JPH02147857A - Spherical carrier for chromatography separation and production thereof - Google Patents

Abstract

PURPOSE:To make a particle shape into a spherical shape and to uniformize the grain size thereof so that a good equil. state is maintained and a pressure drop is minimized by providing the two-layered structure in which a particulate inorg. material is used as a nucleus and a calcium phosphate compd. contg. a Ca-P apatite phase is formed on the surface thereof. CONSTITUTION:This carrier is formed to have the two-layered structure in which the particulate inorg. material is used as the nucleus and the calcium phosphate compd. contg. the Ca-P apatite phase is formed on the surface thereof. This carrier is obtd. by splashing the inorg. material which is in a molten state, rapidly cooling the material to form the spherical solid, immersing this solid into an aq. acidic or alkaline soln. to dissolve away the soluble part on the surface of the solid, then immersing the solid into a liquid contg. P4 ions. The carrier which is spherical and is uniform in grain size is obtd. by this method and the separation power in chromatography is enhanced.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a separation carrier optimal as a packing material for column chromatography and a method for producing the same, and particularly relates to a technique for making the carrier spherical. .

[Prior art] Liquid chromatography using a column method (hereinafter sometimes simply referred to as chromatography) is used for separation and purification of substances, and is used to partition various substances between two phases (stationary phase and mobile phase). Separation and purification are performed based on differences in coefficients.

The solid substance used in the stationary phase is called a carrier,
With the development of high-speed mobile phase technology, this carrier is required to have high strength.

That is, as in high performance liquid chromatography, the mobile phase is
In recent years, there has been a trend to speed up separation by flowing at a high speed of ~30 arm/sec, but in order to handle such high speeds, strength sufficient to withstand high pressure is required.

Furthermore, in order to maintain a high separation ability by chromatography, it is necessary to maintain the approximate distribution equilibrium state of chromatography as much as possible by loading the carrier. Also, the particle size of the carrier! If the size is about OO to 150 μm, it is difficult to maintain the equilibrium state under the high speed state of the mobile phase. Therefore, in order to achieve the above-mentioned equilibrium state under high-speed conditions, it is necessary to make the particle size of the carrier sufficiently fine and to make the particle size uniform.

The carrier materials that have been used so far include silica,
Examples include glass, alumina, and highly cross-linked resins, and silica and alumina are said to have relatively good properties. However, even silica and alumina are difficult to obtain as fine bead-like substances, and conventionally, when used as a carrier, they are crushed to make them fine and of uniform particle size. However, since the particle shapes vary, and the particle size distribution is still wide, there are drawbacks such as large pressure loss.

By the way, chromatography is also used for the separation and purification of biologically related substances such as proteins, and when the purpose is to separate such substances, the carrier is required to have good affinity with the living body. With silica-based carriers that have been widely used so far, substances are generally eluted from the carrier by chemically modifying the carrier surface and adding chemicals to the eluate. However, these conditions are harsh for biological substances, leading to degeneration such as loss of activity of the substances. Furthermore, when cleaning proteins, bacteria, etc., cleaning with an acidic solution alone is insufficient, and it is necessary to also perform cleaning with an alkaline solution. However, when a silica-based carrier is used, the carrier itself or the carrier is washed with an alkaline solution. There is a drawback that the surface modification group is leached out.

For these reasons, hydroxyapatite (hereinafter sometimes referred to as HAP) has come to be used as a carrier for chromatographic separation. That is, HAP not only has good affinity with biological substances, but also has ion exchange ability itself, and is particularly suitable for carboxyl groups, phosphoric acid groups, and
It has the property of adsorbing ε-amino groups, guanidino groups, etc.
Furthermore, it is stable against alkalinity.

[Problem to be solved by the invention] HAP used as a carrier for chromatography separation
is generally synthesized by a wet method and used as it is or in the form of granules.

However, the yield is low when producing fine particles with a particle size of 10 μm or less, and the aforementioned problems in terms of shape, particle size distribution, etc. remain unsolved.

The present invention was made to solve these technical problems, and its first purpose is to make the particle shape spherical and uniform in particle size, maintain a good equilibrium state, and minimize pressure loss. HAP-based chromatography separation carrier that can increase the number of theoretical plates,
and to provide a method for producing such a carrier. A second object of the present invention is to achieve better separation of biologically related substances, in particular, by increasing the ion exchange capacity of the HAP carrier itself.

[Means for Solving the Problems] The carrier of the present invention that achieves the above object has a core of a powdery inorganic substance, and a calcium phosphate compound containing a Ca-P apatite phase is formed on the surface of the carrier. The main point is that it has a two-layer structure.

The above-mentioned carrier is produced by scattering and rapidly cooling the molten inorganic substance to form a spherical solidified substance, and immersing this solidified substance in an acidic or alkaline aqueous solution to leach out the soluble portion on the surface of the solidified substance, which contains PO4 ions. Obtained by immersion in liquid.

Further, the above-mentioned carrier is made by rolling a particulate inorganic substance in a semi-molten state to form a spherical solidified substance, and after immersing this solidified substance in an acidic or alkaline aqueous solution to leach out the soluble portion on the surface of the solidified substance, It can also be obtained by immersion in a solution containing PO4 ions.

[Function] The present invention is constructed as described above, but in short, the inorganic substance is solidified into a spherical shape, the solidified material is immersed in an acidic or alkaline aqueous solution, the soluble portion on the surface is leached, and then the solidified material is made into powder and granules. By immersing it in a solution containing PO4 ions,
The basic gist is to form a calcium phosphate compound containing Ca-P and % apatite phase on the surface. Since the surface of the carrier obtained in this way is coated with a calcium phosphate compound containing at least an apatite phase, the original properties of the apatite phase such as biocompatibility are not impaired, and the overall shape Since a substantially spherical shape can be obtained, pressure loss is less likely to occur and an approximate distribution equilibrium state can be maintained, making it possible to separate substances at a higher flow rate. Moreover, since the particle size distribution can be narrowed, the number of theoretical plates can be increased, and the pressure and flow rate of liquid feeding can be easily adjusted. Furthermore, by leaching soluble components and forming fine pores, the surface area can be increased and the ion exchange ability of the carrier itself can be increased, and the separation ability in chromatography can be improved.0 Used as a core in the present invention The substance may be any compound that precipitates a calcium phosphate compound containing a Ca-P apatite phase when immersed in a solution containing PO4 ions, but it is required to be at least an inorganic substance. Various materials can be considered as such materials, but inorganic glass is an example of ease of melting and softening.

A method for turning an inorganic substance into a spherical solidified substance is a method of rolling granulation of powder, but if you want to obtain a particle size of 100 μm or less (substantially 40 μm or less), the raw material must first be sized to 1 μm. - It is necessary to grind the powder into the following fine powder and then granulate it, which increases the grinding cost and makes it difficult to handle the powder, making it impractical. (referred to as a molten state), a method is adopted in which the powder is rolled into a spheroid in order to prevent the powder from sintering with each other and to apply rotational force to each particle [Claim (3)
], However, as a method other than this, for example, a liquid such as water or oil or air, N2. Ar
An atomization method in which a gas such as the above is sprayed to rapidly cool the material by scattering, or a method in which the material is dropped onto a rotating disk and dispersed by centrifugal force to rapidly cool the material are also effective for spheroidization [Claim (2)].

As a method for forming a phosphoric acid compound on the surface of a spherical solidified product, for example, a sputtering method, a spray drying method, a one-rotation granulation method, etc. are known (for example, JP-A-63-16
044 Publication), the Shikashi sputtering method requires a special equipment, and it is very difficult to completely coat the surface of each particle of 100μ or less. It is difficult to coat the surface, and even in the rolling granulation method, there is a problem with the fineness of the particles.

For this reason, in the present invention, we focused on a method in which particles are immersed in a solution to precipitate a calcium phosphate compound on the surface, and we investigated the possibility of adopting this method. However, according to the research conducted by the present inventors, many calcium phosphate compounds such as apatite are not water-soluble, and most of them precipitate as particles.In order to coat the surface of a solidified product with this compound, a slurry substance must be used. It turns out that it is not easy to use. Therefore, in the present invention, a spherical solidified material is immersed in an acidic or alkaline aqueous solution to leach out the soluble portion on the surface of the solidified material, and a compound that precipitates a calcium phosphate compound containing an apatite phase when reacted with POa ions is used. A method was adopted to expose a large amount of the material to the surface of the solidified material. This allows the above-mentioned calcium phosphate compound to be deposited on the surface of the solidified product relatively easily. From this point of view, the spherical solidified material obtained by scattering and rapidly cooling the molten inorganic material should be immersed in a solution containing PO4 ions (i.e., before or after the process of leaching the soluble part). ), it is recommended to add a step of crystallizing the surface of the solidified material by heating it at a temperature just before melting. In other words, by adding such a process, calcium phosphate compounds and C are added to the surface of the solidified product.
If as much as possible of a5iOs (wollastonite or precipitated wollastonite) etc. is crystallized in advance before reacting with PO4 ions,4, using this as a starting point, a calcium phosphate compound containing an apatite phase will form on the surface of the solidified product. is likely to precipitate. Also, from this point of view, the inorganic substance that becomes the spherical solidified substance is crystallized to form a calcium phosphate compound containing an apatite phase and carbon dioxide.
It is preferable that the two phases of a5te and a5te are easily precipitated at the same time, but it is not preferable because it inhibits the precipitation of calcium phosphate containing AI and T1. When a step of obtaining a spherical solidified product is adopted, crystallization of the surface will proceed at the same time as the solidified product becomes spherical.

The calcium phosphate compound formed on the surface of the carrier of the present invention must contain at least a Ca-P apatite phase, but may also contain Ca2F207. Ca (PO3)2
, Cat(P 04)s.

Ca+o(PO4)6 F2. Ca+o(PO4)I!
It may contain C1, etc., and a part of Ca may be replaced with S.
r.

It may also include one in which a metal such as Ba is substituted or a part of Po4 is substituted with CO2. These compounds are mainly produced during crystallization depending on the composition of the core inorganic substance. Also, based on these compounds, Po4
Amorphous calcium phosphate, OP C[Caa H2 (P 0
4)a・5 H201゜Ca HP Oa, Ca HP
04”2 H20°Ca (H2PO4)2.ca
It may contain a series of calcium phosphates such as ()12PO4)2.2H20 or Ca3(HF20?)2 produced by heating these.

Regarding the liquid partially containing PO4 ions, there are no limitations as long as the phosphoric acid is in an ionized state, but phosphoric acid, metaphosphoric acid, sodium phosphate,
Examples include liquids in which soluble phosphates such as potassium phosphate and ammonium phosphate are dissolved, as well as liquids in which esters such as trimethyl phosphate and triethyl phosphate, and alkoxides such as phosphoric acid trimethoxide are dissolved.

Hereinafter, the present invention will be explained in more detail with reference to examples, but the following examples are not intended to limit the present invention.
Any design changes for the purposes described below are included within the technical scope of the present invention.

[Example] CaO: 44%, S i 02: 34.6%,
F20°: Reagents were mixed at a ratio of 15.5% and the remainder was sodium borate, and dissolved at 1550°C. This was dropped into water, rapidly cooled, and ground to a size of 44 μm or less using a vibration mill to obtain a powder sample. Pour this into a rotating crucible and
The mixture was kept in a semi-molten state for 1 hour while being rolled at a temperature of 0.degree. C., and then cooled at a rate of 200.degree. C./hour. The solidified product thus obtained had rounded corners and a spherical shape.

Next, 100 g of the above solidified material was added to 0.0 g. It was added to 100 cc of IN acetic acid to leach out the soluble components on the surface. Subsequently, the solidified product was rolled for 24 hours while being heated to 50° C. in a solution containing ammonium phosphate. The obtained particles (carrier) are
When analyzed by SCA, it was confirmed that a calcium phosphate phase was formed on the surface. When high performance liquid chromatography was performed by filling a column with the above carrier, it was possible to separate each moderate protein (and amino acid) contained in the test liquid without causing any problems. The chromatogram at this time is shown in FIG.

[Effects of the Invention] As described above, according to the present invention, a carrier having a spherical shape and uniform particle size could be obtained, and the separation ability in chromatography could be improved. Furthermore, the ion exchange ability of the HAP-based carrier itself can be increased, which is particularly advantageous in the separation of biologically related substances.

[Brief explanation of the drawing]

FIG. 1 is a chromatogram obtained when liquid chromatography was performed using the carrier of the present invention.

Claims (3)

[Claims] (1) The core is a powdery inorganic substance, and the surface is Ca-P.
A spherical carrier for chromatographic separation characterized by having a two-layer structure formed of a calcium phosphate compound containing an apatite phase. (2) The inorganic substance in the molten state is rapidly cooled by scattering to form a spherical solidified substance, and this solidified substance is immersed in an acidic or alkaline aqueous solution to leach out the soluble portion on the surface of the solidified substance, and then PO
A method for producing a carrier for chromatographic separation, which comprises producing the carrier according to claim (1) by immersing the carrier in a liquid containing _4 ions. (3) The powdered inorganic material is rolled in a semi-molten state to form a spherical solidified material, and this solidified material is immersed in an acidic or alkaline aqueous solution to leach out the soluble portion on the surface of the solidified material, and then PO_4 ions are formed. Claims (
A method for producing a carrier for chromatographic separation, which comprises producing the carrier according to 1).