Calcium-Phosphorus-Apatite Having Novel Properties and Process for Preparing the Same

FIG. 1 is a diagram showing an X-ray diffraction pattern of a hydroxyapatite obtained by the process of Example 1 in accordance with the process of the present invention.

FIG. 2 is a diagram showing an X-ray diffraction pattern of a chloroapatite obtained by the process of Example 4 in accordance with the process of the present invention.

FIG. 3 is an X-ray diffraction pattern of a hydroxyapatite obtained in Example 19.

FIG. 4 is an X-ray diffraction pattern of a chloroapatite obtained in Example 20.

FIG. 5 is an X-ray diffraction pattern of calcium chlorophosphate obtained by a heat treatment at 800.degree. C. in Example 21 (a).

FIG. 6 is an X-ray diffraction pattern of a chloroapatite obtained in Example 21 (h).

FIG. 7 is a diagram showing a chromatogram of cytochrome c obtained using a chromatographic packing material according to the present invention, which is obtained by an example of the process of the present invention (Example 51).

The following Examples will illustrate the present invention in more detail, but should not be construed as limiting the scope of the invention.

A solution of 3.11 g of NH.sub.4 H.sub.2 PO.sub.2 dissolved in 35 ml of water was admixed with a solution of 14.76 g of Ca(NO.sub.3).sub.2.4H.sub.2 O dissolved in 50 ml of water, and then further mixed with 100 ml of ethanol by stirring to prepare a raw material solution. Subsequently, the raw material solution and pressurized air was supplied into a two fluid atomization nozzle at rates of 20 ml/min and 10 liter/min, respectively, and atomized into a flame coming from a gas burner. Fine droplets formed from the raw material solution were instantaneously heated by the solvent evaporated and burned in the flame, thereby to be thermally decomposed. The powder formed was collected by a cyclone system. The results of X-ray diffraction measurement of the formed powder are shown in FIG. 1. As is apparent from FIG. 1, the powder formed by the process of the present invention was a hydroxyapatite whose crystals were well grown. Three main peaks observed consist of a diffraction peak (211) at a diffraction angle 2 .theta. or 31.7.degree., a diffraction peak (112) at 2 .theta. of 32.2.degree., and a diffraction peak (300) at 2 .theta. of 32.9.degree..

A solution of 8.59 g of phosphoric acid (H.sub.3 PO.sub.4 content: 85.6%) dissolved in 20 ml of water was added to a solution of 29.52 g of Ca(NO.sub.3).sub.2.4H.sub.2 O dissolved in 40 ml of water and admixed with 5 ml of nitric acid, and then further mixed with 120 ml of ethanol by stirring to prepare a raw material solution. Subsequently, the raw material solution was atomized into a flame in the same manner as in Example 1 to be thermally decomposed. The powder obtained by this process was found, as a result of X-ray diffraction measurement, to be one including a crystalline phase of hydroxyapatite and a small amount of an amorphous phase.

A powder as obtained by the process of Example 2 was heated at a rate of 5.degree. C./min to 1,300.degree. C., at which it was then heat-treated for 1 hour, followed by cooling at a rate of 5.degree. C./min. The results of X-ray diffraction measurement of the product obtained by the heat treatment showed that the product was a hydroxyapatite whose crystals were better grown than those of the hydroxyapatite obtained in Example 2, and decomposition of which was not recognized even at high temperatures. Thus, it was confirmed that the heat treatment provides a hydroxyapatite having a high thermal stability, the crystals of which are well grown.

A solution of 8.63 g of NH.sub.4 H.sub.2 PO.sub.4 dissolved in 20 ml of water and admixed with 5 ml of hydrochloric acid was added to a solution of 18.38 g of CaCl.sub.2.2H.sub.2 O dissolved in 35 ml of water and admixed with 5 ml of hydrochloric acid, and then further mixed with 120 ml of ethanol by stirring to prepare a raw material solution. Subsequently, the raw material solution was atomized into a flame in the same manner as in Example 1 to be thermally decomposed. The results of X-ray diffraction measurement of the powder obtained by this process are shown in FIG. 2. As is apparent from FIG. 2, the powder shows a diffraction pattern of chloroapatite. Main peaks observed are a diffraction peak (211) at a diffraction angle 2 .theta. of 31.4.degree. and superposed diffraction peaks (112) and (300) at a diffraction angle 2 .theta. of 32.2.degree..

A solution of 4.31 g of NH.sub.4 H.sub.2 PO.sub.4 dissolved in 35 ml of water was admixed with a solution of 14.76 g of Ca(NO.sub.3).sub.2.4H.sub.2 O dissolved in 50 ml of water and admixed with 5 ml of nitric acid to prepare a raw material solution. Subsequently, the raw material solution was atomized into a reaction tube (30 .phi..times.1,000 mm) of an electric furnace, which was preliminarily heated to 1,200.degree. C., from one end of the reaction tube at a rate of 2 ml/min. Fine droplets formed from the raw material solution were heated during passage thereof through the reaction tube to be thermally decomposed. The product was collected in a container connected to the other end of the reaction tube, while a gas and a water vapor formed by the thermal decomposition were aspirated by an aspirator to be discharged out of the system. As a result of X-ray diffraction measurement of the powder obtained by this process, the conjoint presence of a crystalline phase of hydroxyapatite and an amorphous phase, and a slight amount of calcium carbonate were recognized by the peaks assigned thereto.

3.08 g of phosphorus acid was dissolved in 190 ml of methanol. 6.94 g of CaCl.sub.2 was dissolved in the resulting solution to prepare a raw material solution. Subsequently, the raw material solution was atomized into a flame in the same manner as in Example 1 to be thermally decomposed. The powder formed was heated up in an atmosphere of air at a rate of 5.degree. C./min to 1,300.degree. C., at which it was heat-treated for 1 hour, followed by cooling at a rate of 5.degree. C./min. As a result of detection of chlorine by a silver nitrate solution and X-ray diffraction measurement of the powder obtained, it was confirmed that it was an apatite solid solution of hydroxyapatite with chloroapatite.

Various calcium-phosphorus-apatites were obtained in Examples 7 to 17 in which the procedure of Example 1 or 5 was repeated using a raw material and a formulation as listed in Table 1. The heat decomposition temperature, the heat treatment conditions, and the main component are listed for each product in Table 1.

15.58 g of (C.sub.2 H.sub.5 O).sub.3 P was added to a solution 36.90 g of Ca(NO.sub.3).sub.2.4H.sub.2 O dissolved in about 200 ml of ethanol, and the resulting mixture was stirred to effect dissolution thereof. Ethanol was added to the resulting solution to total 250 ml to prepare a homogeneous raw material solution. Subsequently, the raw material solution and pressurized air were supplied into a two fluid atomization nozzle from the liquid supply side and the gas supply side thereof at rates of 10 ml/min and 10 liter/min, respectively, and atomized into a flame coming from a gas burner. Fine droplets formed from the raw material solution were instantaneously heated by the solvent evaporated and burned in the flame, thereby to be thermally decomposed. The temperature during the thermal decompositon reaction was 1,050.degree. to 1,105.degree. C. The powder thus formed was collected by a cyclone system. It was observed as a fine powder of hollow globules through an electron microscopy. Subsequently, the formed powder was found, as a result of X-ray diffraction analysis thereof, to be one including a crystalline phase of hydroxyapatite and an amorphous phase, and a residual unreacted CaO.

A powder as obtained by the process of Example 18 was heated up in an atmosphere of air at a rate of 5.degree. C./min to 1,300.degree. C., at which it was heat-treated for 1 hour, followed by cooling at a rate of 5.degree. C./min. The results of X-ray diffraction measurement of the product obtained by the heat treatment are shown in FIG. 3. As is apparant from FIG. 3, the product obtained in this Example was a hydroxyapatite whose crystals (hexagonal) were well grown, and decompositon of which was not recognized even at high temperatures.

31.16 g of (C.sub.2 H.sub.5 O).sub.3 P was added to a solution of 45.94 g of CaCl.sub.2.2H.sub.2 O dissolved in about 200 ml of ethanol, followed by mixing by stirring to effect dissolution thereof. Ethanol was added to the resulting solution to total 250 ml to prepare a homogeneous raw material solution. Subsequently, the raw material solution was atomized and heat-decomposed in the same manner as in Example 18. The results of X-ray diffraction measurement of the resulting powder are shown in FIG. 4. As is apparent from FIG. 4, it was confirmed that the formed powder included both of a crystalline phase of chloroapatite and an amorphous phase.

(a): A powder as obtained by the process of Example 20 was heated up in an atmosphere of air at a rate of 5.degree. C./min to a given temperature, at which it was then kept for 1 hour, followed by cooling at a rate of 5.degree. C./min. In such a heat treatment process, the experiments were conducted at 400.degree., 600.degree., 800.degree., and 1,000.degree. C. as that given temperature. As a result of X-ray diffraction measurement of the product heat-treated at 400.degree. C., substantially the same diffraction pattern of chloroapatite as in FIG. 4 was obtained. The diffraction intensities were slightly larger to demonstrate growth of crystallites. On the other hand, the results of X-ray diffraction measurement of the products heat-treated at 600.degree. and 800.degree. C. concurred with the characteristic diffraction peaks of calcium chlorophosphate (Ca.sub.2 PO.sub.4 Cl) disclosed in ASTM Card 19-247. The results of X-ray diffraction measurement of the product heat-treated at 800.degree. C. are shown in FIG. 5. As a result of X-ray diffraction measurement of the product heat-treated at 1,000.degree. C., there was obtained a diffraction pattern in which the diffraction peak assigned to calcium chlorophosphate decreased and a diffraction line attributed to formation of chloroapatite was recognized. It was confirmed by this series of experiments that, in the course of heating, the powder including both phases, a chloroapatite phase and an amorphous phase, (powder as obtained in the process of Example 20), the chloroapatite remained as such at 400.degree. C., changed into calcium chlorophosphate at 600.degree. to 800.degree. C., and changed into chloroapatite again at 1,000.degree. C. or more.

(b) A powder as obtained in Example 20 was heated up in an atmosphere of air at a rate of 5.degree. C./min to 1,200.degree. C., at which it was kept for 1 hour, followed by cooling at a rate of 5.degree. C./min. The product obtained by this treatment was dissolved in nitric acid, and subjected to a test of examining whether or not a milky turbidity is made with a silver nitrate solution, thereby to confirm formation of a silver chloride precipitate. The results of X-ray diffraction measurement of the above-mentioned product are shown in FIG. 6. As is apparant from FIG. 6, this product was chloroapatite. Main peaks consist of a diffraction peak (211) at a diffraction angle 2 .theta. of 31.4.degree. and superposed diffraction peaks (112) and (300) at 2 .theta. of 32.3.degree..

15.58 g of (C.sub.2 H.sub.5 O).sub.3 P was added to a solution of 36.90 g of Ca(NO.sub.3).sub.2.4H.sub.2 O dissolved in about 180 ml of ethanol, followed by mixing by stirring to effect dissolution. Subsequently, an aqueous ammonium bromide solution prepared by dissolving 4.90 g of NH.sub.4 Br in 25 ml of water was well admixed with the resulting solution, and then additionally admixed with ethanol to total 250 ml to prepare a homogeneous raw material solution. Subsequently, the raw material solution was atomized and thermally decomposed in the same manner as in Example 18. The obtained powder was heat-treated in the same manner as in Example 21. As a result of X-ray diffraction measurement of the product obtained, the same diffraction pattern of hydroxyapatite (hexagonal) as in FIG. 3 was obtained, and it included a residual peak of liberated CaO. The product was dissolved in nitric acid, and subjected to detection of bromine with a silver nitrate solution. A silver bromide precipitate was formed so that the presence of bromine was confirmed. Thus, it was understood that the product obtained in this Example was an apatite solid solution of hydroxyapatite with bromoapatite.

15.58 g of (C.sub.2 H.sub.5 O).sub.3 P was added to a solution of 36.87 g of CaBr.sub.2.2H.sub.2 O dissolved in about 180 ml of ethanol, followed by mixing by stirring to effect dissolution. Ethanol was added to the resulting solution to total 250 ml to prepare a homogeneous raw material solution. Subsequently, the raw material solution was atomized and thermally decomposed in the same manner as in Example 18. The resulting powder was heat-treated in the same manner as in Example 21 (b). As a result of X-ray diffraction measurement of the product obtained, the same diffraction pattern of hydroxyapatite (hexagonal) as in FIG. 6 was obtained. The presence of bromine was confirmed with a silver nitrate solution. Thus, it was understood that the above-mentioned product was bromoapatite.

Ca(NO.sub.3).sub.2.4H.sub.2 O, CaCl.sub.2, CaCl.sub.2.2H.sub.2 O, CaBr.sub.2.2H.sub.2 O, NH.sub.4 Cl, (CH.sub.3 O).sub.3 P, (C.sub.2 H.sub.5 O).sub.3 P, [(CH.sub.3).sub.2 CHO].sub.3 P, [CH.sub.3 (CH.sub.2).sub.3 O].sub.3 P, (C.sub.6 H.sub.5 O).sub.3 P, (C.sub.6 H.sub.5).sub.3 PO, (C.sub.2 H.sub.5 O).sub.2 POH, methanol, ethanol, isopropanol, acetone, H.sub.2 O, etc. were used as raw materials. Raw material formulations as listed in Table 2 were prepared. They were each subjected to the procedure of atomization and thermal decomposition under the same conditions as in Example 18 except for the conditions as listed in Table 2 to synthesize various calcium-phosphorus-apatites.

A test was conducted in accordance with the following procedures (a) and (b). The effect of addition of aqueous ammonia to a raw material solution could be confirmed by the test.

(a): 11.63 g of (CH.sub.3 O).sub.3 P was admixed with a solution of 36.90 g of Ca(NO.sub.3).sub.2.4H.sub.2 O dissolved in about 150 ml of methanol to effect dissolution, and then admixed with 16.7 ml of aqueous ammonia (28%). Thereafter, methanol was added to the resulting mixture to total 250 ml to prepare a homogeneous raw material solution. Subsequently, the raw material solution was atomized and thermally decomposed in the same manner as in Example 18.

(b): Preparation, and atomization and thermal decomposition of a raw material solution were made under the same conditions as in the above-mentioned procedure (a) except that no aqueous ammonia was added to the raw material solution. As a result of X-ray diffraction analysis of powders formed by the procedures (a) and (b), both of the powders comprised hydroxyapatite as the main component, while the powder obtained by the procedure (a) involving addition of aqueous ammonia to the raw material solution has a main peak diffraction intensity 4 times larger than that of the powder obtained by the procedure (b). In addition to the peak assigned to hydroxylapatite, a peak assigned to CaO was scarcely recognized in the powder obtained by the procedure (a), while a clear peak assigned to CaO was recognized in the powder obtained by the procedure (b). Concerning the intensity ratio, the CaO peak diffraction intensity for the powder obtained by the procedure (a) was as low as about 1/6 of that of the powder obtained by the procedure (b). These facts demonstrate that addition of aqueous ammonia effectively increases the hydroxyapatite yield and effectively suppresses the liberation formation of CaO.

A powder as obtained by the process of Example 36 (a) was heated up in an atmosphere of air at a rate of 5.degree. C./min to 600.degree. C., at which it was then heat-treated for 2 hours, followed by cooling at a rate of 5.degree. C./min. As a result of X-ray diffraction analysis of the product obtained by this heat treatment, there was recognized only a peak assigned to further grown hydroxyapatite, crystals of which were further grown. A peak assigned to CaO as scarcely recognized in the powder formed in Example 36 (a) disappeared and was not recognized at all.

Ca(NO.sub.3).sub.2.4H.sub.2 O, (CH.sub.3 O).sub.3 P, (C.sub.2 H.sub.5 O).sub.3 P, [CH.sub.3 (CH.sub.2).sub.3 O].sub.3 P, (C.sub.2 H.sub.5 O).sub.2 POH, methanol, ethanol, aqueous ammonia (28%), a 30% aqueous trimethylamine solution, triethylamine, tri-n-butylamine, etc. were used as components of raw material solutions. Raw material formulations as listed in Table 3 were prepared. They were each subjected to the procedure of atomization and thermal decomposition under the same conditions as in Example 36 except for the conditions as listed in Table 3 to synthesize hydroxyapatites. In every Example, it was confirmed that the hydroxyapatite yield increased as compared with that in the case where neither aqueous ammonia nor amine was added to the raw material solution.

(1) Preparation of Fine Spheres of Hydroxyapatite

Ca(NO.sub.3).sub.2.4H.sub.2 O and H.sub.3 PO.sub.4 in such amounts as will provide an atomic ratio (Ca/P) of calcium to phosphorus of 1.67 were dissolved in methanol to prepare a solution having a whole solute concentration of 2 mol/liter. 25 ml of HNO.sub.3 was added to 1 liter of the resulting solution to prepare a homogeneous solution. Subsequently, the solution was atomized into a flame coming from a gas burner at a rate of 8 ml/min (atomizing air pressure: 0.7 kg/cm.sup.2) by using a two fluid atomization nozzle to cause a thermal decomposition reaction. The resulting powder was collected by a cyclone. As a result of X-ray diffraction analysis of the powder, it was found to be hydroxyapatite. As a result of scanning electron microscope (SEM) observation, it was found to be a powder including spherical particles of about 1 to 37.mu.m. The formed powder was classified by a pneumatic classification machine to extract only regular hydroxyapatite spheres of 5 to 15 .mu.m.

(2) Packing Pressurization Test

The hydroxyapatite particles obtained in the procedure (1) were dispersed in pure water. The resulting dispersion was subjected to a packing pressurization test under the varied pressures of 100 to 500 kg/cm.sup.2 with a stainless high-performance liquid chromatography column of 4 mm in inner diameter and 250 mm in length according to the slurry packing method. After the pressurization test, the packed column was dismantled, and the packed hydroxyapatite was observed by a SEM. Deformation and destruction of the particles were not recognized even after application of a pressure as high as 500 kg/cm.sup.2. The hydroxyapatite particles recovered by dismantling the packed column were subjected again to a packing test according to the same procedure and method as described above. The packing work could be made in the same way as in the first work. The hydroxyapatite particles recovered by dismantling the column were observed by a SEM to find neither deformation nor destruction thereof.

(3) Column Chromatography

The spherical hydroxyapatite particles prepared by the procedure as described in (1) were dispersed in an aqueous NaH.sub.2 PO.sub.4 solution (1/10 M), and packed in a stainless column of 4 mm in inner diameter and 150 mm in length according to the slurry packing method (packing pressure: 170 kg/cm.sup.2).

Egg lysozyme was subjected to a measurement with this column by using a sodium phosphate buffer solution having a pH of 6.8 according to the linear gradient method (gradient: 3.49.times.10.sup.-2 M/ml). When the liquid flow rate was varied from 0.5 to 2.0 ml/min (3.13 to 12.5 ml/min as converted in a column of 1 cm in inner diameter), the measurement pressure varied from 19 kg/cm.sup.2 (at 0.5 ml/min) to 81 kg/cm.sup.2 (at 2.0 ml/min). Under various conditions, the measurement was conducted stably without any troubles such as blocking. This proves that the packing material according to the present invention is suitable for chromatography at high pressures around 80 kg/cm.sup.2. This also proves that the packing material according to the present invention allows a low viscosity solution of a protein such as lysozyme to be flowed at a high flow rate of 10 ml/min or more as converted in a column of 1 cm in inner diameter, and hence provides a superior characteristic of a column packed therewith to those of conventional columns.

The hydroxyapatite particles prepared in Example 47 (1) were heat-treated in air at 800.degree. C. for 2 hours. The analysis of particles obtained showed an increase in the X-ray diffraction intensity of hydroxyapatite with growth of crystals and no proof of decomposition of hydroxyapatite. No destruction of the shape of the spherical particles was also recognized in SEM observation. Subsequently, the hydroxyapatite particles thus heat-treated were packed in a stainless column of 4 mm in inner diameter and 150 mm in length by the same procedure as in Example 47 (3).

Egg lysozyme and cytochrome c were subjected to a measurement with the resulting column at random to carry out a repeated service test of the column for a total of 30 times with egg lysozyme and a total of 20 times with cytochrome c for a grand total of 50 times. Blocking or non-blocking of the column and repeatability of the retention time thereof were examined. Every measurement was carried out at a flow rate of 0.5 ml/min according to the linear gradient method using a sodium phosphate buffer solution having a pH of 6.8 (gradient: 3.49.times.10.sup.-2 M/ml). The pressure in the measurement was 20 to 23 kg/cm.sup.2. No blocking of the column occurred until the last time of the test, and very good chromatograms were obtained. As for the repeatability, a variation of the egg lysozyne retention time in the measurement was examined to find a minimum value of 22.05 minutes and a maximum value of 22.37 minutes throughout the measurement. Although this measurement was not intended to involve such severe test contents as to find the repetition accuracy and repeatability accuracy of the retention time by statistically processing the measured data, the variation was within 2% as the common yardstick of evaluation of the repeatability as described in manuals or the like concerning liquid chromatography, thus proving that the column used in the measurement had a high repeatability. After the repetition test, the column was dismantled to carry out SEM observation of the spherical particles of hydroxyapatite. None of abnormalities such as deformation and destruction of the particles were recognized.

Ca(NO.sub.3).sub.2.4H.sub.2 O and H.sub.3 PO.sub.4 in such amounts as will provide an atomic ratio of calcium to phosphorus of 1.67 were dissolved in 2-methoxyethanol to prepare a solution having a whole solute concentration of 1.0 mol/liter. The solution was subjected to fast thermal decomposition of atomized solution in the same manner as in the process of fast thermal decomposition of atomized solution as performed in Example 47 (1) to obtain a product which was in the form of a mixture of spherical hydroxyapatite particles of 1 to 6 .mu.m and clumpy hydroxyapatite particles consisting of aggregates of fine crystals. The product was classified by a pneumatic classification machine to extract only spherical hydroxyapatite particles of 3 to 6 .mu.m. The spherical hydroxyapatite particles thus obtained by classification were packed in a stainless column (inner diameter: 4 mm, length: 100 mm) under 400 kg/cm.sup.2 by the slurry packing method. A test evaluation of them as a packing material was carried out in the same manner as in Example 48. The hydroxyapatite packing material obtained in this Example provided good results of mechanical strength, durability, and repeatability as in Example 48.

A solution of 220.25 g of Ca(CH.sub.3 COO).sub.2.H.sub.2 O dissolved in 450 ml of 6N HNO.sub.3 was mixed with a solution of 85.85 g of H.sub.3 PO.sub.4 (content:85.6%) dissolved in 300 ml of methanol to prepare a homogeneous solution. The solution was subjected to fast thermal decomposition of atomized solution in the same manner as in Example 47 (1) to obtain spherical hydroxyapatite particles of about 1 to 25 .mu.m. The product was classified by a pneumatic classification machine to extract hydroxyapatite particles of 5 to 15 .mu.m, which were then heat-treated at 600.degree. C. for 2 hours. Subsequently, column packing and a liquid chromatography evaluation test were effected in the same manner as in Example 49.

The hydroxyapatite particles obtained in this Example did not have as regularly spherical a shape as the particles obtained in Example 49, and included some distorted or uneven spheres. However, the same results as in Example 49 were obtained for the chromatographic properties.

Ca(NO.sub.3).sub.2.4H.sub.2 O and (C.sub.2 H.sub.5 O).sub.3 P in such amounts as will provide an atomic ratio of calcium to phosphorus of 1.67 were dissolved in ethanol to prepare a solution having a whole solute concentration of 1 mol/liter. 500 ml of the solution was admixed with 34 ml of aqueous ammonia (concentration: 28%) to prepare a homogeneous solution. Subsequently, the solution was subjected to fast thermal decompositon of atomized solution in the same manner as in Example 47 (1) to obtain a product consisting of spherical hydroxyapatite particles of about 1 to 8 .mu.m. the product was heat-treated at 800.degree. C. for 1 hour, and then packed in a stainless column of 4 mm in inner diameter and 50 mm in length under a packing pressure of 500 kg/cm.sup.2 by the same slurry packing method as in Example 47 (3).

Cytochrome c was subjected to a measurement with the resulting column and with a sodium phosphate buffer solution having a pH of 6.8 at a liquid flow rate of 0.5 ml/min according to the linear gradient method (gradient: 3.49.times.10.sup.-2 M/ml). The chromatogram in the measurement is shown in FIG. 7. In view of the fact that cytochrome c includes at least two kinds of molecular species, one of which contains an iron atom in an oxidized state, and another one of which contains an iron atom in a reduced state, two mutually clearly separated peaks in FIG. 7 are believed to be assigned to these molecular species (the left peak assigned to the oxidized form of molecules and the right peak assigned to the reduced form of molecules), thus proving that the column in this Example has a high separating capacity.

Ca(NO.sub.3).sub.2.4H.sub.2 O and H.sub.3 PO.sub.4 in such amounts as will provide an atomic ratio of calcium to phosphorus of 1.67 were dissolved in methanol to prepare a solution having a whole solute concentration of 0.5 mol/liter. The solution was subjected to fast thermal decomposition of atomized solution in the same manner as in Example 47 (1) to obtain a product consisting of spherical hydroxyapatite particles of about 1 to 9 .mu.m. The product was heat-treated at 600.degree. C. for 2 hours, and then packed in a 50 mm column in the same manner as in Example 51. Cytochrome c was subjected to measurement with the resulting column in the same manner as in Example 51. The separating capacity of the column was as high as in Example 51.

Ca(NO.sub.3).sub.2.4H.sub.2 O and (CH.sub.3 O).sub.3 P in such amounts as will provide an atomic ratio of calcium to phosphorus of 1.67 were dissolved in methanol to prepare a solution having a whole solute concentration of 1 mol/liter. 1 liter of the solution was admixed with 68 ml of aqueous ammonia (concentration: 28%) to prepare a homogeneous solution. Subsequently, the solution was subjected to fast thermal decompositon of atomized solution in the same manner as in Example 47 (1) to obtain a product consisting of spherical hydroxyapatite particles of about 0.5 to 5 .mu.m. The product was classified by a pneumatic classification machine to remove particles of 1 .mu.m or less. The residual product was then packed in a stainless column of 4 mm in inner diameter and 30 mm in length under a packing pressure of 500 kg/cm.sup.2 by the same slurry packing method as in Example 47 (3).

Lysozyme was subjected to a measurement with the resulting column under the same conditions as in Example 51. It was found that the hydroxyapatite packing material obtained in this Example had good chromatographic properties such as mechanical strength and repeatability as in Example 48.

A solution having a whole solute concentration of 0.5 mol/liter was prepared in substantially the same manner as in Example 53 except that (C.sub.2 H.sub.5 O).sub.2 POH and ethanol were used as the phosphorus compound and the organic solvent, respectively. The solution was subjected to fast thermal decomposition of atomized solution to obtain a product consisting of spherical hydroxyapatite particles of about 0.5 to 3 .mu.m in size. Classification, packing, and measurement of the product were made in the same manner as in Example 53. As a result, good chromatographic properties as in Example 53 were secured.

CaBr.sub.2.2H.sub.2 O and phosphorous acid in such amounts as will provide an atomic ratio of calcium to phosphorus of 1.67 were dissolved in methanol to prepare a solution having a whole solute concentration of 1 mol/liter. The solution was then subjected to fast thermal decomposition of atomized solution in the same manner as in Example 47 (1) to obtain spheres of about 0.5 to 5 .mu.m in size in the form of an apatite solid solution of bromoapatite with hydroxyapatite. The product was heat-treated at 300.degree. C. for 1 hour, and then packed in a 50 mm column in the same manner as in Example 51. Cytochrme c was subjected to a measurement with the resulting column in the same manner as in Example 51. The obtained chromatogram showed that the separating capacity of the column was as high as in Example 51.

Ca(NO.sub.3).sub.2.4H.sub.2 O and phosphoric acid in such amounts as will provide an atomic ratio (Ca/P) of calcium to phosphorus of 1.67 were dissolved in a solution prepared by adding 40 ml of HNO.sub.3 and 150 ml of CH.sub.3 COOH to 1 liter of a mixed solvent of methanol and water (6:1) to prepare a solution having a whole solute concentration of 2 mol/liter. The raw material solution was subjected to fast thermal decomposition of atomized solution in the same manner as in Example 47 (1). The resulting powder was heat-treated in air at 1,000.degree. C. for 1 hour. The subsequent SEM observation of the resulting product revealed a sintered body consisting of hollow particles having a spherical hull network structure of 5 to 26 .mu.m as a whole which was formed by a network-like connection of hydroxyapatite grains of 0.2 to 0.4 .mu.m as well as broken pieces thereof. The powder thus heat-treated was then subjected to a pulverization treatment with a miniature test sample mill. It was capable of being pulverized into hydroxyapatite grains of about 0.4 .mu.m.