JPS60143762A - Hydroxy apatite for chromatography - Google Patents

Abstract

PURPOSE:To obtain an excellent filling agent for chromatography by making phosphoric acid or a calcium salt thereof react with a calcium compound in an organic solvent to form a specified hydroxy apatite easily with a better reproducibility. CONSTITUTION:Phosphoric acid or a calcium salt thereof such as calcium hydrogenphosphate 2 water (CaHPO4.2H2O) is made to react with a calcium compound such as calcium hydroxide [Ca(OH)2] in an organic solvent, for example, n-hexane and water at about 60 deg.C. Here, Ca/P in the number of atoms is set at about 1.3-1.9. After about 2hr of reaction, an reactant is filtered and dewatered to obtain hydroxy apatite [Ca10(PO4)6(OH)2]. The apatite thus obtained is less than 50m<2>/g and forms a hexagonal prism-shaped assembly. Thus, the reaction in the organic solvent enables the manufacture of a filling agent for chromatography of hydroxy apatite excellent in the separation performance in a constant character with a better reproducibility.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to hydroxyapatite for chromatography.

Hydroxyapatite (hereinafter abbreviated as HA) is generally CaXo(PO4)6(
It is a compound represented by OH)2, and has attracted attention in recent years as a material for bioceramics because its composition is similar to the minerals of bones and teeth.

In addition, by utilizing the biocompatibility of catalysts used in the dehydration of alcohol, fluorescent materials, inorganic ion exchangers, and HAp, we can develop technologies that can help improve the production of proteins and nuclear layers. It is expected to be used as a packing material for chromatography when the female is 4 days old.

Among these, HA as a packing material for chromatography
The use of p is on the rise with the recent development of biochemical fields such as gene exchange technology.

Conventional chromatography HAp is A, T, 5e-
Wet synthesis method of reacting a water-soluble calcium salt with a water-soluble phosphate (Arch, Biochem, Biophys,...
65.152 (1956)) with appropriate improvements. These are currently said to have excellent performance for chromatography, and usually exhibit physical properties such as .

Oa / P ratio 1.4 ~ 155 X-ray diffraction Diffraction peak is weak.

Specific surface area 6o~12oTr? /2 Grain shape: Laminar crystals However, the conventional method for producing such HAp involves sample preparation, pH'e pressure, oxidation, oxidation, and subsequent post-treatment, etc. requires extremely complicated operations.

Moreover, the reproducibility of this method is poor, and it is also true that it is extremely difficult to even synthesize HAp. Furthermore, when this method is scaled up industrially, its reproducibility is significantly poorer than in flask experiments, making it unsuitable for industrial implementation.

Moreover, the obtained HAp with the above-mentioned physical properties cannot maintain its physical properties unless it is stored in a cold place in a sodium phosphate buffer.

As mentioned above, H for chromatography has excellent physical properties.
Ap is difficult to synthesize quantitatively by conventionally known methods 1 and is expensive due to manufacturing difficulties. Therefore, although HAp is known for its excellent adsorption properties as a filler for biopolymers, it is used extremely less frequently than other fillers for biopolymers, and is only used in limited fields. The reality is that it is being used.

As a result of intensive study on HAp with excellent quality for use in chromatography, the present inventors found that it is completely different from conventionally known HAp, that is, it has excellent crystallinity in terms of X-ray diffraction, and has a specific surface area of S On, Despite showing a small value of 2/1 or less, HAp was found to have excellent performance for chromatography, leading to the present invention.

That is, the present invention is a hydroxyapatite for chromatography with a specific surface area of 50 tr? / fl or less, and is characterized by being a hexagonal columnar crystal aggregate. The physical properties of HAp of the present invention are as follows.

CaZP ratio 1.30-1.90 X-ray diffraction: Strong diffraction peak.

Specific surface area 50d/? The HAp of the present invention exhibits good crystallinity as mentioned above,
It also has a small specific surface area. Moreover, there is little change in physical properties over time. These characteristics are similar to conventional HA for chromatography.
In X-ray diffraction, p has a weak diffraction peak and a specific surface area of 6.
0m'? In this way, it is different from a thin plate crystal.

Conventional HAp for chromatography utilizes the fact that the larger the specific surface area, the larger the adsorption amount. In this way, in the past, H
Ap has a specific surface area of less than 50m71, which is rare.
It is known that the adsorption amount and recovery rate of IAI with a small specific surface area are greatly reduced compared to those with a large specific surface area (M, 5pence).
, etol, J., Ohromato7. .

166.423-434 (197B)).

However, although the HAp of the present invention is considered unsuitable as a packing material for chromatography based on conventional wisdom due to its small specific surface area, it exhibits excellent performance.

The HAp for chromatography of the present invention has phosphoric acid and/or
Alternatively, it can be obtained by a method in which a calcium salt thereof and a calcium compound are reacted in a reaction medium containing an organic solvent.

Phosphoric acid and/or used as raw materials in this method
Or its calcium salt is H3P0. .

Ca (H2PO4) 2・H2O, CaHPO,,C
aHPO, 2H20.

Ca3(PO4)2, etc., and calcium compounds such as CaCt2. CaO, C'a(OH)z, C
a(No3)2. CaCo3°Ca(COO)3 and the like are preferred. However, these compounds are only examples of usable raw materials, and there is no need to be limited thereto. Further, the higher the purity of the raw materials used, the easier it is to obtain a highly pure product, and is preferable as a packing material for chromatography, but this is not intended to be particularly limiting.

These raw material compounds have a calcium to phosphorus ratio during the reaction in terms of Ca/P atomic ratio of 1.6 to 1.9, preferably 1.
Two or more kinds of compounds a are used so that the number is in the range of 45 to 175.

Organic solvents used in the reaction include, for example, benzene, toluene, xylene, pentane, octane, tecane, aromatic and aliphatic hydrocarbons such as various petroleum naphthas and industrial gasoline, and alcohols such as iso/butanol and n-amyl alcohol. Examples include ethers such as ethyl ether, ethyl butyl ether, various cellosolves, carpitols, and ketones such as methyl ethyl ketone and methylinobutyl ketone. Such organic solvents are appropriately selected and used depending on the reaction conditions and purpose.

The appropriate amount of organic solvent used varies depending on its type, the type of raw material during reaction, the stirring conditions during reaction, etc., or the slurry concentration is 50q under any condition.
It is desirable to add in an amount that is equal to or less than b.

To carry out the reaction, phosphoric acid and/or its calcium salt and calcified islands are charged in advance into a reaction tank at a desired Ca/P ratio, and an appropriate amount of water is added to form a slurry.

Next, add an organic solvent and heat to reflux. A reflux time of about 2 hours is sufficient. Note that the heating reflux time and the order of charging raw materials including organic solvents to the reaction tank are generally carried out as described in 1, but are not necessarily limited thereto.

After the heating is completed, the generated HAp precipitates and becomes a slurry, which can be separated. As for the separation method, the commonly used method using only f separation, and the method in which HAp is separated after evaporating water together with the organic solvent can be applied. Particularly in the latter method, dehydration is carried out while adding an organic solvent in a volume corresponding to the distilled water and the organic solvent, and after the dehydration is completed, the organic solvent and HAp are separated (1). The latter method is preferred in that HAp can be completely dehydrated.

According to this method, it is possible to obtain powdered HAp at a relatively low temperature, in a short time with simple operations, with good reproducibility, and with sufficient reproducibility for scale amplifiers, making it possible to obtain powdered HAp with excellent properties for chromatography. can.

As mentioned above, conventional methods for producing HAp for chromatography lack reproducibility and require complicated operations.
In the above method, the reaction is carried out at low temperature in the coexistence of an organic solvent, and the obtained crystals are separated by f and dried, so the manufacturing equipment is simple and inexpensive, and excellent physical properties can be obtained with simple operations. HAp can be obtained as a powder, which is of great industrial value.

Hereinafter, the present invention will be explained in detail with reference to Examples and Reference Examples.

Example-1 Calcium hydrogen phosphate dihydrate (CaHPO4・2H2
0) 105.2s', calcium hydroxide (ca(on)
2) 29.6 t, 100 liters, n-hexane 65
01 was placed in a 1 t flask, heated while stirring, and heated below the reflux temperature (63°C) for about 2 hours, and then water and n-hexane in the reaction system were evaporated, resulting in distilled n-hexane and water. Dehydration was carried out while adding a volume of n-hexane to the system. As the dehydration progressed, the internal temperature rose, and when the internal temperature exceeded 68°C, heating was stopped, and after cooling, the product was separated from the solvent and dried to obtain a white powder. The X-ray diffraction pattern of the powder is shown in FIG.

As is clear from Figure 1, the diffraction angle 20 = 31.7
．． ASTM card 9 with main peak at 322.52B
This coincided with the characteristic diffraction peak of HAp described in -462.

The grain shape is spherical or similar, and consists of a collection of fine hexagonal columnar crystals. The physical property analysis results are also shown in Table 2.

Examples 2 to 4 Monocalcium phosphate monohydrate, anhydrous dicalcium phosphate, dicalcium phosphate dihydrate, calcium hydroxide, calcium carbonate, water, and organic solvents are shown in Table 1. HAp was obtained by the same operation as in Example-1 except for conditions other than Table 1. Its physical properties are shown in Table 2.

Example-5 Example-1 was scaled up 50 times and its reproducibility was confirmed.

Calcium hydrogen phosphate dihydrate 516Kg, calcium hydroxide 1.48Kg > Water s Kg, rhohexane 17
．． No. 5 garlic was charged into a stainless steel reaction tank No. 507, and the following reaction was carried out in the same manner as in Example-1.

As a result, it gave the physical properties shown in Table 2 and showed good performance as a chromatography packing material.

Comparative Example Conventional HAp synthesis method (A, Ti5elius,
eLat method Arch, Biochem, Bioph
ys, 65.132 (1956)).

Add 1 t of 0.5 movt cact2 aqueous solution and 1 t of 0.5 mot/l Na2HPo4 aqueous solution to a 6 t flask.
t was added dropwise at the same time at a rate of 120 drops per minute, and the reaction was carried out while being vigorously hit with a stirring rod. After the dropwise addition was completed, the supernatant liquid was removed by decantation, and the remaining precipitate was washed four times with 2 tons of distilled water each time. Next, 2 tons of distilled water was added to this precipitate, and 5 tons of 40% by weight NaOH was added.
After adding 0 iJ, it was boiled for 1 hour.

After cooling, the supernatant liquid was again removed by decantation, and the mixture was again washed four times with 2 tons of distilled water. Next, ODl at pH 68
Add 2 mol/7 sodium phosphate buffer and heat until just before boiling. This operation was repeated twice, with boiling times of 5 minutes and 15 minutes, respectively.

Next, 2 ml of sodium phosphate buffer with a pH of 6.8 and 1 mot/t was added and boiled twice (15 minutes).

The HAp obtained in this way was 0.001 at pH 6.8.
It was placed in f'rIOt/l sodium phosphate buffer and stored in the refrigerator.

As a result, HAp having the physical properties shown in Table 2 was obtained.

Reference Example Using the HAp obtained in Examples 1 and 5 and Comparative Example, bovine serum albumin was separated according to the column chromatography procedure shown in the afternoon.

Table 1: Take Cellosolve-based solvent (Mitsui Toatsu Chemical Co., Ltd. product) HAp 1 Of in a beaker of sooml!, gently add ion-exchanged water (abbreviated as water), and stir gently.10 After standing for 1 minute, decant. Repeat this operation until the supernatant becomes clear. Then, layer the suspended HAp on an i cm glass column for chromatography (height: 10 to 12 cr IL). Wash with water and measure the flow rate at the same time (
Fill to approximately 1011 Ll/hr). Next, add 1ml of bovine serum albumin dissolved in water to a concentration of 1% by weight.
Adsorb onto Ap column. All operations at this time are performed at room temperature. Next, in order to separate the adsorbed bovine serum albumin, development is performed with a phosphate-strength thorium buffer (pH 6, 8).

The eluate is collected in a gravimetric fraction collector (g 2. i y eluate per tube). The absorbance of the obtained eluate was measured in an ultraviolet 280 chamber. The results obtained are shown in FIGS. 6-5. 6 and 4, the HAp of the present invention shows excellent resolution and excellent adsorption/desorption, whereas the HAp synthesized according to the conventional method as a comparative example
It is clear from FIG. 5 that the recovery rate is inferior.

[Brief explanation of drawings]

FIGS. 1 and 2 show X-ray diffraction patterns of HAp obtained in Example 1 and Comparative Example, respectively. FIG. 3 shows the separation of bovine serum from HAp obtained according to Example 1 according to the procedure of Reference Example. FIG. 4 shows the separation of bovine serum using the procedure of the HAp reference example obtained in the comparative example. FIG. 5 shows the separation of bovine serum from HAp obtained in Example 5 by the procedure of the reference example. Patent applicant Mitsui Toatsu Chemical Co., Ltd. Figure 3 Figure 4 Fraction number Fraction number Figure 5 Fraction number Procedural amendment (voluntary) November 24, 1981 Commissioner of the Patent Office Kazuo Wakasugi 1; Display of the case 1983 Patent Application No. 183161 Title of the invention Hydroxyapatite 6 for chromatography, Person making the amendment Relationship to the case Patent applicant address 3-2-5 Kasumigaseki, Chiyoda-ku, Tokyo Name (31
2) Mitsui Toatsu Chemical Co., Ltd. 1) Specification, page 2, line 6: rA, T, 5e-J
Corrected it to rA, Ti5e-J. 2) Similarly, on page 4, the phrase ``1st diffraction peak at the 2nd line'' is corrected to ``diffraction peak.'' 3) Similarly, on page 5, line 9, r(M, 5penc
Er. etol, J and r (M, 5pence
, et, at J. 4) Similarly, page 5, top 1st line to page 6, 1st line "H3PO4,Ca(112P04)2・H2O" [H8Po4゜1-IP On, H4P2O7,P2
Corrected as O5, Ca (H4F 04 )2 ・l-120J. 4) Similarly, on page 6, top 4th line, "Iso-butanol" is corrected to "1-iso-butanol." 5) Same (, page 7, line 6, "...is different?" is corrected to "...is different, but". 6) Same, page 10. , ``etatJ'' in the 5th item above is corrected as [et, all. 7) Similarly, replace Table 2 on page 13 with the following table. Procedural amendment (voluntary) December 2, 1982 ? - Mr. Manabu Shiga, Commissioner of the Japan Patent Office 1, Indication of the case, Patent Application No. 183161 of 1983, 2, Title of invention: hydroxyapatite for chromatography 3, Person making the amendment 4, Invention of 1 of the specification subject to amendment "Detailed Description" column and drawings ■) Specification, page 4, line 10: "..."
1-IA, p of the present invention is
It can be used as a packing material for ordinary open columns and high performance liquid chromatographs. ” he corrected. 2) Similarly, in the 11th line, the 4th step of the "reference example" is changed to "
Reference Example 1". 3) Similarly, add the following example between the first and second lines of page 15. "Reference Example 2 1 (Ap) obtained in Example-1 was transferred to a column for high performance liquid chromatography with an inner diameter of 8 mm and a length of 10 CwL.
kl? The column was filled at a pressure of /i, and after the filling was completed, the column was attached to a high performance liquid chromatography device. The separation ability of this column was evaluated by separating bovine serum albumin in the same manner as in Reference Example 1. The eluent used was sodium phosphate buffer (pH 6, 8), and the pressure was adjusted to 1.5 kgZ so that the flow rate was 1 ml/min.
Adjusted to CRL. The absorbance of the obtained eluate is in the ultraviolet region 2
Measured at 80+++m. The chromatogram obtained from this is shown in FIG. It is clear from this that the HAp of the present invention exhibits excellent resolution even in high performance liquid chromatography. ”, 4) Similarly, page 15, lines 6-7 [Reference example 1
is corrected to "Reference Example 1". 7s) Similarly, change "1 reference example" on page 15, line 9 to "
Reference example-1” is corrected. 6) Similarly, on page 15, lines 11-12, "Reference example" is corrected to "Reference example-1." 7) Similarly, add the following sentence below the 13th line on page 15. "Figure 6 is a separated chromatogram of bovine serum albumin obtained in Reference Example 2." 8) Add the attached drawing, Figure 6. Written amendment to the above procedure (method) Manabu Shiga, Director General of the Japan Patent Office, 1985, Indication of the case, Patent Application No. 183161 filed in 1983, 2, Title of the invention: Hydroxyapatite for chromatography 3, Amendments are made. Relationship with the Patent Case Patent Applicant Address 3-2-5 Kasumigaseki, Chiyoda-ku, Tokyo Name (3
12) Mitsui Toatsu Chemical Co., Ltd. February 5, 1986 (shipping date) 5. Subject of amendment ``Subject of amendment'' and ``Contents of amendment'' of the procedural amendment submitted on December 24, 1980 Column 6: Correct according to the content of the correction. (2) Regarding the "Contents of Amendment" column, in Contents of Amendment (2), "Similarly, in the 11th line, the lower 4th line...
``...'' [Also, page 11, bottom 4th line...''
"..." I corrected myself. Written amendment to the above procedure (voluntary) December 24, 1980 Manabu Shiga, Commissioner of the Patent Office1, Indication of the case: Patent Application No. 183161 of 19832, Name of the invention: Hydroxyapatite for chromatography 3, Amendments made Complainant 4, "Detailed Description of the Invention" and "Brief Description of Drawings" columns of the specification to be amended, Drawing 5, Contents of the amendment

Claims (1)

[Claims] 1) Hydroxyapatite for chromatography, which has a specific surface area of 50 or less and is a hexagonal columnar crystal aggregate.