JPH06300746A - Filler for liquid chromatography - Google Patents

Abstract

PURPOSE:To obtain a filler composed of hydroxyapatite(HPA) which is porous and whose durability is excellent by a method wherein slurry which contains an HPA particle A synthesized in a specific temperature range and an HPA particle B synthesized in a higher temperature region is granulated and fired. CONSTITUTION:A granulated substance is prepared from slurry which contains an HPA particle A synthesized in a temperature region of less than 800 deg.C and an HPA particle B synthesized in a high temperature region of 800 deg.C or higher and in which the mixture ratio of the particle B is 30 to 60% of the total weight. The granulated substance is fired at 800 deg.C or higher and formed. When the particle A is heated at its firing temperature of 800 deg.C or higher, its volume is contracted due to a sintering reaction. On the other hand, the particle B is a thermally stable single crystal or polycrystal whose volume is not contracted by the same heating. Consequently, when, e.g. the porous particle B having a pore whose diameter is larger than that of the particle A is used, the particle A which has crept into the pore of the particle B is crystallized at the inside and the outside of the pore of the particle when it is fired, a firm bond is produced in such a way that the particle B is taken into due to the sintering reaction, and it is possible to obtain a filler whose pore volume is kept and whose mechanical strength is large.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a packing material for liquid chromatography.

[0002]

BACKGROUND ART Liquid chromatography using hydroxyapatite (hereinafter referred to as HAP) as a packing material has an excellent feature that biopolymers such as proteins and nucleic acids can be separated and purified under mild conditions. Is extremely desirable as a packing material for chromatography which is industrially collected in large quantities. In addition to such excellent chromatographic properties, HAP for a packing material for chromatography in order to reduce the fractionation cost has a high load capacity for biopolymers, a large mechanical strength, and an excellent durability. Must be one. Generally, in chromatography using HAP, HAP as a packing material
It is known that the load capacity for proteins, nucleic acids and the like can be increased by making the porous. As a conventional technique relating to a method for producing a porous HAP as a filler, for example, only a slurry of wet-synthesized HAP is used to dry / granulate at a temperature of 100 to 250 ° C. by a spray drying method or the like, followed by heating and baking. There is known a method of imparting mechanical strength at the same time as making it porous. However, in this method, the volume shrinkage and the densification due to the sintering of HAP proceed as the firing temperature increases, and in particular, when the temperature is higher than 800 ° C., the porosity decreases remarkably due to the disappearance of the pores. For this reason, firing must be performed at a temperature of 800 ° C. or lower, but such a temperature is insufficient for the purpose of increasing mechanical strength. In order to prevent a decrease in porosity due to densification, a process of mixing heat-decomposable particles such as hydrogen peroxide water and a blowing agent such as hydrogen peroxide and styrene resin into the slurry of HAP and heating it is incorporated, and large pores are chemically prepared in advance. However, this method is limited to relatively simple shaped pores, and not all of them act as open pores, and the pores become closed pores. The particles become trapped inside the particles and become meaningless, and also weaken the mechanical strength. Further, as another method for suppressing the decrease in porosity due to densification, there is a method in which hydroxyapatite prepared by a solid reaction at a high temperature of 900 to 1400 ° C. is crushed into fine particles, and the resulting particles are re-granulated and fired. However, this method has low activity for the sintering reaction because the hydroxyapatite particles have already been prepared at a high temperature, and the granulated particles are
Even if it is baked at 0 to 1400 ° C, the bond between the particles is weak. Therefore, in order to increase the mechanical strength, it is necessary to perform firing at a temperature higher than this temperature range. However, firing at such a high temperature causes a decrease in porosity again or decomposition of HAP. Therefore, it was practically difficult to heat at a higher temperature in order to sufficiently increase the mechanical strength.

[0003]

SUMMARY OF THE INVENTION In view of the above-mentioned current situation, an object of the present invention is for liquid chromatography comprising a porous HAP which has excellent durability and is suitable for separation / preparation of biopolymer samples such as proteins and nucleic acids. To provide a filler. Furthermore, an object of the present invention is to provide a method for producing the above packing material for liquid chromatography.

[0004]

DISCLOSURE OF THE INVENTION According to the present invention, the following novel packing materials for liquid chromatography are provided. The novel packing material for liquid chromatography according to the present invention comprises hydroxyapatite particles synthesized in a temperature range of less than 800 ° C (hereinafter referred to as HAP particles A) and hydroxyapatite particles synthesized in a temperature range of 800 ° C or more. (Hereinafter, referred to as HAP particles B) is dried and granulated, and the granulated product is formed by firing the granulated material at a temperature of 800 ° C. or higher, and is made of porous HAP. To do.

The present inventors have earnestly studied and found that HAP
Since the particles A are prepared at a low temperature, they have a high activity for the sintering reaction, and when the HAP particles A alone are heated to a high temperature range of about 800 ° C., the aggregates of the HAP particles A leave many gaps (open pores). However, it can be seen that rice-shaped HAP particles grow and sinter in a continuous manner and that they develop porosity with shrinkage of the volume as a whole. Contribution HAP particles A
On the other hand, it was found that by mixing the HAP particles B, it is possible to suppress the degree of decrease in the pore volume even when fired at a high temperature, and as a result, it is possible to keep the pore volume large.
The present invention has been made based on such findings.

The present invention will be described in detail below. The present invention relates to hydroxyapatite particles (HAP particles A) synthesized in a temperature range below 800 ° C and hydroxyapatite particles (HAP particles B) synthesized in a temperature range above 800 ° C.
And a mixing ratio of HAP particles B is 3 to 60% by weight based on the total weight of HAP particles A and HAP particles B to prepare a granulated product, and the granulated product is 800 ° C. or higher. The present invention provides a packing material for liquid chromatography composed of porous hydroxyapatite, which is produced by firing at the temperature of 1. The HAP particles A according to the present invention are prepared at a temperature of less than 800 ° C. Amorphous or crystalline particles of HAP, which have high activity for crystal growth and sintering reaction when heated at a calcination temperature of 800 ° C. or higher, so that large volume shrinkage occurs with these reactions. It means particles. Such HAP particles may be produced by any manufacturing method and are not particularly specified. To give an example, the Tizerius method [A.
Tiselius, et al, Arch. Bioch
em. Biophys. , 65 , 132-155 (19
56)], and various known methods that cause precipitation of HAP fine particles by direct reaction of ions from an aqueous solution, so-called wet synthesis method. HA in the slurry prepared by mixing HAP particles A and B with HAP particles A
P particles B are evenly covered, and when HAP particles B are porous, HAP particles A are also allowed to penetrate into the pores thereof.
In order to obtain the effect that the HAP particles A are sintered together during the firing, and at the same time, the HAP particles A are firmly bonded to each other so as to wrap the HAP particles B inside and outside the pores.
The size of the P particles A should be as small as possible. The size of the pores generated in the preparation of HAP particles B described below varies depending on the manufacturing method, and smaller pores easily produce pores of 1 micron or less, but in order to obtain the effect of the present invention by experiments, , HAP particles A have been found to be more desirable to be able to penetrate these small pores. Therefore, HA
The size of the P particles A is preferably at least smaller than the size of the pores present in the HAP particles B, and in many cases,
One micron or less is sufficient.

The above HAP particles B are HAP crystal particles which have been sufficiently heated at a temperature of 800 ° C. or higher and have undergone sufficient volume shrinkage due to crystal growth and sintering reaction. It means thermally stable, single-crystal or polycrystalline particles of HAP in which no new large volume contraction occurs upon heating under the conditions. The shape may be any shape such as spherical shape, plate shape, columnar shape, and indefinite shape, and is not particularly specified. Also, this HAP
The particles B may be porous or dense. As an example, when using porous HAP particles B having open pores (pores) having a larger diameter than HAP particles A, HA
The P particles A not only cover the surface of the HAP particles B, but also HA
The HAP particles A that have penetrated into the open pores of the P particles B are crystallized inside and outside the pores of the HAP particles B by firing after drying and granulation, and are solid so that the HAP particles B are taken in with the sintering reaction. Since a strong bond is generated, a filler having high mechanical strength can be obtained. Further, when columnar HAP particles B are used, HA
By mixing with the P particles A, the HAP particles A that fill the entangled gaps between the HAP particles B undergo volumetric shrinkage due to crystallization and a sintering reaction due to heating after drying and granulation. Because it is tightly coupled so that it is entwined with
A filler having high mechanical strength can be obtained.

The size of the HAP particles B is not particularly specified, but even if the HAP particles A undergo volume contraction, it is said that the size of the HAP particles B is sufficiently larger than that of the HAP particles A. It is possible to suppress a decrease in the porosity of the granulated particles as a whole. HAPs that are usually easy to prepare
Since the size of the particles A is as small as about 0.1 micron, it is desirable that the size of the HAP particles B is at least 1 micron or more.

The aforementioned HAP slurry in the present invention means a suspension in which HAP particles A and HAP particles B are dispersed in a solution such as water or an organic solvent. A slurry preparation method therefor is not particularly specified. As an example of the preparation method, a suspension containing only HAP particles A and a suspension containing only HAP particles B may be prepared and then mixed, or a mixture of HAP particles A and HAP particles B may be used. It may be dispersed in water or an organic solvent, and HAP particles B may be mixed with a solution in which a precipitate of HAP particles A is produced in a wet synthesis method such as the Tizellius method. Can be used as a method for preparing the slurry of the above HAP.

H composed of HAP particles A and HAP particles B
The concentration of the AP slurry is not particularly specified, and the drying / granulating method used, the degree of shrinkage and porosity of particles due to firing,
The concentration is adjusted to be suitable for achieving the object of the present invention in consideration of the pore volume and the like.

In order to achieve the object of the present invention, the mixing ratio of the above HAP particles A and HAP particles B is HAP particles B.
Is preferably in the range of 3 to 60% by weight,
15-30% by weight is preferred. When the amount is 3% by weight or less, HAP particles A are obtained by firing at a high temperature of 800 ° C or higher.
Since a large volume shrinkage occurs due to the sintering of and the densification progresses, there is almost no effect of suppressing the decrease in the porosity of the filler due to the coexistence of the HAP particles B, and in the case of 60 wt% or more, However, the decrease in porosity can be suppressed, but the effect of strengthening the bonding of the entire granulated particles by covering the HAP particles B with the HAP particles A becomes small, and the increase in mechanical strength can be further increased even if firing at high temperature. It is not remarkable, and neither of them can sufficiently achieve the object of the present invention.

The method of drying and granulating the slurry containing the above HAP particles A and HAP particles B is not particularly specified. Examples of the method include the following known methods. (1) Put in a heat-resistant container, heat to dry and solidify, and then pulverize to obtain particles having a desired particle size. (2) Drying and granulation are performed simultaneously by the spray drying method.
According to this method, it is possible to obtain spherical particles that are extremely preferable as the shape of the packing material for chromatography. Further, if the drying temperature in the spray drying method is raised to, for example, 700 ° C., not only the drying but also the partial sintering reaction can be caused. Such a method is also a dry granulation method in the method of the present invention. It can be preferably used.

The pore volume of porous HAP varies depending on the preparation method and conditions of HAP slurry, heating and firing conditions, and the like. As an example, when a slurry containing HAP particles A produced by the reaction of calcium nitrate and phosphoric acid is dried, pulverized, and heated to 1000 ° C., sintering progresses and the porosity remarkably decreases, and The volume is 0.18cc
/ G, and the loading capacity of biological samples such as proteins is about 1 compared to porous HAP prepared at 800 ° C.
It decreased to / 5. Therefore, it is desirable to increase the firing temperature in order to increase the mechanical strength, but on the other hand, in order not to significantly reduce the load capacity of proteins and the like,
The pore volume of porous HAP is at least 0.2 cc /
It is desirable that it is g or more, preferably 0.35 cc / g or more.

According to the present invention, since the slurry containing the HAP particles B which have been previously sintered at a high temperature and sufficiently subjected to the sintering and the volume shrinkage and the HAP particles A having a high activity for the sintering reaction, the HAP particles are used. The crystallization and sintering reaction of the HAP particles A encapsulating B occur around the HAP particles B without volume shrinkage, and at the same time, the HAP particles A shrink as H
While firmly enclosing the AP particles B, they are sintered so as to be assimilated into the HAP particles B. HA like this
By inhibiting the uniform shrinkage of only P particles A, the effect of suppressing the decrease in the porosity of the entire particles and the effect of increasing the mechanical strength are produced. In addition, porous H having open pores
By using the AP particles B and the columnar HAP particles B to select the mixing ratio of the HAP particles A and the HAP particles B and the concentration of the slurry, the open pores of the HAP particles B and the HAP particles B can be obtained.
It is also possible to cause the HAP particles A in the slurry filling the gaps between them to undergo a large volume contraction by heating, resulting in the reopening of open pores. Naturally, these methods reduce the porosity of the HAP fired body. Helps control.

The present invention will be specifically described below with reference to Examples of the present invention and Comparative Examples. However, the present invention is not limited to the following examples.

[0016]

【Example】

Example 1 0.1 to 0.
A suspension was prepared in which 7 micron sized HAP particles A were dispersed. On the other hand, the suspension in which the HAP particles A are dispersed is dried at 150 ° C. to obtain a powder, which is then calcined at 1000 ° C. for 4 hours to be pulverized to obtain H containing particles of 2 to 15 μm.
AP particles B were prepared. Next, using the HAP particles A and the suspension in which the HAP particles A are dispersed,
Were prepared to prepare slurries of four kinds of HAPs containing 5, 10 25, and 40% by weight, respectively.
Each slurry was dried at 150 ° C. The four types of dried slurries were calcined at 800 ° C. and 1000 ° C. for 4 hours each. Each of the eight types of fired bodies obtained in this way was crushed to obtain a porous H of about 0.5 mm in size.
I got AP. The pore volume of these eight types of porous HAP was measured by the mercury porosimetry. The results are shown in Table 1. The numerical values in Table 1 are (cc / g).

From the results shown in Table 1, the firing temperature was 80.
At 0 ° C., the pore volume of porous HAP becomes smaller as the mixing ratio of HAP particles B increases, but the degree of decrease in the pore volume when the calcination temperature is increased is large at the mixing ratio of HAP particles B. As a result, the higher the mixing ratio of the HAP particles B is, the higher the mixing ratio of the HAP particles B at the firing at 1000 ° C.
It can be seen that the remaining pore volume is also large. After all, when the firing temperature is increased for the purpose of increasing the mechanical strength of the porous HAP, the degree of reduction of the pores can be suppressed to a low level by mixing the HAP particles A with the HAP particles B,
It can be seen that even when firing is performed at a temperature of 800 ° C. or higher, porous HAP having a large pore volume can be obtained.

[0018]

[Table 1]

Comparative Example 1 A suspension in which only HAP particles A are dispersed (see Example 1).
Was dried at 150 ° C. to form a powder, and then 800, and
It heated at each baking temperature of 1000 degreeC for 4 hours. Obtained 2
Each of the types of fired bodies was crushed to obtain two types of porous HAP sintered particles having a size of about 0.5 mm.
The measurement results of the pore volumes of the two types of HAP sintered particles are shown in Table 1. The pore volume of the porous HAP produced using only HAP particles A sharply decreases as the firing temperature increases. When it is fired at 1000 ° C. to increase the mechanical strength, its pore volume is compared with that of any sample when 10 to 40 wt% of HAP particles B are mixed and fired at 1000 ° C. small.

Example 2 HAP produced by the reaction of calcium hydroxide and phosphoric acid
HAP particles B (Example 1) were added to the suspension in which the particles A were dispersed.
20% by weight, and mixed and stirred, and spray-dried at 300 ° C. to obtain spherical particles having an average particle size of 100 μm. The particles are calcined at 850 ° C. to give a pore volume of 0.38 c
C / g of spherical and porous HAP was obtained.

This porous HAP was pressure-filled into a glass tube having a diameter of 10 mm and a length of 10 cm, and 0.01 M to 0.3 M was prepared using a sodium phosphate buffer solution (pH 6.8).
By the linear gradient measurement mode of up to 60 minutes,
A separation test was conducted on a mixed sample of bovine serum albumin, rhizotium, and cytochrome C under a flow rate of 2 ml / min, and good separation results were obtained. In addition, the column back pressure at the time of measurement was 0 kg / cm ² , and repeated measurement of 500 times or more was possible. Furthermore, the result of examination using lysothium to evaluate the loading capacity of the biological sample was 15 mg /
It was g. From these results, it can be seen that the HAP packing according to the present invention has high durability and liquid chromatographic characteristics suitable for separation / preparation of biopolymer samples.

Comparative Example 2 HAP produced by the reaction of calcium hydroxide and phosphoric acid
The suspension obtained by separating the particles A is spray-dried at 300 ° C.,
Spherical particles with an average particle size of 100 microns were obtained. Then
The particles were fired at 850 ° C. to obtain porous HAP.
This porous HAP was filled in a glass tube of the same size as the glass tube used in Example 2, and the characteristics as a packing material for liquid chromatography were evaluated under the same conditions as in Example 2. In the separation test of the mixed sample, the same separation result as in Example 2 was obtained, but the resistance to the passage of the liquid during the measurement was large, and the column back pressure was as high as 20 kg / cm ² . In addition, the loading capacity of Rhizotium was 11 mg / g.

Example 3 Alginate Method [Hiromichi Iwasaki, Yasunari Kaneko, Materials, 37 , 6
0-64 (1988)], the gelled fiber is heat treated at 900 ° C. for 1 hour, pulverized and treated with a 1% ammonium chloride solution to remove CaO as a by-product, and to have a diameter of 30. ~ 40 μm, length about 100 ~ 30
A HAP particle B composed of 0 μm columnar HAP was prepared. Then, react calcium hydroxide and phosphoric acid to generate H
A suspension of AP particles A was prepared and added to the above-mentioned columnar HAP.
The particles B were added in an amount of 30% by weight, mixed and stirred, dried at 150 ° C., and then calcined at 900 ° C. Particles having a size of 0.5 to 1 mm were selected as a filler by crushing the obtained fired product and dividing it with a sieve. The selected HAP was a porous HAP having a pore volume of 0.36 cc / g.

A glass tube having a diameter of 10 mm and a length of 10 cm was filled with this porous HAP, and a mixed sample of bovine serum albumin, rhizotium, and cytochrome C was prepared by using a sodium phosphate buffer (pH 6.8). 0.07M,
Separation was carried out by a stepwise method with concentrations of 0.14M and 0.25M, and good results were obtained. In addition, the result of measurement using lysodium as the evaluation of the loading capacity of the biological sample was 14 mg / g.

Claims (7)

[Claims] 1. A hydroxyapatite particle (a) synthesized in a temperature range of less than 800 ° C. and a hydroxyapatite particle (b) synthesized in a temperature range of 800 ° C. or higher,
Produced by firing a granulated product prepared from a slurry having a mixing ratio of the particles (b) of 3 to 60% by weight based on the total weight of the particles (a) and the particles (b) at a temperature of 800 ° C. or higher. A packing material for liquid chromatography, which comprises porous hydroxyapatite. 2. The packing material for liquid chromatography according to claim 1, wherein the hydroxyapatite particles (a) synthesized in the temperature range of less than 800 ° C. are particles of 1 micron or less. 3. The packing material for liquid chromatography according to claim 1, wherein the hydroxyapatite particles (b) synthesized in the temperature range of 800 ° C. or higher are particles of 1 micron or higher. 4. The packing material for liquid chromatography according to claim 1, wherein the hydroxyapatite particles (b) synthesized in the temperature range of 800 ° C. or higher are porous. 5. A granulated product from a slurry containing hydroxyapatite particles (a) synthesized in a temperature range of less than 800 ° C. and hydroxyapatite particles (b) synthesized in a temperature range of 800 ° C. or more. The packing material for liquid chromatography according to claim 1, which is prepared by a spray drying method. 6. The packing material for liquid chromatography according to claim 1, wherein the pore volume of the porous hydroxyapatite is 0.2 cc / g or more. 7. The packing material for liquid chromatography according to claim 1, wherein the porous hydroxyapatite is a spherical body.