JPH0414748B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a packing material for chromatography comprising hydroxyapatite, Ca ₅ (PO ₄ ) ₃ OH, having novel properties. More specifically, the present invention provides a chromatographic packing material comprising microspherical hydroxyapatite particles. The chromatography packing material according to the present invention is made of microspherical hydroxyapatite that has high mechanical strength, excellent durability, and reproducibility, and is particularly suitable as a packing material for column chromatography. The above usability is extremely great.

Recently, high-performance liquid chromatography and electrophoresis equipment are often used for the separation and purification of chemical and biochemical substances. It is widely used because it is easy to handle as it only requires passing the material to be separated. Until now, silica and alumina have been the mainstream filler materials used in such applications. However, with existing high-performance liquid chromatography using ion exchange type or molecular sieve type packing materials, there is a limit to the separation of biopolymers with large molecular weights, and the separation of biopolymers with a large molecular weight is limited to approximately 10 to the fifth power Dalton. It is considered impossible to separate and purify proteins with higher molecular weights. On the other hand, with the recent progress in biotechnology, there is a strong desire to develop technical means to break through these separation limitations.

By the way, recently, columns filled with hydroxyapatite have become commercially available, and it has become possible to separate and purify biopolymers with a size of 10 5 Daltons or more, which was previously impossible.
It is attracting attention as a response to the industrialization of biotechnology, which requires the separation and purification of nucleic acids, enzymes, etc.

However, conventional hydroxyapatite fillers have low mechanical strength, are easily pulverized, have poor reproducibility of filling properties and chromatographic properties, are poor in durability, have a short lifespan, and are expensive. Due to various drawbacks, it has not been widely used despite its excellent properties for separating and purifying biopolymers, and there is a strong desire to resolve these drawbacks.

In other words, until now hydroxyapatite has been produced using the Teiselius method [A.Tiselius, S.Hjerte´n
and O¨.Levin, Arch.Biochem.Biophys., 65 ,
132-155 (1956)], Ca/P
It is difficult to control the ratio, and because the phosphoric acid content cannot be removed sufficiently, it has the disadvantage of being sensitive to strong alkalis and organic solvents.Furthermore, the shape of the filler particles is a collection of easily broken plate-shaped crystals or microcrystals. Since it consists of a solid body and has low mechanical strength, it has the disadvantage of being easily pulverized during filling or measurement operations under high pressure. Furthermore, since the particles are composed of aggregates of plate-shaped or columnar microcrystals, they have poor fluidity during filling, and as mentioned above, their mechanical strength is low, so it is difficult to separate the filler particles by applying high pressure. It was extremely difficult to pack the particles densely and to make the gaps between the particles uniform. In addition, there were many practical difficulties, such as the need to take special care in handling the packed column so as not to change the packing state of the column.

Packing materials for high performance liquid chromatography must have extremely small particle sizes. That is,
The diffusion coefficient of substances in liquids is significantly smaller than that of gases, so in order to reduce the effects of non-equilibrium partitioning in liquid chromatography, it is necessary to make the mass transfer distance in the mobile phase sufficiently small. . Therefore, in order to increase the speed of analysis in liquid chromatography, a packing material with an extremely small particle size is required compared to that in gas chromatography. Recently, the development of various fillers has progressed, and as their performance has improved, particle sizes have also progressed, and particles with a particle size of about 3 to 5 μm have come into practical use.

As for hydroxyapatite, particle miniaturization has been achieved to some extent, but as mentioned above, hydroxyapatite particles produced by conventional technology have the drawback of having low mechanical strength and being easily pulverized. Since it consists of an aggregate of plate-shaped or columnar microcrystals, when used as a filler, it is inevitable that the particle shape will be irregular.

In addition to reducing the particle size of the filler, making the filler particles spherical not only has the practical advantage of facilitating the packing operation, but also reduces the cause of peak broadening in the chromatogram. It is possible to reduce the variation in the gap between packed particles, which is one of the
This is extremely important from the viewpoint of improving chromatographic properties. However, regarding the spheroidization of hydroxyapatite fillers using conventional technology, only spherical molded particles with a size of about 100 μm, which are technically easy to granulate, have been produced.
Although production of extremely small spherical hydroxyapatite of several μm to 10-odd μm, which is suitable for high-performance liquid chromatography, is strongly desired, due to technical difficulties, it has not been possible to date to date. , had not been realized.

The present inventors previously proposed a novel method for producing hydroxyapatite, in which water or a water-compatible organic solvent was used alone as a solvent, or a water-compatible organic solvent and a water-compatible organic solvent were used as a solvent. Using a mixed solvent of We provide a method for producing microspherical hydroxyapatite by causing a decomposition reaction, and by selecting the manufacturing conditions at that time, we can directly produce microspherical hydroxyapatite from submicrons to several tens of micrometers without any molding operation. It has been discovered that it is possible to produce hydroxyapatite particles having a micron spherical shape (Japanese Patent Application No. 1983-
No. 265270 [Unexamined Japanese Patent Publication No. 146704], Patent Application No. 146704, Patent Application No. 1983-
No. 38655 (see Japanese Patent Application Laid-Open No. 61-201612). Further, through subsequent research, the present inventors found that the spherical particles obtained in this way are not only the most desirable particle shape for chromatography filler particles, but also have extremely excellent chromatographic properties. It was found that In other words, hydroxyapatite particles with this novel property not only have the advantage of being able to directly produce spherical particles of several micrometers without any molding operation, which was impossible with conventional techniques, but also have the advantage of being able to produce Ca/ Because the manufacturing method allows for easy control of the P ratio, the produced hydroxyapatite spherical particles have excellent chemical stability, extremely high mechanical strength, and excellent durability, which makes them ideal for fillers. It was found that it has excellent properties. The present invention has been made based on this knowledge.

The present invention will be explained in detail below.

In the present invention, the hydroxyapatite spherical particles of several micrometers to several tens of micrometers produced by the above-mentioned method of the present inventors are used as a column packing material either as they are or after further classification to make the particle size uniform. .

Since the above-mentioned hydroxyapatite has a spherical particle shape, it has good fluidity during filling and does not cause the bridging phenomenon that tends to occur when filling angular particles, and the filling operation can be performed based on normal operating procedures. A uniformly packed column can be easily manufactured by simply doing the following. Furthermore, when filling well-shaped spherical particles with uniform particle diameters, not only is the filling operation easier, but also the interparticle gaps formed by the uniformly filled hydroxyapatite particles This also eliminates variations in the gaps between packed particles, which is one of the causes of decreased column separation performance due to the vortex flow that causes differences in the linear velocity of solute flow in the column axis direction. It has the advantage of being able to

Minimization of particle size and uniformity of particle size have been achieved to some extent with hydroxyapatite packing materials produced by conventional techniques, and when packing into columns, variations in the gaps between packed particles can be reduced. Despite these efforts, hydroxyapatite fillers produced by conventional techniques have a variety of particle shapes, such as plate-like or columnar aggregates of microcrystals. Moreover, since the mechanical strength is low, it has not been possible to apply sufficient pressure to achieve uniform filling and completely eliminate variations in the interparticle gaps.

As is well known, there are three factors that reduce the separation power of column chromatography: (1) diffusion, (2) non-equilibrium, and (3) vortex flow. All of these have the effect of increasing the band broadening of the solute and therefore increasing the height of the theoretical plate. (1) is the diffusion of solute in the column axis direction, but pumps that can stably pump liquid at high pressure are now in practical use, so chromatographic separation can be performed at a high flow rate where solute diffusion can be ignored. By doing so, the effect of (1) can actually be eliminated. (2) is an effect that occurs when equilibrium is not necessarily established in each part of the column, and is more pronounced as the particle size of the packing material becomes larger. In addition, (3) is an effect in which a vortex is generated because the packing material is positioned to block the flow, causing a difference in the linear velocity of the solute in the column axis direction. The effect of this phenomenon becomes greater as the particle size becomes larger, the particle shape is irregular, or the filling method is uneven. A corollary of the well-known above theory is that in order to reduce the height of the theoretical plate and increase the resolution of the column, one should use a packing material with as fine particles as possible, and the particles should be as uniformly arranged as possible. It can be seen that it is important to fill the material evenly.
From this, it is clear that the microspherical hydroxyapatite used in the present invention satisfies the properties conventionally desired as a filler.

In addition, the microspherical hydroxyapatite produced by the above-mentioned method has extremely high mechanical strength, with the densely packed particles not breaking even when a filling pressure of 500 kg/cm ² is applied, for example. Therefore, it has a practically important advantage in that the filling operation and the handling of the filled column are extremely easy. In column chromatography, increasing the separation power is achieved by making the particle size of the packing material extremely small, as mentioned above, but on the other hand, by packing fine particles, The interparticle gaps also become narrower, thus resulting in a significant reduction in flow rate. In practice, in order to solve this problem, a method is used in which high pressure is applied to the column to increase the flow rate, but in this case, considerable pressure is applied to the packing material.

Therefore, if the mechanical strength of the packing particles used is low, the packed particles will be destroyed and pulverized during packing or measurement, which will cause column clogging and shorten the column life. becomes. While conventional hydroxyapatite is composed of plate-shaped or aggregates of microcrystals and has the disadvantage of being easily broken, the filler of the present invention is composed of particles with extremely high mechanical strength. , the problems in this regard can be solved all at once.

Furthermore, whether a packing material consisting of extremely small particles ranging from several micrometers to several tens of micrometers can be easily packed into a column with good reproducibility is a matter of practical importance as a characteristic that the packing material should have. be. However, the conventional hydroxyapatite fillers have poor fluidity and are difficult to fill uniformly because they have a variety of particle shapes, such as plate shapes and aggregates of microcrystals. , which required special ingenuity. Therefore, uniform packing of large columns with larger diameters is made even more difficult due to the low mechanical strength of the particles and the technical problems of suppressing the variation in interparticle spacing. However, the ease of filling work was not as good as could be expected.

However, since the microspherical hydroxyapatite used in the present invention has a well-ordered spherical shape,
Since it has good fluidity during filling and high mechanical strength, it is possible to easily perform filling operations with high reproducibility. In addition, the packing operation can be easily performed not only for packing small-diameter analytical columns but also for manufacturing large-diameter preparative columns without worrying about the destruction of packed particles or uneven packing. The advantage is that it can be done.

Furthermore, the hydroxyapatite used in the present invention is not only a spherical particle with high mechanical strength as described above, but also has a high chemical stability because the Ca/P ratio can be easily controlled. Excellent, resistant to organic solvents and strong alkalis, and does not become pulverized during use. Because it uses hydroxyapatite, which is extremely stable both physically and chemically, the reproducibility of filling is high, and therefore the reproducibility of chromatographic properties such as retention time and retention capacity is high, making it suitable for repeated use. It is possible to provide highly durable columns that can withstand This makes it possible to provide a hydroxyapatite column with a long life, and has the effect of greatly reducing the cost of using the column, which is of extremely great practical significance.

Next, a method for producing microspherical hydroxyapatite used in the present invention will be specifically explained. First, as a starting material, water or a water-compatible organic solvent is used alone as a solvent, or a mixed solvent of a water-compatible organic solvent and water is used as a solvent. , a soluble calcium compound and a phosphorus compound are added and mixed, and the whole is dissolved to prepare a raw material solution. The order of mixing the raw materials for preparing the solution at this time is not necessarily limited to the above-mentioned order, and it is sufficient that the raw materials are mixed together in an order that does not cause precipitation. Regarding the above raw material solutions, depending on the combination of calcium compounds, phosphorus compounds, and solvents, it may be difficult to prepare a uniform solution, or the solution once prepared may become cloudy over time. However, in some cases, these problems can be solved by adding an acid. Furthermore, when an organic compound is used as the phosphorus compound, adding aqueous ammonia to the solution may have the effect of suppressing the production of by-products and free CaO and improving the production rate of hydroxyapatite. In this way, acid or aqueous ammonia can be added to the solution, if necessary. The time for mixing varies depending on the raw materials selected, but sufficient stirring is performed until all the raw materials are dissolved and a solution of uniform composition is obtained. Next, the solution prepared in this way is sprayed into a fire such as a gas burner using a pressurized nozzle or a rotating disk using a public spraying method, or in a gas furnace, electric furnace, etc.
The raw material solution is sprayed into a heating zone that has been preheated to a desired temperature within the temperature range where thermal decomposition occurs using high-temperature gas or the like. When a flammable organic solvent that is compatible with water or a mixed solvent of a flammable organic solvent that is compatible with water and water is used as a solvent, the combustion of the flammable organic solvent itself The heat of combustion can also be used as a heat source for the pyrolysis reaction. In addition, it is used to promote evaporation of a mixed solvent of water and an organic solvent that is compatible with water without burning the organic solvent,
If water is used alone as a solvent, a heat source such as a gas furnace as described above is required. Alternatively, both the combustion heat of the organic solvent and a heating source such as a gas furnace can be used in combination. Note that even when an organic solvent that is compatible with water is used alone as a solvent, depending on the combination of raw material compounds, the reaction system may be mixed with water due to residual moisture or crystal water in the raw material compounds. In some cases, the system uses a mixed solvent with an organic solvent, but in any of these cases, as long as the raw material solution can be prepared without precipitation, it can be used as a solvent in this method. I can do it.

Calcium compounds and phosphorus compounds used in this method include Ca(NO ₃ ) _{2.4H 2} O,
Ca(CH ₃ COO) ₂・H ₂ O, CaCl ₂・2H ₂ O,
CaBr ₂・2H ₂ O, CaHPO ₄・2H ₂ O, NH ₄ H ₂ PO ₄ ,
NH ₄ H ₂ PO ₃ , NH ₄ H ₂ PO ₂ , H ₃ PO ₄ , H ₃ PO ₃ , H ₃
PO ₂ , (CH ₃ O) ₃ P, (C ₂ H ₅ O) ₃ P, [(CH ₃ ) ₂
CHO] ₃ P, [CH ₃ (CH ₂ ) ₃ O] ₃ P, (C ₆ H ₅ ) ₃ P,
Examples include (C ₂ H ₅ O) ₂ POH and (C ₆ H ₅ ) ₃ PO.
In addition, as the organic solvent, it is preferable to use alcohols such as methanol, ethanol, isopropanol, and acetone, but they are not particularly limited to these. By mixing, a homogeneous solution that does not produce a precipitate can be obtained. Any combination of raw materials may be used as long as it can prepare the following. For this purpose, nitric acid and hydrochloric acid are suitable as acids to be added as necessary.

The powder produced by the spray pyrolysis reaction by the above method can be made up of submicron particles that are densely or coarsely aggregated and adhered and sintered to each other, depending on the type and combination of raw material compounds and the spray pyrolysis conditions. , as a whole, it is a hollow spherical micro powder, a micro spherical powder packed to the inside, or a spherical powder that is broken into powders of various shapes, etc.
You can change it in many ways. Among these, well-shaped spherical particles can be suitably used as the filler for chromatography of the present invention, regardless of whether they are hollow or not.

In addition, when the powder produced by the above-mentioned spray pyrolysis reaction is further placed in various atmospheres such as air, air with water vapor added, and an inert atmosphere, the temperature range in which hydroxyapatite can stably exist without decomposition is determined. By heat-treating at a desired temperature, it is possible to promote the degree of crystallinity and sintering and further increase the mechanical strength.

If necessary, the microspherical hydroxyapatite produced as described above can be classified by various known methods in order to make the particle size more uniform.

EXAMPLES Below, the present invention will be specifically explained with reference to Examples. However, the present invention is not limited to the following examples.

Example 1 1 Production of microspherical hydroxyapatite Ca(NO ₃ ) _{2.4H 2} O and H ₃ PO ₄ were dissolved in methanol so that the atomic ratio of calcium to phosphorus (Ca/P) was 1.67. , the total solute concentration is 2mol/
A solution was prepared as follows, and 25 ml of HNO ₃ was added to this solution 1 to prepare a homogeneous solution. Next, this solution was sprayed into the flame of a gas burner at a rate of 8 ml per minute (spraying air pressure = 0.7 Kg/cm ² ) using a two-fluid spray nozzle to cause a thermal decomposition reaction, and the resulting powder was was collected using a cyclone. X-ray diffraction analysis of this powder revealed that it was hydroxyapatite, and electron microscopy (SEM) observation revealed that particles of 0.1 to 0.5 microns were tightly adhered and sintered, making the powder approximately 1 to 37 microns in size as a whole.
It was found that the powder contained powder that had become spherical particles. The resulting powder was classified using an air classifier, and only well-shaped spherical hydroxyapatite with a particle size of 5 to 15 μm was taken out.

2 Pressure test during filling The hydroxyapatite particles obtained in 1 were dispersed in pure water, and using a stainless steel column for high performance liquid chromatography with an inner diameter of 4 mm and a length of 250 mm,
A filling test was carried out using a slurry filling method at various pressures ranging from 100 to 500 Kg/cm ² . After the pressure test, the packed column was disassembled and the packed hydroxyapatite was observed using SEM, and it was found that it was 500Kg/cm ²
No deformation or destruction of the particles was observed even after application of high pressure. Therefore, we performed a packing test using the same method as above again using the hydroxyapatite particles recovered by disassembling the packed column.
It was possible to perform the same packing as the first time, and furthermore, the column was disassembled and SEM observation was performed, and no changes such as deformation or destruction were observed in the hydroxyapatite particles.

3 Column chromatography The hydroxyapatite spherical particles produced by the method described in 1 were treated with an aqueous solution of NaH ₂ PO ₄ (1/10
M) with an inner diameter of 4 mm by slurry filling method.
It was packed into a stainless steel column with a length of 150 mm. (Filling pressure = 170Kg/cm ² ).

Using ^this column, the PH
6.8 Egg white lysozyme was measured using a sodium phosphate buffer. The measurement pressure was 19Kg/cm ² (0.5
ml/min) to 81Kg/cm ² (at 2.0ml/min), but measurements were stable under each condition, and no problems such as column clogging occurred. . This shows that the packing material of the present invention is suitable for chromatography at a high pressure of about 80 kg/cm ² . Also,
This means that when the packing material of the present invention is used for a low viscosity protein solution such as lysozyme, the flow rate is 10 ml/1 cm in column inner diameter.
This shows that it has an excellent feature compared to conventional columns, which allows solutions to flow at a high flow rate of min or more.

Example 2 The hydroxyapatite particles produced in Example 1-1 were heat-treated at 800° C. for 2 hours in the air. As a result of analyzing the obtained particles, the X-ray diffraction intensity of hydroxyapatite increased with crystal growth, but no decomposition was observed at all, and no destruction of the shape of the spherical particles was observed by SEM observation. Next, the heat-treated hydroxyapatite particles were packed into a stainless steel column with an internal diameter of 4 mm and a length of 150 mm by the same method as the filling operation in Example 1-3.

Using this column, measurements of egg white lysozyme and cytochrome C were carried out in random order, and the test was repeated 30 times in total for egg white lysozyme, 20 times in total for cytochrome C, and 50 times in total. The presence or absence of clogging and the degree of reproducibility of retention time were investigated. All measurements were PH6.8
Linear gradient method using sodium phosphate buffer (gradient = 3.49 x 10 ^-2 M/ml), the flow rate was
The rate was 0.5ml/min. Pressure during measurement is 20-23Kg/
It was warm in ^cm2 . An extremely good chromatogram was obtained without clogging of the column until the end of the test. Regarding reproducibility, the variation in the retention time of egg white lysozyme in this measurement was investigated, and the minimum and maximum values were 22.05 minutes and 22.37 minutes, respectively, throughout the measurement. This measurement is not intended to be a rigorous test that involves statistical processing of measurement data to determine retention time repeatability or retention time reproducibility.
Usually, it is within 2%, which is the guideline for reproducibility evaluation described in liquid chromatography instruction manuals, etc., indicating that the column used for this measurement has high reproducibility. Recognize. After repeated tests, the column was disassembled and the spherical hydroxyapatite particles were observed by SEM, but no abnormalities such as deformation or destruction of the particles were observed.

Example 3 Ca(NO ₃ ) _{2.4H 2 O} and H ₃ PO ₄ were dissolved in 2-methoxyethanol so that the atomic ratio of calcium to phosphorus was 1.67, and the concentration of the whole solute was
A solution was prepared so that the concentration was 1.0 mol/. Using this solution, spray pyrolysis was carried out in the same manner as the spray pyrolysis method used in Example 1-1, resulting in a hydroxyapatite lump powder consisting of spherical hydroxyapatite particles of 1 to 6 μm and aggregates of microcrystals. A product was obtained which was a mixture of the following substances. This product was classified using an air classifier, and only spherical hydroxyapatite with a size of 3 to 6 μm was taken out. The spherical hydroxyapatite particles obtained by this classification were packed into a stainless steel column (inner diameter 4 mm, length 100 mm) at 400 kg/cm ² using the slurry filling method, and an evaluation test as a filler was conducted in the same manner as in Example 2. Ivy. The hydroxyapatite filler obtained in this example gave good results in terms of mechanical strength, durability, reproducibility, etc., as in Example 2.

Example 4 6 Add Ca(CH ₃ COO) ₂・H ₂ to 450 ml of N HNO ₃
A solution containing 20.25g of O2 and 300ml of methanol
and a solution in which 85.85 g of H ₃ PO ₄ (content 85.6%) was dissolved to prepare a homogeneous solution. This solution,
Spray pyrolysis was carried out in the same manner as used in Example 1-1 to obtain spherical hydroxyapatite with a size of approximately 1-25 μm. This product was classified using an air classifier, hydroxyapatite particles with a particle size of 5 to 15 μm were taken out, and after heat treatment at 600°C for 2 hours, column filling and liquid chromatography evaluation were carried out in the same manner as in Example 3. I did a test.

The shape of the hydroxyapatite particles obtained in this example was not as well-organized as the particles obtained in Example 3, and some spherical particles were somewhat distorted or uneven. However, the same results as in Example 3 were obtained regarding the chromatographic properties.

Example 5 Ca(NO ₃ ) ₂ 4H ₂ O and (C ₂ H ₅ O) ₃ P were dissolved in ethanol so that the atomic ratio of calcium to phosphorus was 1.67, and the concentration of the whole solute was 1 mol/. A solution was prepared, and 34 ml of ammonia water (concentration 28%) was added to 500 ml of this solution and mixed to obtain a homogeneous solution. This solution was then used to carry out spray pyrolysis in the same manner as used in Example 1, part 1 to obtain a product consisting of spherical hydroxyapatite with a size of approximately 1-8 μm.
After heating this product at 800℃ for 1 hour,
Using the same slurry filling method as used in Example 1-3, a filling pressure of 500 Kg/cm ² was applied and the inner diameter was 4.
packed in a stainless steel column with a length of 50 mm.

Using ^this column, the PH
6.8 Using sodium phosphate buffer, liquid flow rate = 0.5ml/
Cytochrome C was measured under conditions of min. A chromatogram obtained during this measurement is shown in FIG. Cytochrome C contains at least two types of iron atoms, one in an oxidized state and one in a reduced state, so the two clearly separated peaks in Figure 1 are due to these two types. This is presumed to be due to the molecular species (the peak on the left is due to oxidized molecules, and the peak on the right is due to reduced molecules), and it can be seen that the column of this example has a high degree of separation.

Example 6 Ca(NO ₃ ) 2.4H _{2 O} and H ₃ PO ₃ were dissolved in methanol so that the atomic ratio of calcium and phosphorus was 1.67, and the concentration of the whole solute was 0.5 mol/ A solution was prepared. This solution was then subjected to spray pyrolysis in the same manner as used in Example 1, 1, to obtain a product consisting of spherical hydroxyapatite with a size of approximately 1-9 μm. This product
After heat treatment at 600° C. for 2 hours, it was packed into a 50 mm column in the same manner as in Example 5. Cytochrome C was measured using this column in the same manner as in Example 5, and showed the same high degree of separation as in Example 5.

Example 7 Ca(NO ₃ ) _{2.4H 2} O and (CH ₃ O) ₃ P are dissolved in methanol so that the atomic ratio of calcium to phosphorus is 1.67, and the concentration of the entire solute is 1 mol/ A solution was prepared as described above, and 68 ml of ammonia water (concentration 28%) was added to this solution 1 and mixed to obtain a homogeneous solution. This solution was then used to carry out spray pyrolysis in the same manner as used in Example 1, part 1 to obtain a product consisting of spherical hydroxyapatite with a size of approximately 0.5-5 μm. This product was classified using an air classifier to remove particles of 1 μm or less, and then the same slurry filling method as used in Example 1-3 was used to apply a filling pressure of 500 Kg/cm ² to produce an inner diameter of 4 mm. , packed into a 30 mm long stanless column.

Using this column, lysozyme was measured under the same conditions as in Example 5. The hydroxyapatite packing material obtained in this example had a high mechanical strength,
It was found that it had good chromatographic properties, such as reproducibility, similar to Example 2.

Example 8 The same method as in Example 7 was used, except that (C ₂ H ₅ O) ₂ POH was used as the phosphorus compound and ethanol was used as the organic solvent, so that the total solute concentration was 0.5 mol/
A solution was prepared and subjected to spray pyrolysis to obtain a product consisting of spherical hydroxyapatite with a size of approximately 0.5-3 μm. Classification of this product,
Both filling and measurement were performed in the same manner as in Example 7. As a result, good chromatographic properties similar to Example 7 were obtained.

[Brief explanation of drawings]

FIG. 1 is a diagram showing a chromatography of cytochrome C performed using the packing material for chromatography of the present invention obtained in one example (Example 6) of the present invention.

Claims (1)

[Claims] 1. Water or a water-compatible organic solvent is used alone as a solvent, or a mixed solvent of a water-compatible organic solvent and water is used as a solvent; A raw material solution is prepared by adding and mixing a soluble calcium compound and a phosphorus compound, and the raw material solution is sprayed into a flame or heating zone to produce microspherical hydroxy A chromatography packing material made of apatite. 2 Water or a water-compatible organic solvent is used alone as a solvent, or a mixed solvent of a water-compatible organic solvent and water is used as a solvent, and a calcium compound that is soluble in the used solvent is used. and a phosphorus compound are added and mixed to prepare a raw material solution, and the raw material solution is sprayed into a flame or a heating zone to generate microspherical hydroxyapatite through a thermal decomposition reaction, which is then further heat-treated. A packing material for chromatography consisting of the obtained hydroxyapatite particles.