcohol, and these mixed solvents are used preferably. Thus, by heating the particle / polyethyleneimine dispersion, the hydroxyapatite and polyethyleneimine can be reacted, and the dispersion medium is removed from the particle / polyethyleneimine dispersion by heating at a relatively low temperature. be able to.

  Although it does not specifically limit as alcohol, For example, methanol, ethanol, isopropanol etc. are mentioned, Among these, it can use combining 1 type (s) or 2 or more types.

  Among these, the dispersion medium is preferably composed mainly of methanol. Hydroxyapatite and polyethyleneimine can be efficiently reacted by using a dispersion medium composed mainly of methanol. That is, the reaction between the phosphate group contained in hydroxyapatite and the amino group contained in polyethyleneimine can be efficiently advanced. As a result, the coverage with polyethyleneimine on the surface of the particles can be improved more reliably.

  Moreover, when using what comprised methanol as a main component as a dispersion medium, it is preferable that the content rate of methanol in a dispersion medium is 50 wt% or more, and it is more preferable that it is about 65-85 wt%. As a result, the effect obtained when methanol composed mainly of methanol is used as the dispersion medium.

  Furthermore, the coating amount of polyethyleneimine can be accurately controlled, and the surface of the particles can be reliably coated with polyethyleneimine with a desired coating amount.

  Moreover, since the usage-amount of the polyethyleneimine required in order to obtain a desired coating amount can be suppressed, material cost can be reduced.

  The mixing ratio (weight ratio) of particles and polyethyleneimine in the dispersion is slightly different depending on the type of the dispersion medium, the specific surface area of the particles, etc., but for example, the dispersion medium is composed mainly of methanol. Is preferably about 500: 1 to 10: 1, more preferably about 100: 1 to 50: 1. If the mixing ratio of polyethyleneimine is too small, it may be difficult to coat the surface of the particles with polyethyleneimine with a desired coating amount. Moreover, when there are too many mixing ratios of polyethyleneimine, there exists a possibility that it may become difficult for particles to make a big aggregate by binding of polyethyleneimine and to obtain a covering particle with a large specific surface area.

  Further, the content of the hydroxyapatite particles in the particle / polyethyleneimine dispersion is not particularly limited, but is preferably about 200,000 to 2,000,000 particles / mL, and preferably about 500,000 to 1,500,000 / mL. More preferred. As a result, when the particle / polyethyleneimine dispersion (droplet) is heated in the heating chamber 2 and the reaction between the hydroxyapatite and the polyethyleneimine is carried out, the polyethyleneimine is connected so that the adjacent hydroxyapatite particles are connected to each other. It is possible to reliably prevent the coupling. As a result, it is possible to suitably prevent the formation of aggregates of the coated particles.

  Next, the droplets are heated by supplying the particle / polyethyleneimine dispersion as droplets in the heating atmosphere. As a result, hydroxyapatite and polyethyleneimine react to obtain coated particles in which the hydroxyapatite particles are coated with polyethyleneimine, and the dispersion medium in the particles and polyethyleneimine is removed to heat the coated particles. Obtained as a dry product in the atmosphere.

  Thus, by setting it as the structure which supplies and heats as a droplet in a heating atmosphere, the reaction of a hydroxyapatite and a polyethyleneimine is performed stably, and while a hydroxyapatite particle is obtained, a coated particle is dried. In this case, it is possible to accurately suppress or prevent the formation of aggregates due to the aggregation of the coated particles, and the yield of the resulting coated particles can be improved.

  Such heating of the droplets in the heating atmosphere is easily performed by the above-described spray drying method using the spray dryer apparatus 1.

  Here, in order to supply the particle / polyethyleneimine dispersion as droplets into the heating chamber (heating atmosphere) 2 using the spray dryer apparatus 1, as described above, the spray platen 4 is rotated. The particle / polyethyleneimine dispersion liquid stored in the storage tank 6 is supplied to the spray board 4 through the liquid feeding section 3.

  When the particle / polyethyleneimine dispersion supplied to the spraying plate 4 is pushed out of the spraying plate 4 by the centrifugal force generated by the rotation of the spraying plate 4, it becomes a state of being subdivided by the columnar bodies 43. , 42 scatters from the outer peripheral direction to form droplets, and is ejected (sprayed) as droplets from the ejection hole 45. In such a droplet formation method using centrifugal force, droplets can be formed without applying a compressive force to the particles contained in the particle / polyethyleneimine dispersion. Therefore, it is possible to accurately suppress or prevent the particles from being destroyed.

  The rotational speed of the spray platen 4 when supplying the droplets into the heating chamber 2 is slightly different depending on the viscosity of the particle / polyethyleneimine dispersion, but is preferably about 3000 to 60000 rpm, and about 5000 to 50000 rpm. More preferably, it is about 10,000 to 40,000 rpm. The particle / polyethyleneimine dispersion can be reliably supplied as droplets over almost the entire heating chamber 2.

  In addition, the amount of droplets supplied into the heating chamber 2, that is, the amount of one droplet of the particle / polyethyleneimine dispersion is preferably about 0.1 to 8000 pL, more preferably about 1 to 400 pL. preferable. Thereby, it can suppress or prevent that the produced | generated covering particle aggregates and the aggregate of a covering particle is formed.

  Moreover, although the heating temperature which heats the droplet supplied in the heating chamber 2, ie, the temperature of a heating atmosphere, changes a little also with the kind of solvent, it is preferable that it is about 30-200 degreeC, and about 55-95 degreeC is preferable. It is more preferable that If the heating temperature is too low, the reaction between hydroxyapatite and polyethyleneimine becomes insufficient, and it may be difficult to coat the surface of the particles with polyethyleneimine with a desired coating amount. Moreover, when heating temperature is too high, there exists a possibility that a polyethyleneimine may change and deteriorate, and it is unpreferable.

[S4: Step of recovering coated particles]
Next, the coated particles in which the hydroxyapatite particles obtained in the step [S3] are coated with polyethyleneimine are collected from the heating chamber 2.

  Here, the coated particles obtained in the step [S3] are obtained as a dried product in the heating chamber 2, and are stored under the heating chamber 2 by their own weight. Further, the fine coated particles mixed in the gas exhausted from the exhaust unit 8 are also stored below the storage chamber 82 by its own weight. Therefore, in the present embodiment, the coated particles are collected through the through hole 21 and the through hole 83 provided in the heating chamber 2 and the storage chamber 82, respectively.

  Next, the collected coated particles are placed on a filter, and the coated particles are washed by passing distilled water through the coated particles and the filter. Thereafter, the coated particles are collected from the filter and dried using a desiccator or the like.

  This washing of the coated particles is preferably performed until the colored reaction is not confirmed when the coated water and the distilled water after passing through the filter react with the ninhydrin reagent. Thereby, the unreacted polyethyleneimine adhering to the coated particles can be surely removed. As a result, it is possible to prevent the remaining unreacted polyethyleneimine from impairing the adsorption ability and separation ability of the coated particles, or from altering the adsorbate. Furthermore, when the coated particles are applied to an adsorbent provided in an adsorbing device described later, it is possible to reliably prevent polyethyleneimine from leaking into the eluate.

  Through the steps described above, coated particles in which the surface of hydroxyapatite particles is coated with polyethyleneimine can be produced.

  In the present embodiment, the case where the particle / polyethyleneimine dispersion is used as a droplet and the droplet is supplied into a heated atmosphere using the above-described spray dryer apparatus has been described. The method for forming droplets is not particularly limited. For example, using a nozzle having an ejection hole, and supplying the dispersion liquid with pressure applied to the ejection hole, the dispersion liquid may be formed into droplets, or piezoelectric It is also possible to use a method in which a dispersion liquid is formed into droplets by using vibration of the piezoelectric element (inkjet method).

<Adsorption device>
Next, an adsorption apparatus in which the coated particles produced by the method for producing coated particles of the present invention is applied as an adsorbent will be described.

  FIG. 4 is a longitudinal sectional view showing an example of an adsorption device in which the coated particles produced by the method for producing coated particles of the present invention are applied as an adsorbent. In the following description, the upper side in FIG. 4 is referred to as “inflow side” and the lower side is referred to as “outflow side”.

  Here, when the target adsorbate is separated (purified), the inflow side refers to, for example, liquid such as sample liquid (liquid containing adsorbate), elution buffer (eluate), etc. On the other hand, the outflow side refers to the side opposite to the inflow side, that is, the side from which the liquid flows out from the adsorption device.

  The adsorption device 400 shown in FIG. 4 has a column 500, an adsorbent 100 filled with granular coated particles as an adsorbent, and two filter members 600 and 700.

  The column 500 includes a column main body 510 and caps (lid bodies) 520 and 530 attached to the inflow side end portion and the outflow side end portion of the column main body 510, respectively.

  The column main body 510 is composed of, for example, a cylindrical member. Examples of the constituent material of each part (each member) constituting the column 500 including the column main body 510 include various glass materials, various resin materials, various metal materials, various ceramic materials, and the like.

  Caps 520 and 530 are screwed into the inflow side end portion and the outflow side end portion of the column main body 510 in a state where the filter members 600 and 700 are disposed so as to block the inflow side opening and the outflow side opening, respectively. It is attached by the match.

  In the column 500 having such a configuration, an adsorbent filling space 560 is defined by the column main body 510 and the filter members 600 and 700. Then, at least a part of the adsorbent filling space 560 (almost full in the present embodiment) is filled with the coated particles of the present invention as the adsorbent 100.

  The volume of the adsorbent filling space 560 is appropriately set according to the volume of the sample solution, and is not particularly limited, but is preferably about 0.05 to 10 mL, more preferably about 0.5 to 2 mL with respect to 1 mL of the sample solution. .

  By setting the size of the adsorbent filling space 560 as described above and setting the size of the coated particles as described above, a plurality of types of adsorbents can be separated from each other reliably.

  Further, in a state where the caps 520 and 530 are attached to the column main body 510, the liquid-tightness between them is ensured.

  An inflow pipe 540 and an outflow pipe 550 are fixed (fixed) in a liquid-tight manner at substantially the centers of the caps 520 and 530, respectively. The liquid is supplied to the coated particles through the inflow pipe 540 and the filter member 600. Further, the liquid supplied to the coated particles passes between the coated particles (gap) and flows out of the column 500 through the filter member 700 and the outflow pipe 550. At this time, when the coated particles have an average particle size of more than 1 μm, a gap sufficient for the liquid to flow is formed between the coated particles. Therefore, the liquid can easily flow between the coated particles, and the liquid can be suitably prevented from staying in the column main body 510. In the course of the liquid passing through the column main body 510 in this way, the coated particles have an excellent adsorbing ability for both a negatively charged substance and a positively charged substance. The multiple types of adsorbate contained in the are surely separated based on the difference in adsorptivity to the coated particles and the difference in affinity to the buffer solution.

  Each of the filter members 600 and 700 has a function of preventing the coated particles from flowing out from the adsorbent filling space 560. These filter members 600 and 700 are, for example, non-woven fabrics and foams (sponge-like porous bodies having communication holes) made of synthetic resin such as polyurethane, polyvinyl alcohol, polypropylene, polyether polyamide, polyethylene terephthalate, polybutylene terephthalate, etc. ), Woven fabric, mesh or the like.

  The average pore diameter of each filter member 600, 700 is usually set to about 0.2 to 0.8 μm. Therefore, when the coated particles have an average particle size of more than 1 μm, in the adsorption device 400 provided with the coated particles as the adsorbent 100, the coated particles enter the pores of the filter members 600 and 700 and become clogged. Or the coated particles are surely prevented from flowing out through the filter members 600 and 700.

  In the adsorption apparatus configured as described above, since the coated particles have excellent adsorption ability for both negatively charged substances and positively charged substances, various substances are coated mainly by electrostatic interaction. Based on the difference in the adsorptivity to the adsorbent 100 and the difference in the affinity to the buffer solution, the particles can be separated from each other. In other words, both the negatively charged substance and the positively charged substance contained in the sample liquid can be reliably separated and recovered.

  In addition, for example, by changing the salt concentration of the elution buffer continuously or stepwise, multiple types of adsorbate can be separated with high resolution, so that a negatively charged substance is introduced into the eluate at an early stage. The problem of leakage is also eliminated.

  When the coated particles are almost fully filled in the adsorbent filling space 560 as in the present embodiment, the coated particles have almost the same composition in each part of the adsorbent filled space 560. preferable. Thereby, the adsorption device 400 has particularly excellent ability to separate (purify) various adsorbates.

Alternatively, a part of the adsorbent filling space 560 (for example, a part on the inflow pipe 540 side) may be filled with coating particles, and the other part may be filled with another adsorbent.
Next, an example of a separation method using such an adsorption device 400 will be described.

[1] Preparation Step First, a sample solution is prepared by mixing a sample containing a plurality of types of adsorbate and a buffer solution.

  The buffer used for the preparation of the sample solution preferably has a salt concentration equal to or lower than the salt concentration of the elution buffer described later. Thereby, the adsorbate can be reliably adsorbed on the coated particles filled as the adsorbent 100.

  The amount of the buffer used when preparing the sample solution is not particularly limited, but is preferably about 5 to 50000 times, more preferably about 50 to 1000 times the mass of the adsorbate.

  Further, the pH of this buffer solution varies depending on the type of adsorbate contained in the sample. For example, when the adsorbate is a biological substance such as protein, nucleotide, and nucleic acid, it is preferably about 6 to 8, More preferably, it is about 6.5 to 7.5.

  Furthermore, the temperature of the buffer solution is not particularly limited, but is preferably about 20 to 50 ° C, and more preferably about 25 to 45 ° C.

  By using a buffer solution in such a pH range and temperature range, when the adsorbate is a biological substance, its alteration can be reliably suppressed, and the adsorbate can be efficiently attached to the coated particles. As a result, it is possible to improve the recovery rate of the target adsorbate.

  In addition, when a solid substance is contained in the prepared sample liquid, it is preferable to remove the solid substance from the sample liquid. Thereby, clogging of the column 500 can be reliably prevented. The method for removing the solid matter is not particularly limited. For example, after centrifuging the sample solution, the supernatant solution is recovered, and the solid matter remaining from the supernatant solution is filtered off using a filter. It is done.

[2] Supplying Step Next, the sample liquid is supplied to the coated particles through the inflow pipe 540 and the filter member 600, and is passed through the column 500 (adsorbing device 400), so that the adsorbent (coated particles) 1 Contact.

  As a result, a component having a low adsorbability with respect to the coated particles flows out of the column 500 through the filter member 700 and the outflow pipe 550. A component having a high adsorbability with respect to the coated particles is held in the column 500.

[3] Fractionation step Next, an elution buffer (eluent) for eluting the adsorbate is supplied from the inflow pipe 540 into the column 500 and flows out from the column 500 through the outflow pipe 550. Fractionate (collect) the effluent by a predetermined amount.

  In this embodiment, the salt concentration of the elution buffer is changed continuously or stepwise. As the elution buffer, the same type as the buffer used in the preparation step is preferably used.

  Here, when a plurality of kinds of adsorbents are adsorbed on the coated particles, when the elution buffer comes into contact with the coated particles, first, the adsorbent having a low adsorbing ability with respect to the coated particles is detached from the coated particles, and the outflow pipe 550. Spill from. Thereafter, the other adsorbates adsorbed on the coated particles are separated from the coated particles according to the salt concentration of the elution buffer, from those having a low adsorbability for the coated particles. Then, it is mixed in the elution buffer and collected in the effluent that flows out from the effluent pipe 550. Therefore, if the effluent flowing out from the effluent pipe 550 is fractionated by a predetermined amount, the specific adsorbate can be separated from the sample liquid containing plural kinds of adsorbents.

  Here, the coated particles can control the charge state, that is, the adsorption ability for each adsorbate by changing the coating amount of the polyamine compound. For this reason, for example, when the coating amount of the polyamine compound is set by giving priority to the adsorption ability for the adsorbate to be collected, the recovery rate of the adsorbate can be improved.

  Furthermore, when the polyamine compound coating amount is set so that the difference between the adsorption capacity for the adsorbate to be collected and the adsorption capacity for other adsorbates is larger, the elution time of the adsorbate to be collected is set. And the elution time of other adsorbents can be made longer, and a specific adsorbate can be separated from the sample liquid containing a plurality of types of adsorbates with high resolution.

  The flow rate of the buffer solution is not particularly limited, but is preferably about 1 to 10 mL / min, and more preferably about 1 to 5 mL / min.

  The passing time of the buffer solution is not particularly limited, but is preferably about 5 to 60 minutes, and more preferably about 10 to 30 minutes.

  As mentioned above, although the manufacturing method of the covering particle | grains of this invention was demonstrated, this invention is not limited to this.

  For example, the method for producing coated particles of the present invention can add one or more steps for any purpose.

Next, specific examples of the present invention will be described.
1. Production of coated particles (Example)
First, 140 g of calcium hydroxide was dispersed in 1200 mL of pure water to prepare a dispersion, and 700 mL of an aqueous phosphoric acid solution (phosphoric acid concentration: 10 wt%) was added dropwise to this dispersion while stirring. Thus, calcium hydroxide and phosphoric acid were reacted to synthesize hydroxyapatite, and a slurry containing hydroxyapatite aggregates was obtained.

  Next, the slurry was spray-dried and further classified to obtain hydroxyapatite granules (HAp particles having an average particle size of 40 μm), and then the hydroxyapatite granules were fired at 700 ° C.

  Next, 1 kg of calcined hydroxyapatite particles was mixed with 9 L of a 1.5 wt% polyethyleneimine (MW 70000) aqueous solution to obtain a particle / polyethyleneimine dispersion.

  Next, the particle / polyethyleneimine dispersion was supplied and heated as droplets in a heating atmosphere using a spray dryer (Okawara Chemical Co., Ltd., “OC-20”). As a result, the hydroxyapatite constituting the particles reacts with polyethyleneimine to coat the surface of the hydroxyapatite particles with polyethyleneimine, and the dispersion medium contained in the dispersion is removed to obtain the particles as a dried product. It was.

  The heating conditions for heating the droplets of the particle / polyethyleneimine dispersion in a heating atmosphere are as follows.

<Heating conditions>
・ Heating chamber temperature: 90 ℃
-Volume of droplet: 40 pL

Next, the obtained particles were placed on a filter, and the particles were washed by passing distilled water through the particles and the filter. The particles were washed until the colored reaction was not confirmed when the particles and distilled water after passing through the filter reacted with the ninhydrin reagent.
Thereafter, the particles were collected from the filter and dried using a desiccator.

  As described above, particles having hydroxyapatite particles coated with polyethyleneimine (coated particles: PEI-HAp particles) were obtained.

(Comparative Example 1)
First, hydroxyapatite particles (HAp particles having an average particle size of 40 μm) were obtained in the same manner as in the above example, and then the hydroxyapatite particles were fired at 700 ° C.

  Next, 100 g of calcined hydroxyapatite particles were mixed with 150 mL of 1.0 wt% polyethyleneimine (MW 70000) aqueous solution to obtain a particle / polyethyleneimine dispersion.

  Next, the dispersion liquid was accommodated in a rotary evaporator, and the dispersion liquid in the evaporator was heated using a bathtub. Thus, the surface of the hydroxyapatite particles was coated with polyethyleneimine, and the dispersion medium contained in the dispersion was removed to obtain particles as a dried product.

  The heating conditions for heating the droplets of the particle / polyethyleneimine dispersion in the container are as follows.

<Heating conditions>
-Bathtub temperature: 80 ° C
・ Evaporator speed: 78-80 times / min
・ Cooling water temperature: 8-9 ° C
・ Vacuum degree of evaporator: 22.25 hPa

  Next, in the same manner as in the above Example, the obtained particles were washed and then dried to obtain coated particles (PEI-HAp particles).

(Comparative Example 2)
Particles (HAp particles) were obtained in the same manner as in Example 1 except that the coating of the particles with polyethyleneimine was omitted.

2. Evaluation 2-1. Confirmation of presence / absence of aggregation of coated particles The coated particles produced in Examples and Comparative Example 1 were each confirmed for the presence / absence of aggregation.

  As a result, as shown in FIG. 5, almost no aggregates were observed in the coated particles of the example. On the other hand, in the coated particles of Comparative Example 1, many aggregates were observed due to the aggregation of adjacent coated particles.

2-2. Examination of Protein Elution Pattern Each of the particles produced in Examples and Comparative Examples 1 and 2 was packed in a column (40 × 100 mm). In addition, about the particle | grains manufactured by the comparative example 1, what formed the aggregate was removed, and it filled in the column.

  Thereafter, 400 mM sodium phosphate buffer (pH 6.8) was passed for 10 minutes, and further 10 mM sodium phosphate buffer (pH 6.8) was passed for 20 minutes at a rate of 1.0 mL / min. The particles packed in the column were washed.

  Further, as a sample solution, 10 mM sodium phosphate buffer (pH 6.8), ovalbumin (Ova) 10 mg / mL, myoglobin (Myo) 5 mg / mL, α-chymotrypsinogen-A (α-Chymo) 5 mg / ML, a protein mixture solution in which cytochrome-C (Cyto) was dissolved at 5 mg / mL was prepared.

  Then, 50 μL of this protein mixture is supplied into each column at a rate of 1.0 mL / min, and then 10 mM sodium phosphate buffer (pH 6.8) is supplied at a rate of 1.0 mL / min for 1 minute. did.

  Subsequently, after each sodium phosphate buffer solution (pH 6.8) was supplied to each column for 15 minutes while being changed from 100% at 100 mM to 75% at 400 mM. 400 mM sodium phosphate buffer (pH 6.8) was supplied for 5 minutes.

  Then, an absorbance curve at 280 nm was observed for the effluent flowing out from each column. The observed absorbance curve for each column is shown in FIG.

  Referring to FIG. 6, in the column using the particles of Comparative Example 2 (HAp particles), the protein in the sample solution is eluted in the order of ovalbumin, myoglobin, α-chymotrypsinogen-A, and cytochrome-C.

  On the other hand, in the column using the particles of Examples and Comparative Example 1 (PEI-HAp particles), myoglobin, α-chymotrypsinogen-A, and cytochrome-C were eluted with almost the same elution time as one peak. Only ovalbumin was observed as a separate peak eluting later than the other proteins.

  From this, in the coated particles of Examples, like the coated particles of Comparative Example 1, the hydroxyapatite particles are coated with polyethyleneimine, and the protein elution pattern of the column using this as an adsorbent can be changed. I understood.

  That is, in the column using the coated particles (PEI-HAp particles) of the example, since the elution time of ovalbumin (acidic protein) is particularly delayed as in Comparative Example 1, the adsorption to acidic protein is particularly important. It was found that the ability to improve the ability to separate acidic protein from other proteins (separation ability for acidic protein) can be improved.

2-3. Examination of nucleotide elution pattern Each of the particles produced in Examples and Comparative Examples 1 and 2 was packed in a column (40 × 100 mm). In addition, about the particle | grains manufactured by the comparative example 1, what formed the aggregate was removed, and it filled in the column.

  Thereafter, 400 mM sodium phosphate buffer (pH 6.8) was passed for 10 minutes, and further 10 mM sodium phosphate buffer (pH 6.8) was passed for 20 minutes at a rate of 1.0 mL / min. The particles packed in the column were washed.

  As a sample solution, a nucleotide solution prepared by dissolving 1 mg / mL of adenosine, AMP, ADP, and ATP in a 10 mM sodium phosphate buffer (pH 6.8) was prepared.

  Then, 50 μL of this nucleotide solution was supplied into each column at a rate of 1.0 mL / min, and then 10 mM sodium phosphate buffer (pH 6.8) was supplied at a rate of 1.0 mL / min for 1 minute. .

  Subsequently, after each sodium phosphate buffer solution (pH 6.8) was supplied to each column for 15 minutes while being changed from 100% at 100 mM to 75% at 400 mM. 400 mM sodium phosphate buffer (pH 6.8) was supplied for 5 minutes.

  Then, an absorbance curve at 260 nm was observed for the effluent flowing out from each column. The absorbance curves observed for each column are shown in FIG.

  FIG. 7 shows that four peaks are observed in the absorbance curves of the column of Example and Comparative Example 1, and peaks corresponding to adenosine, AMP, ADP and ATP are observed in this order. In contrast, in the absorbance curve of Comparative Example 2, only three peaks were observed, and it is assumed that the peaks of adenosine and AMP overlapped and were observed as one peak.

  From these facts, in the coated particles of Examples, like the coated particles of Comparative Example 1, the hydroxyapatite particles are coated with polyethyleneimine, and the peaks corresponding to the nucleotides of the column using this as an adsorbent. I found that I could increase the distance.

  That is, in the column using the coated particles of the example (PEI-HAp particles), as in Comparative Example 1, the retention increases with the increase in the number of phosphate groups. It has been found that it can be improved.

2-4. Summary From the above, as in the examples, the dispersion containing hydroxyapatite particles, polyethyleneimine, and water as the dispersion was heated as a droplet in a heated atmosphere without forming an aggregate. It has been found that coated particles can be produced.

  In addition, the coated particles of Examples manufactured using such a method had the same separation ability as Comparative Example 1 manufactured using a conventional method in which the dispersion was directly heated without forming droplets. From this, it was found that the surface of hydroxyapatite particles was coated with polyethyleneimine.

  Accordingly, it has been found that by producing coated particles by heating the dispersion medium as droplets in a heated atmosphere, coated particles capable of stably obtaining excellent adsorption ability and separation ability can be produced with high yield.

  Further, as a solvent contained in the dispersion, coated particles (PEI-HAp particles) were produced using a solvent containing methanol in addition to water, and the same evaluation as in the above 2-3 was performed. As a result, compared to a column with coated particles obtained using water as the dispersion medium for the dispersion, a column with coated particles obtained with methanol as the dispersion medium for the dispersion is more suitable for each nucleotide. As a result, the distance between the corresponding peaks increased. From this, it was found that by using methanol as a dispersion medium in the dispersion liquid, coated particles having better separation ability can be obtained.

It is sectional drawing which shows an example of the particle | grains which comprise the covering particle | grains of this invention. It is sectional drawing which shows the structure of the spray dryer apparatus used when coat | covering the surface of particle | grains with a polyamine type compound. It is a fragmentary sectional view for showing the composition of the spray board with which the spray dryer apparatus shown in Drawing 2 is provided. It is a longitudinal cross-sectional view which shows an example of the adsorption | suction apparatus with which the coated particle manufactured by the manufacturing method of the coated particle of this invention was applied as an adsorbent. 2 is an optical photograph of coated particles (PEI-HAp particles) produced in Examples and Comparative Example 1. It is an absorbance curve of an effluent when various proteins are made to adsorb about a column using particles (PEI-HAp particle or HAp particle) manufactured in an example and comparative examples 1 and 2. It is an absorbance curve of an effluent when various nucleotides are made to adsorb about a column using particles (PEI-HAp particles or HAp particles) manufactured in Examples and Comparative Examples 1 and 2.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Spray dryer apparatus 2 Heating chamber 21 Through-hole 3 Liquid feeding part 31 High pressure pump 32 Liquid feeding line 4 Spraying board 41 Upper disk 42 Lower disk 43 Columnar body 44 Opening part 45 Ejection hole 5 Heating means 51 Heater 6 Storage tank DESCRIPTION OF SYMBOLS 7 Air supply part 71 Blower 72 Supply line 73 Opening part 74 Filter 8 Exhaust part 81 Exhaust line 82 Storage chamber 83 Through-hole 9 Rotating part 91 Motor 92 Rotating shaft 93 Bearing 100 Adsorbent 200 Particle 210 Base material 220 Calcium phosphate compound layer 230 Microparticle 400 Adsorber 500 Column 510 Column body 520, 530 Cap 540 Inflow pipe 550 Outflow pipe 560 Adsorbent filling space 600, 700 Filter member

Claims (1)

A method for producing a coated particle in which the surface of the particle is coated with a polyamine compound,
After preparing a dispersion containing the particles and the polyamine compound, the dispersion is supplied as a droplet into a heated atmosphere, whereby the droplet is heated to form the polyamine compound on the surface of the particle. A method for producing coated particles, characterized in that :

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