JPH0822377B2 - Column packing material - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial field of application) The present invention is a uniform packing of hydroxyapatite in glass, ceramics, metal or polymer spheres as a packing material for columns used for separation, purification, collection and culturing of substances. The present invention relates to an adsorbent coated with, more specifically, an adsorbent coated with hydroxyapatite without using a binder.

(Prior Art) Hydroxyapatite has been used as a specific adsorption packing material for proteins, cells, bacteria, etc. for substances that are difficult to separate with ion exchange resins, activated alumina, calcium carbonate, etc. Since there was a problem with strength, it was used in a way that it was adjusted at the time of use and discarded after one use. However, in recent years, the separation characteristics of hydroxyapatite have attracted attention, and various column packing materials for chromatography such as single crystal particles, calcined spheroidized particles, agglomerated spherical particles by spray drying, and particles with hydroxyapatite attached to the carrier surface have been developed. in use. For example, JP-A-63-126548 discloses an adsorbent obtained by spraying a suspension containing hydroxyapatite or a binder thereof with a pressure-collapse resistant carrier sphere, and drying or firing the adsorbent. Discloses an adsorbent in which glass spheres or polymer spheres are coated with hydroxyapatite. However, the adsorbents that are agglomerated or coated with hydroxyapatite by these methods are such that the hydroxyapatite is pulverized during use in the column, or is separated from the carrier surface to prevent the separation liquid from flowing, or the binder may be hydroxy depending on the conditions. If the surface of apatite is coated, the separation property of hydroxyapatite is deteriorated. Also, when used in a large column, there is a problem in strength. On the other hand, it is recognized that the calcination enhances the binding force of the hydroxyapatite and makes the atomization thereof less likely, but it cannot be denied that the calcination may impair the separation property of the hydroxyapatite.

(Problems to be Solved by the Present Invention) The present invention eliminates the above-mentioned drawbacks as a column-packed adsorbent, does not impair the adsorption and separation ability, and has high strength. However, for the purpose of providing a hydroxyapatite adsorbent that can obtain a sufficient separation liquid flow rate, without further using a binder, because the hydroxyapatite is directly attached to the carrier without being affected by the binder as in the conventional method, In addition, it aims at providing the hydroxyapatite adsorbent which can be manufactured easily.

(Means for Solving the Problems) In order to obtain the above-mentioned hydroxyapatite adsorbent,
In the present invention, a method described below, that is, a synthetic resin such as polyethylene, polymethylmethacrylate, polystyrene, nylon; ceramics such as titanium oxide, alumina, silica, etc., and a spherical material having a certain particle size (hereinafter referred to as mother particles) ) With a certain particle size of hydroxyapatite,
A method of directly adhering by a ordered mixture method without using a binder such as water or a binder is adopted. The ordered mixture method is a method of adhering two particles by dry mixing without using a solvent, which has been developed as a powder / powder system composite technology and various devices have been proposed. Generally, in order to uniformly attach A particles to particles by the ordered mixture method, the shape, size, and interaction of both particles, that is, the relationship between van der Waals force and electrostatic force is important, and A particles / particles Particle size ratio of 1 /
It is desirable that the particle size is 10 or less, and the smaller the particle size, the more easily the ordered mixture is formed, and the particle size is preferably 100 μm or less. However, when hydroxyapatite is used as in the present invention, the particle size of hydroxyapatite particles / mother particles is preferably 1/10 or less as in general, but if the particle size of the mother particles is 2 mm or less, the ordered mixture is sufficiently It was confirmed that it was formed.

The mother particles and the hydroxyapatite fine powder are put into a mixer and mixed. This mixture is put in a high-speed air stream impact reformer and is operated at high speed to form a hydroxyapatite-coated spherical substance. By using the mother particles depending on the use, coated spherical substances having different particle sizes can be obtained.

(Function) The hydroxyapatite-coated spherical substance thus obtained has high strength, does not peel off hydroxyapatite even when used for a long time under high pressure, and can be used regardless of the scale of the chromatograph. Since there is no effect, excellent adsorption, separation and purification capabilities can be obtained.

 The present invention will be specifically described below with reference to examples.

Example 1. 150 g of polyethylene spheres of 5-8 μm and average particle size of about 0.5 μ
50g of hydroxyapatite powder of m in a mixer
Run for 3 minutes at 1,000 rpm and remove the mixture.

This mixture was put into a high-speed air impact modifier with a diameter of 25 cm, operated at 4,000 rpm for 2 minutes, and then the contents were taken out to obtain polyethylene beads coated with hydroxyapatite. As a result of observing the beads with an electron microscope, it was confirmed that the hydroxyapatite was uniformly attached to the surface of the beads. The results of electron micrograph observation after the beads were washed off with water were also the same.

Example 2 150 g of polyethylene spheres of Example 1 were replaced with 700 g of 4-6 μm titanium oxide spheres, and hydroxyapatite-coated titanium oxide beads were obtained by the same method. As a result of observing the beads with an electron microscope, it was confirmed that the hydroxyapatite was uniformly attached to the surface of the beads. The results of electron micrograph observation after the beads were washed off with water were also the same.

Example 3. In a rotating pan, put 150 g of polyethylene balls of 5-8 μm, spray with acetone solution of acrylonitrile,
100 g of hydroxyapatite powder with an average particle size of about 0.5 μm
Was sprayed and dried at 100 ° C. to obtain beads in which polyethylene beads were coated with hydroxyapatite. As a result of observing the beads with an electron microscope, it was confirmed that the hydroxyapatite was uniformly attached to the surface of the beads. The results of electron micrograph observation after the beads were washed off with water were also the same.

Example 4 150 g of polyethylene spheres of Example 3 were replaced with titanium balls of 4 to 6 μm 70
In place of 0 g, titanium oxide beads coated with hydroxyapatite were obtained by the same method. As a result of observing the beads with an electron microscope, it was confirmed that the hydroxyapatite was uniformly attached to the surface of the beads. The results of electron micrograph observation after the beads were washed off with water were also the same.

Example 5. A stainless steel tube with an inner diameter of 4 mm and a length of 5 cm was filled with the fillers prepared in Examples 1 to 4, and cytochrome C 1 mg / ml solution 2
When 0 μl was treated with a linear gradient method with a potassium phosphate (pH6.80) concentration of 10 mM to 500 mM at a pressure of 20 kg / cm ² and a flow rate of 1 ml / min, the packing material prepared in Example 1 and Example 2 was prepared as shown in FIG. As shown in Fig. 2, cytochrome C was completely separated, and 100% of cytochrome C was recovered. But Example 3. Example
With the filler prepared in 4., cytochrome C was not separated as shown in FIGS. 3 and 4, and the recovery rates of cytochrome C were 59% and 82%, respectively.

(Effects of the Invention) Since the produced hydroxyapatite-coated beads do not contain a binder, there is no deterioration of the adsorption and separation ability due to the deterioration of the binder, and therefore, long-term adsorption and chromatographic operation without deterioration of the separation ability can be performed.
Further, since it is not affected by residual monomers or dissolution of binder, it can be used not only as a packing material for chromatographs but also as microbeads for separating bacteria or viruses or for cell culture.

[Brief description of drawings]

FIG. 1 shows cytochrome C using the filler obtained in Example 1.
Chromatogram of the eluate obtained by column chromatography. Fig. 2 shows cytochrome C using the packing material obtained in Example 2.
Chromatogram of eluate obtained by column chromatography. Fig. 3 shows cytochrome C using the packing material obtained in Example 3.
Chromatogram of the eluate obtained by column chromatography. Fig. 4 shows cytochrome C using the packing material obtained in Example 4.
FIG. 3 is a diagram showing chromatograms of eluates obtained by column chromatography.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-63-16044 (JP, A) JP-A-63-126548 (JP, A) JP-A-61-64326 (JP, A) JP-A-62- 250942 (JP, A) JP 62-83029 (JP, A)

Claims (1)

[Claims] 1. Adsorption characterized in that spherical apatite particles selected from glass, ceramics, metal or polymer are coated with hydroxyapatite by a high-velocity impact method without using a binder and produced without firing. A column packing material with excellent resolution.