JPH08136520A - Column for liquid chromatography - Google Patents

Abstract

PURPOSE: To obtain a column for liquid chromatography which enhances the extent of separation of acid and basic biochemical substances which exhibiting excellent adsorption characteristics for each substance and employed in various biochemical analysis. CONSTITUTION: A column bulking agent layer containing acicular hydroxy apatite crystal having aspect ratio of 10 or above is connected in series with a column bulking layer containing planar hydroxy apatite crystal having aspect ratio of 2 or less to produce a column for liquid chromatography.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a liquid chromatography column which can be used for analysis or fractionation of biochemical substances such as proteins, nucleic acids and saccharides in the fields of medicine, pharmacy and biochemistry.

[0002]

2. Description of the Related Art Liquid chromatography using hydroxyapatite as a column packing material was invented by Tizellius in 1956, and was initially synthesized in the laboratory. As such a commercially available product, for example, a filler obtained by granulating wet- or hydrothermally-synthesized plate-like hydroxyapatite crystals into spherical particles by a spray drying method or the like (Japanese Patent Laid-Open No. 1-230).
No. 413) is known. The adsorption separation mechanism of chromatography using a column packing containing the plate-like hydroxyapatite crystal is, for example, in the case of an acidic biochemical substance, a negatively charged functional group (carboxyl group of acidic protein, DNA And the like) in the polynucleotide of (1) to be adsorbed on the positively charged adsorption site composed of calcium ions existing on the A-face or B-face of the plate crystal. On the other hand, in the case of basic biochemical substances, due to the positively charged functional groups (amino groups of basic proteins, etc.) on the surface of the molecule, a negative ion composed of phosphate ions present on the C-plane of plate crystals Adsorbs on the adsorption site that is charged to the.

However, since the plate-like hydroxyapatite crystal is not a morphologically controlled one and is a hexagonal columnar microcrystal having an ordinary aspect ratio (c / a) of about 3.0, the C-plane surface area of the crystal is Although large, the surface area of the A-face or B-face of the crystal is not relatively large. Therefore, the adsorption binding force of the adsorption site on the C surface for adsorbing the basic biochemical substance is strong, but the adsorption binding force of the adsorption site on the A surface or B surface for adsorbing the acidic biochemical substance becomes weak. FIG. 1 shows the degree of separation between basic biochemical substances and acidic biochemical substances and the adsorption force of each substance when such plate crystals are used as a column packing material.
It is shown in FIG. At this time, the degree of separation can be expressed by the difference between the retention time of the basic biochemical substance and the retention time of the acidic biochemical substance in FIG. 1, and the greater the difference in retention time, the better the separation degree. Indicates. On the other hand, the adsorptive power can be expressed by the value of the absorbance of each substance, and the higher the absorbance, the better the adsorptivity to the substance. As a result, in the case of plate-like hydroxyapatite crystals, as shown in Fig. 1 (a), the adsorptive binding force of the acidic biochemical substance is weak (low absorbance), and its retention time is generally short, but basic. Biochemicals, especially with an isoelectric point of 7.0
Adsorption and binding strength of basic proteins and the like exceeding 5 are good (high absorbance), and their retention time is generally long. Therefore, in this plate crystal, the degree of separation of the basic biochemical substance and the acidic biochemical substance is good, but the adsorptive binding force of the acidic biochemical substance (acidic protein, DNA, etc.) is weak, and therefore the acidic biochemical substance is However, there is a drawback that the adsorption separation property of is poor.

In order to solve such a drawback, therefore, the A surface or B surface having an adsorption site for an acidic biochemical substance is selectively grown, that is, the surface area of the A surface or B surface of the crystal is relatively changed. There has been proposed a column packing material containing needle-shaped hydroxyapatite crystals which has been increased to increase the adsorptive binding force of acidic biochemical substances.

However, in the case of the needle-shaped crystal, as shown in FIG.
As shown in (b), since the adsorptive binding force of the acidic biochemical substance is increased, the retention time of the acidic biochemical substance is increased, and the retention time of the basic biochemical substance is shortened,
There is a drawback that the retention times of both approaches and the peaks overlap each other, resulting in extremely low resolution.

[0006]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to achieve a good balance of separation between an acidic biochemical substance and a basic biochemical substance and an excellent adsorption force for each substance, and to achieve a well-balanced separation. The object is to provide a column for liquid chromatography showing a developing ability.

[0007]

According to the present invention, a column packing layer containing needle-shaped hydroxyapatite crystals having an aspect ratio of 10 or more (hereinafter referred to as column packing layer A),
Column packing layer containing plate-like hydroxyapatite crystals having an aspect ratio of less than 2 (hereinafter referred to as column packing layer B)
There is provided a column for liquid chromatography, characterized in that and are connected in series.

The present invention will be described in more detail below. The liquid chromatography column of the present invention is characterized in that the column packing material layer A and the column packing material layer B are connected in series. By connecting the column packing layers A and B in series as described above, it is considered that the acidic biochemical substance and the basic biochemical substance can be separated and developed in a well-balanced manner due to the following adsorption mechanism. .

[0009] Each packing layer constituting the column of the present invention,
For example, when the column packing material layer B is connected in series so that the column packing material layer A is arranged in an upper layer and the column packing material layer A is arranged in a lower layer, the acidic biochemical substance is first adsorbed to the upper column packing material layer B by gradient elution. And immediately desorbed at a low solvent concentration, but then re-adsorbed on the lower column packing material layer A,
After a higher solvent concentration is reached, the lower layer is desorbed and eluted. Therefore, at this time, the retention time of the acidic biochemical substance is shown in FIG.
As shown in (b) and (c), the length is about the same as in the case of the column packing layer A (acicular hydroxyapatite crystals) alone, and the adsorption force is also good. On the other hand, the basic biochemical substance is adsorbed to the column packing material layer B in the upper layer, and then is retained until the acidic biochemical substance has a higher solvent concentration than that in the lower layer. It passes through without adsorbing to the lower column packing material layer A. Therefore, at this time, the retention time of the basic biochemical substance is as shown in FIGS.
(Plate shaped hydroxyapatite crystal) The length is about the same as the case of using it alone, and the adsorption force is also good. That is, in the column of the present invention, by connecting the column packing material layer A and the column packing material layer B in series, the drawbacks of using each packing material layer alone are improved, and good separation and excellent separation for each substance are achieved. It also functions as an adsorption force and exhibits a well-balanced separation and development ability. It is to be noted that, similar to the above example, the same effects can be obtained by connecting the column packing layer A to the upper layer and the column packing layer B to the lower layer in series, which is the reverse of the above example.

The column packing material layer A contains needle-like hydroxyapatite crystals having an aspect ratio of 10 or more, preferably 15 or more. At this time, the aspect ratio is a relative ratio (c / a) when the crystallite diameter in the c-axis direction of the hydroxyapatite primary crystal is the major axis and the crystallite diameter in the a-axis or the b-axis direction is the minor axis. It is preferable to adjust the crystallite diameter in the c-axis direction to 50 to 2000 nm and the crystallite diameter in the a-axis direction to 10 to 100 nm.

To prepare the acicular hydroxyapatite crystals, for example, EDTA or the like is added to a hydroxyapatite slurry obtained by a wet synthesis method,
Hydrochloric acid is added to and dissolved in a hydroxyapatite slurry obtained by a hydrothermal treatment method or a wet synthesis method to obtain pH = 4.
After adjusting to ~ 6, it can be obtained by a known method such as hydrothermal treatment.

In order to prepare the column packing material layer A, the acicular hydroxyapatite, preferably having a particle size of 1 to
It can be obtained by, for example, a method of granulating into a spherical aggregate of 100 μm and packing in a column. The specific surface area of the granulated spherical aggregates is preferably 10 to 100 m ² / g.

The column packing layer B also contains plate-like hydroxyapatite crystals having an aspect ratio of less than 2, preferably less than 2. At this time, it is preferable to adjust the crystallite diameter in the c-axis direction of the plate-like hydroxyapatite crystal to 1 to 200 μm and the crystallite diameter in the a-axis direction to 10 to 100 μm.

To prepare the plate-like hydroxyapatite crystals, for example, a method of hydrothermal treatment after adding sodium chloride or the like to a hydroxyapatite slurry obtained by a wet synthesis method or a hydroxyapatite slurry obtained by a wet synthesis method is added. It can be obtained by a known method such as a method of adding and dissolving sodium hydroxide to adjust the pH to 10 to 14, and then performing hydrothermal treatment.

To prepare the column packing layer B, the plate-like hydroxyapatite crystals preferably have a particle size of 1
It can be obtained by, for example, a method of granulating into spherical aggregates of ˜100 μm and packing in a column. The specific surface area of the granulated spherical aggregates is preferably 10 to 100 m ² / g.

In the liquid chromatography column of the present invention, the mixing ratio of the column packing layers A and B is
A: B = 1: 10 to 10: 1 is preferable, and especially 1 to 2
2: 1 is preferable.

The liquid chromatography column of the present invention comprises the column packing material layer A and the column packing material layer B connected in series, and the biochemical substances to be purified are tryptophan, albumin, Amino acids and proteins such as lysozyme and cytochrome C; single-stranded DNA,
Nucleic acids such as double-stranded DNA can be preferably mentioned.

[0018]

INDUSTRIAL APPLICABILITY The liquid chromatography column of the present invention has a column packing layer containing needle-shaped hydroxyapatite crystals and a column packing layer containing plate-shaped hydroxyapatite crystals connected in series. It provides good separation of biochemical substances and basic biochemical substances, and exhibits excellent adsorption characteristics for each substance. Therefore, it can be used for various biochemical analysis and the like.

[0019]

Example 1 7.37 g of calcium hydroxide and 85% by weight
500 ml of a 2 wt% hydroxyapatite slurry obtained by wet synthesis with 6.89 g of phosphoric acid was mixed with 5 ml of sodium chloride.
0 g was added, and hydrothermal treatment was performed under the conditions of 200 ° C., 2 MPa, and 5 hours to obtain plate-shaped hydroxyapatite primary crystals. The aspect ratio of this crystal was measured by an X-ray diffraction method and found to be 1.7. The crystallite diameter in the C-axis direction was 100 nm. Next, calcium hydroxide 7.37
ED in 500 ml of a 2 wt% hydroxyapatite slurry obtained by wet-synthesizing g and 85 wt% phosphoric acid 6.89 g.
25 g of TA was added, and hydrothermal treatment was performed under the conditions of 200 ° C., 2 MPa, and 5 hours to obtain acicular primary hydroxyapatite crystals. When the aspect ratio of this crystal was measured by the X-ray diffraction method, it was 12.9. The crystallite diameter in the C-axis direction was 300 nm. The obtained plate-shaped and needle-shaped hydroxyapatite primary crystals were granulated by a spray drying method, and then calcined at 900 ° C. for 1 hour,
Spherical aggregates each having an average particle size of 6 μm were obtained. 4φ x 1
Two 50 Lmm stainless steel columns were separately filled with column packings composed of these two types of crystals. Next, the two columns were connected in series with a joint and attached to a liquid chromatograph. Then tryptophan, albumin, lysozyme and cytochrome C
After injecting four kinds of biochemical substance mixed aqueous solution of No. ₃ into the column and adsorbing the mixture, a development elution test (flow rate: 0. 0%) was performed with a linear gradient (30 minutes) of 10 to 350 mM KH ₂ PO ₄ aqueous solution.
2 ml / min, detection: UV (280 nm)).
The result is shown in FIG. The degree of separation between albumin, which is an acidic biochemical substance having the closest retention time, and lysozyme, a basic biochemical substance, was calculated and compared. Table 1 shows the results. From these results, it was found that both the acidic chemical substance and the basic chemical substance were adsorbed and separated in a good balance in the column of the present invention.

[0020]

[Comparative Example 1] The spherical agglomerates of the plate-like hydroxyapatite having an average particle size of 6 µm obtained in Example 1 were 4φ x 300.
It was packed in an Lmm stainless steel column, and a development dissolution test was conducted in the same manner as in Example 1. The result is shown in Figure 1.
It shows in (a) and Table 1. From these results, it was found that although the degree of separation between albumin which is an acidic biochemical substance and lysozyme which is a basic biochemical substance is good, the adsorptive binding force of an acidic biochemical substance is poor.

[0021]

Comparative Example 2 The spherical agglomerates of the acicular hydroxyapatite having an average particle size of 6 μm obtained in Example 1 were 4φ × 300.
It was packed in an Lmm stainless steel column, and a development dissolution test was conducted in the same manner as in Example 1. The result is shown in Figure 1.
It shows in (b) and Table 1. From these results, the adsorptive bond strength of acidic biochemical substances and basic biochemical substances is good,
It was found that the degree of separation was poor.

[0022]

[Table 1]

[Brief description of drawings]

FIG. 1 (a) is a graph showing the results of a development dissolution test of a column containing plate-like hydroxyapatite crystals and Example 1. FIG. 1 (b) is a graph showing the results of the deployment elution test of the column containing acicular hydroxyapatite and Comparative Example 1. FIG. 1 (c) is a graph showing the results of the deployment dissolution test of a column containing plate-like hydroxyapatite crystals and needle-like hydroxyapatite crystals and Comparative Example 2.

Claims (1)

[Claims] 1. A column packing layer containing acicular hydroxyapatite crystals having an aspect ratio of 10 or more and a column packing layer containing plate-shaped hydroxyapatite crystals having an aspect ratio of less than 2 are connected in series. A column for liquid chromatography.