JPH0516368B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] With the recent development of the field of biochemistry, the present invention has excellent properties for chromatography, such as high adsorption and chemical and physical stability of crystals, as a means of purifying biological substances. This is related to hydroxyapatite (hereinafter abbreviated as HAP). [Prior art] The method for producing HAP for chromatography is a wet method in which CaHPO ₄ 2H ₂ O is generated from water-soluble Ca salt and phosphate, and then reacted under basic conditions such as NaOH to convert it to HAP. Synthesis methods are known,
It is common to make appropriate improvements to this. However, these manufacturing methods have the drawbacks of complicated operations and poor reproducibility. Also, generate
HAP is a plate-shaped crystal with weak mechanical strength, and particles break due to abrasion during column operation, resulting in smaller spaces between filler particles, which causes pressure fluctuations in the mobile phase. [Means for Solving the Problems] The present invention is intended to solve the problems of the prior art described above. That is, the present invention
NH ₃ as an alkali source in the CaHPO ₄ −H ₂ O system,
Add at least one of NH ₄ OH, NaOH, and KOH to make CaHPO ₄ /H ₂ O slurry concentration 20-60%.
Specific surface area 20 characterized by the reaction occurring at
This is a method for producing hydroxyapatite for chromatography with a density of m ² /g or more. Generally, it is known that CaHPO ₄ .2H ₂ O is used as a starting material and an alkali source such as NH ₄ OH or NaOH is added to react to obtain HAP, and HAP for chromatography also uses CaHPO ₄ .2H ₂ O as a starting material. or manufactured through this process. Also, if you add an alkaline source to anhydrous CaHPO ₄ ,
It is also known that HAP can be obtained. However, the reaction is extremely difficult to proceed compared to CaHPO _{4.2H 2} O, and the HAP produced has an extremely small specific surface area compared to HAP produced from CaHPO _{4.2H 2} O, making it unsuitable as a chromatographic packing material. Ta. However, the present inventors found that
It has been found that there is a significant difference in the shape of HAP obtained depending on the CaHPO ₄ /H ₂ O slurry concentration (ratio of CaHPO ₄ to H ₂ O at the time of preparation). In particular, when the slurry concentration is less than 2%, the needle-like crystals are coarsely intertwined and aggregated polycrystalline.
When it is 20% or more, it becomes an extremely dense polycrystalline body, and its specific surface area is significantly large. This material has high mechanical strength and exhibits extremely high adsorption/desorption ability as a packing material for chromatography. In the present invention, the upper limit of the slurry concentration is preferably about 60%. If it exceeds 60%, the viscosity of the reaction solution becomes high, the operability becomes poor, and it is difficult to obtain homogeneous HAP. Further, the reaction temperature is preferably 5 to 100°C, more preferably 70 to 90°C, because the reaction rate is extremely slow at 5°C or lower, and uneconomical in terms of thermal cost at 100°C or higher. In addition, considering the operability and the fact that the slurry concentration does not change, NH ₃ can be used as an alkali source.
(gas) is sufficient. It is also possible to use aqueous solutions such as NaOH, NH ₄ OH, KOH, Na ₂ CO ₃ , etc. as other alkali sources. In this case, it is preferable to use a slurry concentration solution during the reaction. Hereinafter, the present invention will be explained in more detail with reference to Examples. Examples 1 to 8, Comparative Examples 1 to 3 Two four-necked flasks with a stirrer, NH ₃ blowing tube,
Set up a PH meter and reflux condenser, and make sure there is no water inside.
CaHPO ₄ was charged together with water and then heated, and when the desired temperature was reached, alkali was gradually supplied while adjusting the pH to carry out the reaction. After the reaction was completed, the product was separated, washed with sufficient water to remove the alkali, and dried. Table 1 shows the reaction conditions and results. Also, the product obtained in Example 1 (from high slurry concentration)
Figure 1 shows a SEM photograph of the particle structure of NH ₃ decomposition). Furthermore, a SEM photograph of the particle structure of the product obtained in Comparative Example 1 (NH ₃ decomposition from a low slurry concentration) is shown in FIG. As is clear from this photograph, the state of aggregation of the crystals is completely different, and the one of the example is a very dense polycrystalline body.

【table】

[Table] (Note) BSA adsorption capacity was determined by the method shown below. 0.001M sodium phosphate buffer (PH=
6.8) Mix 200mg of HAP in 10ml, 25℃ 1.25Hr,
After stirring, centrifuge at 3000 rpm for 30 minutes, collect 1 c.c. of supernatant, add 4 c.c. of coloring solution, and measure absorbance at 550 nm (Biuret method). Reference Example The HAP obtained in Example 1 and the HAP obtained in Comparative Example were used to image bovine serum albumin according to the following operating procedure. The results are shown in Figure 3 (Example 1) and Figure 3 (Example 1). It is shown in Figure 4 (Comparative Example 1). Approximately 10 g of HAP was dispersed in 0.01M phosphate buffer, immersed for one day and night, and then layered for 10 cm on a 1 cmφ glass column. Next, after washing with a starting buffer solution twice the column volume, 1 ml of 1% BSA was added, and the column was developed with a phosphate buffer solution using a stepwise elution method. As is clear from this result, HAP of the example
There is a big difference in resolution.

[Brief explanation of the drawing]

FIGS. 1 and 2 show SEM photographs of the particle structures of HAP obtained in Examples and Comparative Examples.
Figures 3 and 4 show bovine serum albumin separated from HAP obtained in Examples and Comparative Examples in accordance with the procedure of Reference Examples.

Claims (1)

[Claims] 1 As an alkali source in anhydrous CaHPO ₄ −H ₂ O system
A specific surface area of 20 m ² characterized by adding at least one of NH ₃ , NH ₄ OH, NaOH, KOH, and Na ₂ CO ₃ and carrying out the reaction at a CaHPO ₄ /H ₂ O slurry concentration of 20 to 60%. /g or more of hydroxyapatite for chromatography.