JP2959111B2 - Acicular hydroxyapatite - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (A) Object of the Invention [Industrial Application Field] The present invention relates to a wet-synthesized needle-like hydroxyapatite which is particularly effective for adsorption and separation of biopolymers and the like.

(Prior art)

There are a wet method and a dry method for the synthesis of hydroxyapatite, but the wet method is advantageous as an inexpensive synthesis method.

Hydroxyapatite is classified into plate-like or needle-like according to its shape.
Although it has excellent adsorptivity for basic biopolymers, it has poor adsorbability for acidic biopolymers, and has poor liquid permeability when packed into a column.

On the other hand, needle-like hydroxyapatite has good liquid permeability when packed in a column and is excellent in the adsorptivity of acidic biopolymers, but has a problem of poor adsorbability of basic biopolymers.

In addition, any of the plate-like and needle-like hydroxyapatite synthesized so far cannot be adsorbed and separated with good reproducibility when adsorbing and separating biopolymers and the like by filling in a column. That is, once a biopolymer or the like is once adsorbed to hydroxyapatite in the column, the eluent is passed through the column while changing the concentration from a dilute state to a high concentration at a constant rate, thereby desorbing the biopolymer or the like. The concentration of the eluent at which separation starts (hereinafter referred to as the starting concentration) should be constant, but if the liquid is passed for a long time or the same operation is performed several times, the starting concentration will change, and the lack of reproducibility will result. There's a problem.

[Problems to be solved by the invention]

The present invention relates to a wet-synthesized needle-like hydroxyapatite which is particularly effective for the adsorption and separation of biopolymers and the like. It is another object of the present invention to provide a material exhibiting good adsorption / separation characteristics and having excellent reproducibility.

(B) Configuration of the Invention [Means for Solving the Problems] Wet-synthesized needle-like hydroxyapatite is mainly composed of calcium ion which is a cation and phosphate ion and hydrogen phosphate ion which are anions. Although a compound containing a hydroxyl ion and a carbonate ion as an ionic component, as a result of intensive studies by the present inventors, a compound in which the content of an anionic component such as a hydroxyl ion and a carbonate ion is in a specific range, Compared to conventional hydroxyapatite, it has been found that the adsorption / separation properties are extremely good,
The present invention has been completed.

That is, the present invention provides an anionic component other than a phosphate ion and a hydrogen phosphate ion (hereinafter referred to as a specific anion component).
Is a needle-shaped hydroxyapatite (hereinafter, simply referred to as hydroxyapatite) synthesized by a wet method, characterized in that the content is 7.0 to 9.0 wt%.

 Hereinafter, the present invention will be described in detail.

First, a wet synthesis method of hydroxyapatite will be described.

Any known method for wet synthesis of hydroxyapatite can be employed, but the following method is particularly preferred in that a method having excellent adsorption / separation characteristics can be obtained.

That is, a method of reacting hexagonal columnar or needle-like monetite with potassium hydroxide and / or lithium hydroxide under heating.

To obtain hexagonal columnar or needle-like monetite, phosphoric acid 2
A method is preferred in which sodium or dipotassium phosphate and calcium chloride are mixed and stirred at high temperature to synthesize. In this case, the molar ratio of calcium in the calcium compound, which is a raw material of monetite, to phosphorus in the phosphorus compound is preferably in the range of 0.9 to 1.1. The concentrations of the calcium compound and the phosphorus compound, which are generally used as an aqueous solution and are raw materials of monetite, are each preferably in the range of 0.05 to 2.0 mol /, more preferably 0.1 to 1.0 mol /. If hydroxyapatite is produced using monetite produced outside of this range, by-products other than hydroxyapatite may be produced, and as a result, adsorption / separation properties may be reduced.

The reaction temperature for obtaining monetite is desirably 75 ° C. or higher, which is the dehydration rearrangement temperature of brushite, more desirably 90 ° C. or higher, and particularly desirably 95 ° C. or higher. If the temperature is lower than 90 ° C, unreacted brushite remains, and it may be difficult to obtain pure water monetite.

Next, by reacting the obtained monetite with potassium hydroxide and / or lithium hydroxide under heating,
Hydroxyapatite can be obtained. The heating temperature at this time is preferably equal to or higher than the translocation temperature of brushite, and more preferably in the range of 90 to 100 ° C., as in the case of the above-mentioned reaction of monetite. If the heating temperature is lower than the brushite transition temperature, the particle size of the resulting hydroxyapatite tends to be small.

As the concentration of potassium hydroxide and / or lithium hydroxide used as the aqueous solution is lower, a hydroxyapatite aggregate having a large particle size can be obtained. However, if the concentration is too low, the production efficiency deteriorates. When it is added to water, it is preferably in the range of 0.1 to 5 mol /, more preferably in the range of 0.2 to 2 mol /.

The amount of potassium hydroxide and / or lithium hydroxide to be used is preferably 0.4 mol or more per mol of monetite.
It is preferably at least 6 mol.

If the amount of the alkali used is too large, washing the hydroxyapatite obtained as a result of the reaction with water takes a long time and is not efficient.
Mol or less.

Since the hydroxyapatite obtained as described above contains too much specific anion, the content of the specific anion must be set to 7.0 to 9.0 wt% by the method described later.

The specific anion is an anion other than a phosphate ion and a hydrogen phosphate ion in hydroxyapatite, and specifically, is an anion such as a hydroxyl ion, a carbonate ion or a fluorine ion.

Among the specific ions, carbonate ion and fluorine ion are
It is known that it is easily introduced as an anion of hydroxyapatite by synthesis, and when a small amount of these anions is introduced, it exhibits excellent adsorption / separation properties, but the synthesis reaction can be easily performed. In view of the fact that, in the present invention, a hydroxyl ion is a main anion, and as other specific anions, carbonate ions originating from carbon dioxide gas in the atmosphere are obtained during or after wet synthesis. Only a small amount is introduced.

When the content of the specific anion component is less than 7.0 wt%,
Although the reproducibility of adsorption and separation is considerable, there is a problem that the adsorption capacity of basic biopolymers is small, and at the same time, it is impossible to separate acidic biopolymers and basic biopolymers from each other. If the content of the component exceeds 9.0 wt%, there is a problem that the adsorption capacity for acidic biopolymers is reduced and the reproducibility of adsorption / separation is lost.

The method of adjusting the ratio of the total amount of hydroxyl groups and carbonate groups in hydroxyapatite is not particularly limited, but most commonly, the product obtained by the wet synthesis as described above is subjected to normal pressure at 100 to 300 ° C. It may be dried at a temperature of 2 to 50 hours. As a general tendency, the ratio of the total amount of hydroxyl groups and carbonate groups in hydroxyapatite decreases as the temperature increases or the drying time increases, and the drying temperature and the drying time may be appropriately adjusted within the above ranges.

In addition, the above-mentioned drying after the wet synthesis may be performed under reduced pressure or under increased pressure in addition to normal pressure. Drying under reduced pressure, particularly in vacuum freeze-drying, can be performed at a lower temperature than normal pressure and in a short period of time for desired adjustment. is there.

When storing the hydroxyapatite of the present invention, it is desirable to put it in a vacuum dryer or the like in order to prevent deterioration.

The method of analyzing the content of a specific anion component in hydroxyapatite may be a general analysis method, for example, ICP (high frequency plasma emission) analysis and infrared absorption spectrum analysis.

The content of the specific anion in hydroxyapatite is calculated, for example, as follows.

That is, the total amount (A) of calcium ions and phosphate ions was determined by the above analysis method, and the ratio of the amount obtained by removing the total amount (A) from the sample weight (B) to the sample weight [(B
−A) × 100 / B)].

In the analysis of the specific anion, it is desirable to dry the sample at, for example, 60 ° C. for several hours to 24 hours in order to remove adhering water from the analysis sample.

〔Example〕

 Hereinafter, the present invention will be described more specifically.

Reference Example 1. First, acicular hydroxyapatite was synthesized as follows.

That is, pure water 1 was charged into a 3-flask equipped with a stirrer and a cooling tube, heated to 95 ° C., and then 0.5 mol / aqueous calcium chloride solution and 0.5 mol / aqueous disodium phosphate solution 1 were each added over 5 hours. By dropping, an aggregate composed of monetite of acicular crystals was obtained.

Subsequently, 300 ml of a 1 mol / potassium hydroxide aqueous solution was added dropwise at the same temperature over 1 hour to obtain acicular hydroxyapatite.

Example 1. The acicular hydroxyapatite synthesized in Reference Example 1 was dried at 200 ° C. for 4 hours to obtain an acicular hydroxyapatite having a specific anion content of 8.3 wt%.

Example 2. The acicular hydroxyapatite synthesized in Reference Example 1 was dried at 300 ° C. for 4 hours to obtain an acicular hydroxyapatite having a specific anion content of 7.5 wt%.

Comparative Example 1. The acicular hydroxyapatite synthesized in Reference Example 1 was dried at 60 ° C. for 4 hours to obtain an acicular hydroxyapatite having a specific anion content of 9.7 wt%.

Comparative Example 2. The acicular hydroxyapatite synthesized in Reference Example 1 was dried at 400 ° C. for 4 hours to obtain an acicular hydroxyapatite having a specific anion content of 6.7 wt%.

Comparative Example 3. The acicular hydroxyapatite synthesized in Reference Example 1 was dried at 500 ° C. for 4 hours to obtain an acicular hydroxyapatite having a specific anion content of 6.5 wt%.

(Adsorption Characteristics Test) Using the needle-like hydroxyapatite obtained above, the adsorption amounts of acidic proteins and basic proteins were measured as follows.

That is, first, bovine serum albumin (Bo
vine-Serum-albmin Fraction V, BSA)
[Reagent manufactured by Sigma Co., Ltd.] was mixed with 5 mmol / phosphate buffer (PH 7.0) to obtain a 1 mg / ml sample solution.

5 ml of this sample solution is added to 0.1 g of the acicular hydroxyapatite obtained above, and while keeping the temperature at 4 ° C., 2 times every 15 minutes.
Shake for hours. Preliminary experiments confirmed that the adsorption reached equilibrium after 2 hours of shaking.

As described above, the supernatant of the sample solution sufficiently contacted with hydroxyapatite is filtered, and the absorbance at a wavelength of 280 nm is measured to determine the concentration of BSA remaining in the sample solution, and the adsorption of BSA on the hydroxyapatite is determined. The amount was calculated.

The amount of lysozyme adsorbed was calculated in the same manner as the method for determining the concentration of BSA, except that BSA was changed to lysozyme chloride, a basic protein (hereinafter simply referred to as lysozyme) [Reagent egg white, manufactured by Tokyo Chemical Industry Co., Ltd.] did.

 The results are shown in Table 1.

From Table 1 above, it can be seen that the hydroxyapatite obtained in Examples 1 and 2 has a larger adsorption capacity for acidic proteins and basic proteins than in Comparative Examples 2 and 3.

In addition, the hydroxyapatite obtained in Comparative Example 1 has a large adsorption capacity for acidic proteins and basic proteins, but has a drawback that acidic proteins and basic proteins cannot be separated as described later.

(Protein Elution Test) Using the acicular hydroxyapatite obtained above, the concentration at which the elution of acidic proteins and basic proteins started (hereinafter simply referred to as the starting concentration) was measured as follows.

That is, first, an open column made of glass having an inner diameter of 8 mm and a height of 30 cm was filled with the hydroxyapatite obtained above in a bed volume of 10 cc, and 100 μl of a sample solution of BSA and lysozyme prepared in the above-mentioned adsorption property test was passed through the column. As a result, BSA and lysozyme were adsorbed.

Next, phosphate buffer (PH7.0) was added from 5 mmol / to 300 mmol / using a linear gradient method.
The solution was passed for 0 minutes to elute the adsorbed BSA and lysozyme.

When the phosphate buffer was passed while increasing the concentration as described above, the concentration at which BSA and lysozyme began to elute was measured five times in exactly the same manner. Table 2 shows the first, third and fifth results.

From Table 2 above, when the hydroxyapatite obtained in Example 1 was used, reproducibility was excellent, and BSA was eluted at a lower concentration than lysozyme, which is effective for separation of acidic proteins and basic proteins. You can see that.

On the other hand, when the hydroxyapatite obtained in Comparative Examples 1, 2, and 3 was used, all of them lacked reproducibility. However, when the hydroxyapatite obtained in Comparative Example 1 was used, the reproducibility was slightly good, but it was impossible to separate acidic and basic proteins because BSA and lysozyme were eluted at the same concentration. .

(C) Effects of the Invention The hydroxyapatite of the present invention is particularly effective for adsorption and separation of biopolymers and the like. It also shows good adsorption / separation characteristics and is excellent in reproducibility.

Continuation of the front page Examiner Daiji Daikohara (56) References JP-A-63-64905 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C01B 25/32 A61K 6/033

Claims (1)

(57) [Claims] 1. A wet-synthesized needle-like hydroxyapatite, wherein the content of an anionic component other than phosphate ions and hydrogen phosphate ions is 7.0 to 9.0 wt%.