JP2010208896A - Method of manufacturing plate-like hydroxyapatite single crystal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique for manufacturing a plate-like HAp single crystal with c-surface orientation, which has not been prepared by a conventional technique, in an industrial level. <P>SOLUTION: The method of manufacturing a plate-like hydroxyapatite single crystal includes the steps of: putting an acid aqueous solution containing Ca<SP>2+</SP>ions, PO<SB>4</SB><SP>3-</SP>ions, urea and urease in a vessel; maintaining the acid aqueous solution with outside air in a gas-liquid contact system; generating a crystal nucleus of hydroxyapatite along with the increase of pH of the aqueous solution by hydrolysis of urea by urease; growing the crystal nucleus further in the direction of a-axis and b-axis; separating and collecting precipitation floating on the aqueous solution from the aqueous solution; and then obtaining the plate-like hydroxyapatite single crystal by applying hydrothermal treatment to the precipitation. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing a c-plane oriented plate-like hydroxyapatite single crystal.

  Hydroxyapatite (hereinafter abbreviated as HAp) generally has a crystal system belonging to the hexagonal system, a crystal plane grown in the c-axis direction (hereinafter, this crystal plane is referred to as a-plane), a-axis and b-axis directions. A crystal plane (hereinafter, this crystal plane is referred to as a c-plane). The a-plane is positively charged and the c-plane is negatively charged. Since HAp has two crystal planes with different charges, HAp can adsorb and hold a compound having an acidic group or a basic group. HAp having such adsorption characteristics is used as an adsorbent for physiologically active substances such as proteins (for example, a packing material for chromatography).

  In recent years, attempts have been made to selectively orient the crystal planes of HAp to impart selectivity to its adsorption characteristics. For example, Non-Patent Document 1 synthesizes a-face-oriented fibrous HAp by a uniform precipitation method, and the a-face-oriented fibrous HAp is more acidic protein (albumin, poly-L) than basic protein (lysozyme). -It has been reported that more glutamic acid) is adsorbed.

  Research on c-plane oriented plate-shaped HAp is also underway. Patent Document 1 discloses a method for producing a plate-like HAp, characterized in that calcium phosphate particles are heated in an amino acid aqueous solution to be converted into HAp, and then the HAp is grown in the a-axis and b-axis directions. Has been.

Furthermore, Non-Patent Document 2 discloses that an aqueous solution containing CaCO ₃ , H ₃ PO ₄ , urea and urease is incubated at 50 ° C. for 24 hours to precipitate a uniform product from the solution, and then this precipitate is subjected to hydrothermal treatment. A method of synthesizing c-plane oriented plate-shaped HAp by applying is disclosed.
Unlike the a-plane oriented fibrous HAp, c-plane oriented HAp is expected as an adsorbent that adsorbs more basic protein than acidic protein.

11th Annual Meeting of the Ceramic Society of Japan (1997), p. 11 H. Yamamoto and M. Aizawa, Archives of Bioceramics Reseach, 6 (2006), 212-215

JP 2004-15596 A

HAp used for the analysis or purification / isolation of physiologically active substances such as acidic proteins and basic proteins, using the above-mentioned a-plane oriented fibrous HAp and c-plane oriented HAP as adsorbents Is preferably a single crystal. In the case of polycrystalline HAp, there is a problem that the surface charge is not uniform, the specific adsorption performance with respect to the physiologically active substance is insufficient, and the separation performance is deteriorated.
However, as a result of examining the crystal structure of the plate-like HAp obtained by the prior art disclosed in Patent Document 1 by X-ray diffraction or the like, it has been found that the HAp is mainly polycrystalline.
In addition, the plate-like HAp obtained by the conventional technique disclosed in Non-Patent Document 2 also has a structure close to a single crystal, but it has been found that polycrystalline patterns are mixed.
Thus, in the prior art, a plate-shaped HAp single crystal with c-plane orientation could not be obtained efficiently.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a technique for efficiently producing a c-plane oriented plate-shaped HAp single crystal that has not been obtained by the prior art.

In order to achieve the above object, the present invention puts an acidic aqueous solution containing Ca ²⁺ ions, PO ₄ ³⁻ ions, urea and urease into a container and holds the acidic aqueous solution and the outside air in a gas-liquid contact state. As the pH of the aqueous solution rises due to the hydrolysis of urea, HAp crystal nuclei are generated, the crystal nuclei are grown in the a-axis and b-axis directions, and then the precipitates floating in the aqueous solution are separated from the aqueous solution. A method for producing a plate-like HAp single crystal is provided, which is collected and then subjected to hydrothermal treatment on the precipitate to obtain a plate-like HAp single crystal.

In the method for producing a plate-like HAp single crystal of the present invention, the concentration of Ca ²⁺ ions in the acidic aqueous solution is in the range of 0.0025 to 0.0100 mol · dm ⁻³ , and the concentration of PO ₄ ^3- ions is 0.0015. in the range of ~0.0060mol · ^{dm -3,} the concentration of urea in the range of 1.0~2.0mol · ^{dm -3,} 3 times 0.2 times by equivalent concentrations of urease to said urea 1 eq It is preferable to be in the range of equivalents.

According to the present invention, a plate-like HAp single crystal can be produced efficiently.
The plate-like HAp single crystal obtained by the present invention is a c-plane oriented plate-like HAp single crystal, which is used in many cases when a physiologically active substance such as a basic protein is analyzed or purified / isolated. High adsorption performance and separation performance can be obtained as compared with crystalline HAp.

It is a flowchart which shows the procedure of the HAp synthesis | combination performed in the Example. It is a XRD pattern of the deposit at the gas-liquid interface in the experiment of Ca ²⁺ , PO ₄ ³⁻ concentration fluctuation. Ca ^2+, an SEM image of a deposit of gas-liquid interface in the experiment of _PO ^{4 3-} concentration fluctuations. It is a XRD pattern of a precipitate after hydrothermal treatment in an experiment of Ca ²⁺ , PO ₄ ³⁻ concentration variation. It is a SEM image of the precipitate after the hydrothermal treatment in the experiment of Ca ²⁺ , PO ₄ ³⁻ concentration fluctuation. It is a XRD pattern of the deposit of the gas-liquid interface in the experiment of a urea concentration fluctuation | variation. It is a SEM image of the deposit of the gas-liquid interface in the experiment of urea concentration fluctuation. It is a XRD pattern of a precipitate after hydrothermal treatment in an experiment of urea concentration fluctuation. It is a SEM image of the precipitate after the hydrothermal treatment in the experiment of urea concentration variation. It is an XRD pattern of the deposit of the gas-liquid interface in the experiment of the urease addition amount fluctuation | variation. It is a SEM image of the deposit of the gas-liquid interface in the experiment of the urease addition amount fluctuation | variation. It is a XRD pattern of the precipitate after the hydrothermal treatment in the experiment of urease addition amount variation. It is a SEM image of the deposit after the hydrothermal treatment in the experiment of the urease addition amount fluctuation | variation. It is a XRD pattern of the deposit of the gas-liquid interface in the experiment of a total solution concentration fluctuation | variation. It is a SEM image of the deposit of the gas-liquid interface in the experiment of the total solution concentration fluctuation. It is a XRD pattern of the precipitate after hydrothermal treatment in the experiment of the total solution concentration fluctuation. It is a SEM image of the precipitate after the hydrothermal treatment in the experiment of the total solution concentration fluctuation. Sample No. 2 is a TEM image and an electron diffraction image of product 2 (after hydrothermal treatment). Sample No. 2 is a lattice image of the product 2 (after hydrothermal treatment). Sample No. It is an EDX figure of the product of 2 (after hydrothermal treatment). Sample No. 2 is an FT-IR spectrum of product 2 (after hydrothermal treatment). It is an image which shows a time-dependent change of a gas-liquid interface precipitate. It is a XRD pattern of a time-dependent change of a gas-liquid interface precipitate. It is a FT-IR spectrum of a time-dependent change of a gas-liquid interface precipitate. It is a SEM image of a time-dependent change of a gas-liquid interface precipitate. It is a schematic block diagram of the apparatus for pH fluctuation | variation investigation. It is a time-dependent change figure of pH. It is a XRD pattern after the heating of a gas-liquid interface precipitate. It is an FT-IR spectrum after heating a gas-liquid interface precipitate. It is a SEM image of the phase change by heating of a gas-liquid interface precipitate. It is a TEM image of the surface change by heating of a gas-liquid interface precipitate. Sample No. 2 is an XRD pattern of the lower layer product of No. 2. Sample No. It is a SEM image of 2 lower layer products. Sample No. It is a TEM photograph and electron beam diffraction image of 2 lower layer products (after hydrothermal treatment). Sample No. 2 is an FT-IR spectrum of the lower layer product 2 (after hydrothermal treatment). It is a SEM image of the upper part of an aggregate. It is a SEM image of the lower part of an aggregate. It is a SEM image of the side surface of an aggregate. It is a TEM image of the upper part of an aggregate. It is the schematic for demonstrating step 1 among the mechanisms of a gas-liquid interface synthesis process. It is the schematic for demonstrating step 2 among the mechanisms of a gas-liquid interface synthesis process. It is the schematic for demonstrating step 3 among the mechanisms of a gas-liquid interface synthesis process. It is the schematic for demonstrating step 4 among the mechanisms of a gas-liquid interface synthesis process. It is the schematic for demonstrating step 5 among the mechanisms of a gas-liquid interface synthesis process. It is the schematic for demonstrating step 6 among the mechanisms of a gas-liquid interface synthesis process.

The method for producing the plate-like HAp single crystal of the present invention includes:
A step (1) of preparing an acidic aqueous solution containing Ca ²⁺ ions, PO ₄ ³⁻ ions, urea and urease;
Next, the aqueous solution is put in a container and held in a state where the acidic aqueous solution and the outside air are in gas-liquid contact, and HAp crystal nuclei are generated according to the increase in pH of the aqueous solution due to hydrolysis of urea by urease. A step (2) of growing crystal nuclei in the a-axis and b-axis directions;
A step (3) of separating and collecting the precipitate floating on the aqueous solution from the aqueous solution;
-Next, the said deposit is hydrothermally processed, and the process (4) of obtaining a plate-shaped HAp single crystal is provided.

  In the step (1), the acidic aqueous solution is prepared by weighing and collecting the Ca compound and the P compound so as to have a molar ratio suitable for the synthesis of HAp (for example, Ca / P = 1.67), together with urea. In addition to purified water and mixing, an inorganic acid such as nitric acid or hydrochloric acid or an organic acid such as acetic acid, citric acid or lactic acid is added to this water, and the pH is set to the acidic side to dissolve the Ca compound, P compound and urea. Thereafter, it is preferable to prepare by adding an appropriate amount of urease.

  The Ca compound used for the preparation of the acidic aqueous solution is not particularly limited as long as it dissolves in the acidic aqueous solution, but a Ca compound that does not contain an interfering component that interferes with the precipitation of HAp crystals and the growth of single crystals. Preferably, for example, calcium carbonate, calcium nitrate, calcium chloride, calcium hydroxide, calcium lactate, calcium acetate and the like are preferable.

  The P compound used for the preparation of the acidic aqueous solution is not particularly limited as long as it dissolves in the acidic aqueous solution. For example, orthophosphoric acid, polyphosphoric acid, trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, triphosphate Ammonium, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and the like are preferable.

The ratio of Ca compound and P compound used for the preparation of the acidic aqueous solution is the same as the molar ratio (Ca / P) of Ca and P in hydroxyapatite represented by Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ or It is desirable to weigh and sample by calculating to an approximate value.
Further, the Ca ²⁺ ion concentration and the PO ₄ ^3- ion concentration in the acidic aqueous solution are not particularly limited, but in order to synthesize a beautiful plate-like HAp single crystal, the Ca ²⁺ ion is 0.0025 to 0.0100 mol · dm. ^-3 and PO ₄ ^3- ion are preferably in the range of 0.0015 to 0.0060 mol · dm ⁻³ .

Urea used for the preparation of the acidic aqueous solution has an amount suitable for gradually shifting to a neutral to weak alkaline region suitable for precipitation of HAp because the pH of the acidic aqueous solution rises due to ammonium ions decomposed by urease. Added. The amount of urea added is appropriately set according to the acidity of the acidic aqueous solution, but is usually in the range of 0.5 to 2.0 mol · dm ⁻³ , preferably 1.0 to 2.0 mol · dm ^−3. It is preferable to set it as the range.

The urease used in the preparation of the acidic aqueous solution is hydrolyzed by acting on urea in the acidic aqueous solution to generate ammonium ions, gradually increasing the pH of the acidic aqueous solution, and gradually in a neutral to weak alkaline range suitable for precipitation of HAp. Appropriate amounts are added to make the transition. The amount of urease added is not particularly limited, but in order to synthesize a beautiful plate-like HAp single crystal, it is preferable to add urease in a range of 0.2 to 3 equivalents to 1 equivalent of urea. . Here, 1 equivalent of urease (enzyme activity: 140 unit · mg ⁻¹ ) corresponds to the amount required to hydrolyze all 1 equivalent of urea within 24 hours, and 0.2 equivalent is 0.547 cm ³ . The 3-fold equivalent corresponds to 8.202 cm ³ , respectively.

  After preparing the acidic aqueous solution in the step (1), the acidic aqueous solution is put in a container and held in a state where the acidic aqueous solution and the outside air are in gas-liquid contact, and according to the increase in pH of the aqueous solution due to urea hydrolysis by urease, A step (2) of generating crystal nuclei of HAp and further growing the crystal nuclei in the a-axis and b-axis directions is performed. The details of the container and holding conditions for performing this step (2) are not particularly limited as long as the above-described HAp crystal generation and growth are possible. However, the holding temperature is preferably set in the range of 20 to 55 ° C., preferably in the vicinity of the optimum temperature of urease. The reaction time is preferably in the range of 24 to 120 hours.

  In this step (2), as time elapses, HAp crystals precipitate and grow near the gas-liquid interface of the container and the lower layer of the liquid, but the plate-like HAp that is the object of the manufacturing method of the present invention. The single crystal grows in a state of floating near the gas-liquid interface of the container.

Next, a step (3) of separating and collecting the precipitate floating on the aqueous solution from the aqueous solution is performed. The method for collecting the precipitate is not particularly limited, and can be performed by applying a conventionally known collection method used in the chemical field, the pharmaceutical field, or the like.
The precipitate separated and collected from the container is thoroughly washed with purified water and dried.

  When the precipitate thus obtained is examined by X-ray diffraction or the like, other calcium phosphate compounds such as octacalcium phosphate (OCP) are mixed in addition to the HAp crystals intended for production. Therefore, the precipitate (4) is subjected to a hydrothermal treatment to convert it into a single phase of HAp to obtain a target plate-shaped HAp single crystal.

  The conditions for this hydrothermal treatment are not particularly limited as long as other calcium phosphate compounds such as octacalcium phosphate (OCP) can be converted into a single phase of HAp. Usually, the temperature is in the range of 100 to 200 ° C., and the treatment time is 1 to 1. It is preferable to carry out under conditions of about 3 hours. The target plate-like HAp single crystal is obtained by this hydrothermal treatment.

  The plate-like HAp single crystal obtained by the production method of the present invention has a relatively large and clear plate shape as shown in FIG. 13 and the like, and as seen from the XRD pattern of FIG. Is formed. This plate-like HAp single crystal has a strong feature peak at 26 ° ((002) plane) corresponding to the c-plane of HAp, and was proved to be a c-plane-oriented HAp single crystal.

  The plate-like HAp single crystal obtained by the production method of the present invention is a c-plane oriented plate-like HAp single crystal, which is used for analysis, purification or isolation of physiologically active substances such as basic proteins. In addition, high adsorption performance and separation performance can be obtained as compared with polycrystalline HAp.

1. Experiment (1-1) Preparation Method of Mixed Solution First, a suspension (1000 cm ³ ) composed of calcium carbonate, orthophosphoric acid and urea was prepared and stirred for 2 hours. Nitric acid (1.0 mol · dm ⁻³ ) was added to this mixed suspension so as to have a pH of 3.00. Stir for another hour and add urease.
Table 1 shows the blending ratio of the starting materials calcium carbonate, orthophosphoric acid (H ₃ PO ₄ ), urea and urease (140 units · mg ⁻¹ ). In addition, the addition amount of urease is represented by the addition amount of 0.1 mass% aqueous solution.

(1-2) Synthesis from mixed solution The mixed solution (1000 cm ³ ) obtained in (1-1) above was placed in 8 glass petri dishes having an outer diameter of 12.3 cm (125 cm ^{3 in} each petri dish), and sealed. The reaction was conducted at 50 ° C. for 96 hours. After the reaction, only the product floating on the liquid surface was suction filtered and washed with a suction filter and dried at 110 ° C. for 24 hours to obtain a synthetic powder. An outline of the synthesis here is shown in FIG.

(1-3) Hydrothermal treatment
In the hydrothermal treatment, an autoclave (TVS-1 type (volume: 50 cm ³ ) manufactured by Pressure Glass Industrial Co., Ltd.) having a fluororesin inner cylinder in the reactor was used. The synthetic powder (0.1 g) obtained in (1-2) above was added to 50 cm ³ of pure water and heated at 120 ° C. for 2.5 hours for hydrothermal treatment. Thereafter, the treated product was filtered and washed, and dried at 110 ° C. for 48 hours.

2. Experimental Results (2-1) Characterization of Precipitate at Gas-Liquid Interface (2-1-1) Ca ²⁺ , PO ₄ ^3- Concentration Variation (No. 1-No. 4 in Table 1)
The concentration of urea is 1.0 mol · dm ⁻³ , the volume of the 0.1 mass% urease aqueous solution to be added is fixed to 2.734 cm ³ , which is equivalent, and the concentrations of CaCO ₃ and H ₃ PO ₄ are varied as parameters, Heated at 50 ° C. for 96 hours. The precipitate on the liquid surface and the product on the bottom of the petri dish were separated and filtered and dried, respectively.
In the following description, X-ray diffraction (or apparatus) is abbreviated as XRD, scanning electron microscopy (or apparatus) is abbreviated as SEM, and transmission electron microscopy (or apparatus) is abbreviated as TEM. The energy dispersive X-ray analysis method (or apparatus) is abbreviated as EDX, and the infrared absorption method (or apparatus) is abbreviated as FT-IR.

FIG. 2 shows the XRD pattern of the precipitate at the interface. In the figure, OCP represents octacalcium phosphate and HAp represents hydroxyapatite. In addition to the characteristic pattern of apatite, a characteristic peak of OCP near 4 ° also appeared, so that it was confirmed that all products were a mixed phase of HAp and OCP. Further, in the case of high concentration of Ca ²⁺ , PO ₄ ^3- (No. 3, No. 4), it was found that the main product was OCP because the characteristic peak of OCP was very strong. On the other hand, in the case of Ca ²⁺ , PO ₄ ³ -low concentration (No. 1, No. 2), the characteristic peak of OCP becomes weak, and instead of HAp 26 ° ((002) plane) and 53 ° ((004) It can be inferred that the main product is a HAp with many c-planes exposed due to the development of the characteristic peak of (plane).

In FIG. 1-No. The SEM image of the deposit of the interface of 4 is shown. In the case of high concentration of Ca ²⁺ , PO ₄ ^3- (No. 3, No. 4), the precipitates were in the form of a strip or a plate with the surroundings collapsed, but the Ca ²⁺ , PO ₄ ^3- low concentration In the case (No. 1, No. 2), the precipitate form was agglomerated but became a beautiful plate.

The precipitate was hydrothermally treated at 120 ° C. for 2.5 hours.
The XRD pattern after hydrothermal treatment is shown in FIG. All powders were hydrothermally treated to eliminate OCP and convert to a single phase of HAp. There is a tendency that the characteristic peak at 26 ° ((002) plane) corresponding to the c-plane of HAp becomes stronger as the Ca ²⁺ and PO ₄ ³⁻ concentration is lower. In addition, in the case of high concentration of Ca ²⁺ , PO ₄ ^3- (No. 3, No. 4), it was detected that a characteristic peak of 32 ° ((300) plane) corresponding to the a-plane of HAp was also developed. It was.

FIG. 5 shows No. after hydrothermal treatment. 1-No. The SEM image of the deposit of the interface of 4 is shown. When the Ca ²⁺ and PO ₄ ³⁻ concentrations were low (No. 1, No. 2), the form of the product was slightly collapsed compared with that before the treatment, but it was confirmed that it was plate-like (a-axis orientation). ^Further, when Ca 2+, is _PO ^{4 3-} concentration is high (No.3, No.4), the product is not plate-shaped, has a rectangular form (c-axis orientation). The specific orientation of the product observed by SEM is consistent with the XRD results in FIG.

In Table 2, no. 1-No. The yield and yield in each of the four conditions are shown. Considering the yield and the precipitate form, it can be determined that CaCO ₃ 0.0050 mol · dm ⁻³ , H ₃ PO ₄ 0.0030 mol · dm ⁻³ (No. 2) is the optimum concentration condition for Ca and P.

(2-1-2) Urea concentration fluctuation (No. 2, No. 5-No. 7 in Table 1)
From the experimental result of (2-1-1), No. Since plate-shaped HAp having high orientation was efficiently synthesized under the concentration conditions of 2 CaCO ₃ and H ₃ PO ₄ , the concentration of CaCO ₃ and H ₃ PO ₄ and the amount of urease added here were 0.0050 mol · The sample was fixed at dm ⁻³ , 0.0030 mol · dm ⁻³ and 2.734 cm ³ , and the urea concentration was varied as a parameter and heated at 50 ° C. for 96 hours. In addition, urease is No. No. 2 experimental condition is equivalent addition. In No. 5, urease was doubled excessively, 6 and no. 7 is 2/3 times and 0.5 times excess, respectively. The liquid level precipitate and the petri dish bottom product were separated and filtered and dried.

  FIG. 6 shows a precipitate XRD pattern at the interface. When the urea concentration was too low (No. 5), no precipitate was obtained at the gas-liquid interface. This is thought to be because the pH in the solution is difficult to increase because the concentration of urea is insufficient. No. 2, no. 6, no. As a result of XRD characterization of No. 7 precipitate, an OCP characteristic peak in the vicinity of 4 ° appeared in addition to the characteristic peak of apatite. It could be confirmed.

  FIG. 7 shows an SEM image of precipitates at the interface. All products (No.2, No.6, No.7 precipitates) have an aggregated plate-like precipitate form, and the lower the urea concentration, the larger the size of the precipitate. confirmed.

Hydrothermal treatment was performed on the interface precipitate (No. 2, No. 6, No. 7 precipitate) at 120 ° C. for 2.5 hours.
The XRD pattern after hydrothermal treatment is shown in FIG. All the powders were converted into a single phase of HAp in which OCP disappeared and the characteristic peak at 26 ° ((002) plane) corresponding to the c-plane of HAp became strong by hydrothermal treatment.

FIG. 9 shows an SEM image of each powder after hydrothermal treatment. Although all the powders maintained a plate-like form, there was a tendency that the plate-like form of the generated precipitate collapsed and deteriorated as the urea concentration increased.
Table 3 shows the yield and yield under each condition. Considering the yield and the form of precipitate, 1.0 mol · dm ⁻³ (No. 2) is considered to be the optimum value for urea.

(2-1-3) Variation of urease addition amount (No. 2, No. 8-No. 11 in Table 1)
From the experimental results of (2-1-1) and (2-1-2), Ca ²⁺ , PO ₄ ³⁻ and urea concentrations are fixed to optimum values, and the amount of urease added is varied as a parameter, Heated at 50 ° C. for 96 hours. Filtration and drying were performed separately on the liquid level precipitate and the petri dish bottom product.
FIG. 10 shows the XRD pattern of the product. In addition to the peak characteristic of apatite, the characteristic peak of OCP near 4 ° also appeared, so that it was confirmed that all products were a mixed phase of HAp and OCP. Moreover, it can be inferred that the apatite produced has a lot of c-plane exposed due to the development of characteristic peaks at 26 ° ((002) plane) and 53 ° ((004) plane).

FIG. 11 shows an SEM image of precipitates at the gas-liquid interface. Before the hydrothermal treatment, all the products were in the form of a plate, and as the amount of urease added decreased, the size of the precipitate tended to increase. The addition amount of 0.1 mass% urease when 0.5468cm ³ (No.8), most orientation good and Very Large precipitate.

  FIG. 12 shows an XRD pattern of each precipitate after hydrothermal treatment. By hydrothermal treatment, OCP disappeared and converted to a single phase of HAp in which the characteristic peak at 26 ° ((002) plane) corresponding to the c-plane of HAp became strong.

FIG. 13 shows an SEM image of each precipitate after hydrothermal treatment. After the hydrothermal treatment, it was confirmed that the orientation of all the samples was slightly lowered, which is consistent with the change in the XRD pattern after the hydrothermal treatment in FIG. When the amount of urease added was 0.5468 cm ³ (No. 8), a precipitate having the best orientation and a large size was obtained.

Table 4 shows the yield and yield under each condition. Considering the yield and the form of precipitates, when Ca ²⁺ , PO ₄ ³⁻ and urea concentrations are set to optimum values, 2.734 cm ³ (No. 2), which is equivalent addition to urea in the reaction system, is the optimum for urease. It was judged that the amount was an addition amount.

(2-1-4) Variation in total solution concentration (No. 2, No. 12-No. 14 in Table 1)
From the experimental results of (2-1-1), (2-1-2) and (2-1-3), the optimum ratio of Ca ²⁺ , PO ₄ ³⁻ , urea concentration and urease addition amount is CaCO _{^{3 0.0050mol · dm -3, H 3}} PO 4 0.0030mol · dm -3, urea 1.0 mol · ^{dm -3,} the urease 2.734cm ^3. Here, the ratio of the concentration of each reagent and the ratio of the addition amount was fixed in this way, and the experiment was performed by changing the total concentration of the solution. The liquid level precipitate and the petri dish bottom product were separated and filtered and dried.

  FIG. 14 shows the XRD pattern of the product. In addition to the characteristic peak of apatite, the characteristic peak of OCP near 4 ° also appeared, so all products are a mixed phase of HAp and OCP. As the total concentration increases, the analytical strength of OCP near 4 ° Was also confirmed to be stronger. In addition, it is inferred that a large amount of the c-plane of the apatite is exposed due to the development of characteristic peaks at 26 ° ((002) plane) and 53 ° ((004) plane) of apatite.

  In FIG. 15, the SEM image of the deposit of a gas-liquid interface is shown. All of the products had a plate-like form, and there was a tendency for the size of the precipitate to decrease as the total concentration of the solution increased.

  FIG. 16 shows an XRD pattern after hydrothermal treatment of each of the precipitates. By hydrothermal treatment, OCP disappeared and converted to a single phase of HAp in which the characteristic peak at 26 ° ((002) plane) corresponding to the c-plane of HAp became strong.

  FIG. 17 shows an SEM image of each precipitate after the hydrothermal treatment. As the total concentration of the solution increased, the precipitate size of the product decreased, and the form of the precipitate also tended to collapse and deteriorated.

Table 4 shows the yield and yield of the product under each concentration condition. Taken together the precipitate morphology and _yield, CaCO 3 _0.0050mol ^· dm _-3, the _H 3 PO 4 _0.0030mol · ^{dm -3} and urea 1.0 mol · ^{dm -3,} further 0.1% It was judged that this was the optimum concentration of the solution to which 2.734 cm ^{3 of} urease aqueous solution was added.

(2-1-5) Sample No. 2. From the experimental results of (2-1-1) to (2-1-4), sample No. 2 was characterized. 2 synthesis conditions, _i.e., CaCO 3 _0.0050mol ^· dm _-3, the _H 3 PO 4 _0.0030mol · ^{dm -3} and urea 1.0 mol · ^{dm -3,} further 0.1% by mass urease aqueous solution 2. It was found that the synthesis conditions with the addition of 734 cm ³ were optimal. The product of this concentration condition was further characterized using an apparatus such as TEM, EDX, FT-IR.

  The TEM image and the result of electron beam diffraction are shown in FIG. The TEM image was confirmed to have a plate-like form as with the SEM image. A clear spot was recognized by electron diffraction, and it was confirmed that the precipitate was a single crystal.

  FIG. 19 is a lattice image obtained by further enlarging the precipitate after hydrothermal treatment. A linear or knitted pattern was observed inside the crystal, and a white portion and a black portion were mixed. From this lattice image, the orientation of the crystal was estimated. When the distance of 10 spaces was measured, it was 3.424 nm. According to the JCPDS card search, the mirror index of the closest lattice spacing was (002) plane. If this crystal is oriented in the (002) plane direction, the lattice constant is 0.6848 nm, which is close to the c-axis lattice constant (0.6883 nm), and the measured direction is the c-axis. The vertical direction can be assumed to be the a-axis.

  FIG. It is an EDX figure of 2 precipitates. When characterization was performed using EDX, the peak of the C element was also found in addition to the Ca, P, and O elements, so this final product was judged to contain carbonic acid.

In FIG. 2 shows the FT-IR spectrum of product 2 (after hydrothermal treatment). HAp the characteristic OH ^- absorption based on groups in the vicinity of 3570cm ^-1, The absorption based on _PO ^{4 3-} groups was observed near 1300～900,600 and 570 cm ^-1. In addition to these HAp-related absorptions, absorption based on CO ₃ ²⁻ ions was observed near 1600-1400 and 880 cm ⁻¹ . From these absorption spectra, no. It can be seen that the product of 2 is carbonate-containing (Type AB) HAp. This result is consistent with the EDX result of FIG.

In addition, this No. When the Ca content of the Ca powder of product 2 was quantified by the atomic absorption method and the P content was quantified by the phosphovanadmolybdic acid method, the Ca content was 36.8% by mass and the P content was 17.7% by mass. Met. The Ca / P ratio obtained from these values is 1.59, which is lower than 1.67 which is the Ca / P ratio of the stoichiometric composition of HAp. By combining the above results, this No. HAp synthesized under the condition 2 is a single crystal of aggregated plate-like HAp single phase, and is a calcium deficient hydroxyapatite containing carbonic acid of Ca / P = 1.59.

(2-1-6) Temporal Change of Gas-Liquid Interface Precipitate As shown in FIG. 1, when each material was put in a petri dish and allowed to stand in an incubator at 50 ° C., as the reaction progressed, As shown, a white film-like substance is deposited from the gas-liquid interface. The change with time in the structure and composition of the precipitate was examined.

  FIG. 23 shows an XRD pattern of the change with time of the gas-liquid interface precipitate. Although this precipitate is a mixed phase of HAp and OCP, the diffraction intensity of OCP near 4 ° becomes weaker as the incubation time elapses, and the HAp diffraction intensity near 26 ° tends to become stronger. This is considered to be because urea was hydrolyzed with the progress of the reaction, the pH value in the vicinity of the gas-liquid interface was shifted from acidic to alkaline, and OCP was converted to HAp. Since the XRD patterns of the 96-hour and 120-hour precipitates almost coincided, the incubation time of 96 hours was considered to be the end point of the reaction.

FIG. 24 is an FT-IR spectrum of the same sample used in FIG. From this FT-IR spectrum, it is recognized that the absorption of OH ^{− in} the vicinity of 3570 cm ⁻¹ gradually increases, and over time, since the conversion from OCP to HAp proceeds, the ratio of HAp in the precipitate This is thought to be due to the fact that

FIG. 25 is an SEM image of the same sample used in FIG. From this SEM image, the plate-like precipitate size grew significantly in the 24-48 hour time zone, but almost no change was observed in the precipitate size and morphology in the 48-120 hour time zone.
In this process, since the reaction proceeds in the glass petri dish, it is difficult to measure the change in pH over time in the solution. However, as shown in FIG. 26, the pH change in the solution is measured using a flask. Simulated.

  FIG. 27 shows the change over time of pH in the solution measured as described above. As the reaction proceeds, the pH first rises from around 3.0 to around 6.5 (first stage), then falls to around pH 6.2 (second stage) and then rises again (third stage). There was a trend. In the first stage, the pH in the solution starts to increase due to the hydrolysis reaction of urea, and since crystal nuclei are generated in the second stage, the pH decreases, and in the third stage, the pH increases again and the crystal grows. Conceivable.

(2-1-7) Phase change due to heating of gas-liquid interface precipitate Sample No. The hydrolyzed precipitates 2 were heated to 600 ° C., 700 ° C., 800 ° C., 900 ° C., 1000 ° C., 1100 ° C. and 1200 ° C., and the respective crystal phases and forms were examined.

FIG. 28 shows an XRD pattern in which the phase change due to heating of the precipitate after hydrothermal treatment was examined. The precipitate heated to 600 ° C. showed an XRD pattern of the HAp single phase maintaining the orientation. At 700 ° C., in addition to HAp, a peak of β-tricalcium phosphate (β-Ca ₃ (PO ₄ ) ₂ : β-TCP) was observed at around 31 °. At 800 ° C., 900 ° C. and 1000 ° C., the β-TCP peak was enhanced, but the HAp peak still remained. At 1100 ° C. and 1200 ° C., a peak of high-temperature α-TCP was detected in addition to HAp and β-TCP, confirming the coexistence of the three phases.

FIG. 29 shows an FT-IR spectrum for the same sample as FIG. It was confirmed that the absorption based on CO ₃ ²⁻ ions in the vicinity of 1600 to 1400 and 880 cm ⁻¹ was gradually weakened by heating.

  FIG. 30 is an SEM image of the same sample as FIG. 28 and FIG. When heated to 700 ° C., the form of the precipitate collapsed somewhat, but the plate shape was maintained. Upon further heating, the precipitate surface melted, and a plate-like form could not be observed completely at 1200 ° C. heating.

  FIG. 31 shows TEM images of unheated powder and powder heated to 1200 ° C. It was confirmed that the precipitate surface was uneven by heating at 1200 ° C. with respect to the smooth precipitate surface before heating.

  From the above results, it was confirmed that the plate-like HAp powder obtained from this process is thermodynamically less stable than ordinary stoichiometric HAp. It is thought to be due to calcium deficiency.

(2-2) Characterization of Lower Layer Product The petri dish lower layer product produced under all the concentration conditions shown in Table 1 was a mixed phase of OCP and HAp without orientation. In the following, sample no. Figure 2 shows the characterization results of 2 underlayer products.

  In FIG. 2 shows the XRD pattern of the lower layer product of 2. From FIG. 32, the synthetic powder of this lower layer is a mixed phase of OCP and HAp, and in the XRD pattern, the diffraction intensity of OCP near 4 ° and the diffraction intensity of (300) plane of HAp near 32 ° developed. .

  FIG. 33 shows an SEM image of the synthetic powder. From this SEM observation, it can be seen that the generated precipitates are rod-shaped, spherical, and plate-shaped.

  After hydrothermal treatment, the diffraction of OCP near 4 ° disappeared, the diffraction of the (300) plane of HAp near 32 ° was also weakened, and it was converted into a single phase of HAp. In the SEM image of FIG. 33, the specific orientation was lost.

  FIG. 34 shows a TEM image of the powder after hydrothermal treatment. By this TEM observation, the form of non-regular primary precipitates was observed, and spots were recognized by electron diffraction, but it was found that the crystals were polycrystalline because they were randomly arranged.

FIG. 35 shows the FT-IR spectrum of the powder after hydrothermal treatment. This FT-IR spectrum, characteristic of OH to HAp ^- absorption based on groups in the vicinity of 3570cm ^-1, The absorption based on _PO ^{4 3-} groups was observed near 1300～900,600 and 570 cm ^-1. In addition to these absorptions, absorption based on CO ₃ ²⁻ ions was observed in the vicinity of 1600 to 1400 and 880 cm ⁻¹ . From these absorption spectra, it can be seen that the HAp obtained from the lower layer is a carbonate-containing (Type AB) HAp.

  Moreover, when the Ca content of this HAp powder was quantified by the atomic absorption method and the P content was quantified by the phosphovanadmolybdic acid method, the Ca content was 35.4% by mass and the P content was 18.2% by mass. there were. The Ca / P ratio obtained from these values is 1.51, which is lower than the Ca / P ratio 1.67 in the stoichiometric composition of HAp.

3. Discussion In this example, a film-like HAp containing OCP was precipitated and grown from the gas-liquid interface of the mixed solution, and then the precipitate was subjected to hydrothermal treatment to obtain a single-phase plate-like HAp. Since the theoretical density of apatite is 3.16 g · cm ⁻³ , HAp cannot normally exist stably on the liquid surface. The specific mechanism of this process was examined below.

  FIG. 36 is an SEM image taken from the upper part (above the interface) of the film-like precipitate generated at the gas-liquid interface in the petri dish. A portion like a flower core is seen on the plate-like precipitates orthogonal to each other. It is thought that it corresponds to the base of HAp grown along the liquid surface.

  FIG. 37 is an SEM image of precipitates generated in the lower layer in the petri dish. In the lower precipitate, only the orthogonal plate-shaped HAp was observed, and the HAp base as in FIG. 36 was not observed at all.

  FIG. 38 is an SEM image taken from the side surface of the film-like precipitate generated at the gas-liquid interface in the petri dish. In FIG. 38, the upper part and the lower part of the film-like precipitate were clearly observed.

  FIG. 39 is a TEM image taken from above the precipitate aggregates. It was clarified that the plate-like precipitates inside the agglomerates had a structure perpendicular to each other.

  The mechanism of the gas-liquid interface synthesis process was presumed as follows, considering the difference between the lower part and the upper part of the precipitate and the special environment of the gas-liquid interface.

Step 1: As shown in FIG. 40, the concentration of Ca ²⁺ , PO ₄ ³⁻ and OH ⁻ ions increases at the gas / liquid interface due to evaporation of the solution near the gas / liquid interface.
Step 2: As shown in FIG. 41, when the supersaturation degree of HAp is exceeded, crystal nuclei of HAp are generated, and the concentrations of Ca ²⁺ , PO ₄ ³⁻ and OH ⁻ ions near the gas-liquid interface become low. The ions inside move to the gas-liquid interface and replenish.
Step 3: As shown in FIG. 42, ^Ca 2+ of the liquid surface in solution, _PO ^{4 3-} and OH ^- concentration difference of the ion occurs, a more c-axis direction of the crystal, ^Ca 2+ is in the b-axis direction, PO _{Since a} large amount of ₄ ³⁻ and OH ⁻ ions are present (the pH value is also high), the c-plane of the precipitated apatite grows preferentially along the a and b axes.
Step 4: As shown in FIG. 43, the c-plane charged to the (−) of the HAp deposited from the gas-liquid interface electrostatically attracts the a-plane charged to the (+) of the HAp nucleus in the solution. Aggregates (new bonds may form between each crystal).
Step 5: As shown in FIG. 44, fine HAp crystals present at the bottom of the interface take in Ca ²⁺ , PO ₄ ³⁻ and OH ⁻ ions in the solution and grow large. Here, it is considered that the a-axis direction grows significantly more than the c-axis direction due to the influence of electrostatic repulsion and obstacles in the three-dimensional space.
Step 6: Many small aggregates as shown in FIG. 45 are generated at the gas-liquid interface, and further aggregation occurs with each other, so that they can float stably at the gas-liquid interface like a ship.

  According to the present invention, a plate-like HAp single crystal can be produced on an industrial scale. Since this plate-like HAp single crystal has a widely exposed c-plane charged to (−), it is used as an adsorbent for physiologically active substances such as basic proteins (for example, a packing material for chromatography).

Claims (2)

An acidic aqueous solution containing Ca ²⁺ ion, PO ₄ ^3- ion, urea and urease is put in a container and held in a state where the acidic aqueous solution and the outside air are in gas-liquid contact, and the pH of the aqueous solution is increased by hydrolysis of urea by urease. Accordingly, hydroxyapatite crystal nuclei are generated, the crystal nuclei are further grown in the a-axis and b-axis directions, and then the precipitate floating on the aqueous solution is separated and collected from the aqueous solution, and then the hydrothermal treatment is performed on the precipitate. To obtain a plate-like hydroxyapatite single crystal. The concentration of Ca ²⁺ ions in the acidic aqueous solution is in the range of 0.0025 to 0.0100 mol · dm ⁻³ , the concentration of PO ₄ ³⁻ ions is in the range of 0.0015 to 0.0060 mol · dm ⁻³ , and urea 2. The concentration of urea is in the range of 1.0 to 2.0 mol · dm ⁻³ , and the concentration of urease is in the range of 0.2 to 3 equivalents to 1 equivalent of urea. A method for producing a plate-like hydroxyapatite single crystal according to claim 1.

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