JPH0446383B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a column for high performance liquid chromatography. (Prior art) High performance liquid chromatography (hereinafter abbreviated as HPLC) has rapidly become popular in recent years due to its ability to complete separation in a short time with a small amount of sample compared to ordinary column chromatography. Widely used. Columns used in HPLC have various types of packing materials, such as silica-based, cellulose-based, and ion exchange resin-based, and the packing materials are used depending on the type of analyte. Among these, hydroxyapatite (hereinafter abbreviated as HAp) exhibits excellent properties for separating biopolymers, especially samples that are difficult to separate using other packing materials, such as
Because it can easily separate and purify single-stranded and double-stranded DNA and plasmid DNA with high purity, it is also used as a filler for HPLC. For example, such a conventional HPLC HAp packing material is produced using a method based on the synthesis method published by A. Tiselius et al. (Arch Biochem. Biophys., 65 , 132 (1956)), with appropriate improvements. There are products manufactured by the above method that have been further adjusted in particle size and crystal shape for HPLC use. It generally exhibits the following physical properties. Ca/P ratio (gram atom ratio) 1.40 to 1.55 X-ray diffraction Specific surface area with weak diffraction peaks 60 to 150 m ² /g Crystal shape Thin plate crystal (problem to be solved by the invention) However, the material used for this HPLC Conventional
HAp columns were not yet fully capable of achieving the goal of sharply separating a small amount of sample in a short period of time. Also,
Normally, due to the nature of HPLC, it is desirable to maintain a higher development back pressure, but there is a concern that HAp crystals may break under pressure, so they can only be used with a back pressure of about 5 kg/cm ² at most. Ta. Furthermore, since HAp for HPLC has a thin plate-like crystal shape, it easily breaks down into fine particles when packed into a column using the usual method of filling the column, and has the drawback that it is prone to clogging. Therefore, when using this HAp, while orienting the crystal so as not to destroy it,
It had to be packed into an HPLC column, and this work was very complicated. (Means for Solving the Problems) In view of the above circumstances, the present inventors have conducted intensive research to develop a column suitable for HPLC, and as a result, they have found that it is completely different from the conventionally known HAp for HPLC. , has excellent crystallinity in X-ray diffraction, and has a specific surface area of 50
By using HAp, which has a small value of less than m ² /g, it is possible to pack at a high density of 0.5 g/cm ³ or more, which was previously unattainable, and this highly densely packed column has extremely high separation performance as an HPLC column. Based on this knowledge, the present invention was developed. That is, the present invention provides a column for chromatography using a high-speed liquid, which is characterized by being packed with hydroxyapatite having a specific surface area of 50 m ² /g or less at a high density of 0.5 g/cm ^{3 or} more. In addition, the above-mentioned "packing density of hydroxyapatite" in the present invention refers to the value obtained by drying the hydroxyapatite in the column at a temperature of 110 ° C. for 2 hours and dividing the weight obtained by the effective volume of the column. . In the present invention, in order to achieve the objective, it is necessary to set the packing density of HAp in the column to 0.5 g/cm ³ or more. HPLC if density is less than 0.5g/ ^cm3
It is not possible to achieve sharp separation. The HAp used at this time is preferably a columnar crystal. Here, typical columnar crystals include hexagonal columnar or cylindrical crystals. Note that the lower limit of the specific surface area is not particularly limited, but it is usually preferably about 1 m ² /g or more. There are no particular limitations on the method for producing HAp used in the present invention, but the method previously proposed by the present inventors involves reacting a calcium compound and a phosphorus compound in a reaction medium containing an organic solvent capable of forming two phases with water. A method of reacting phosphoric acid or/and its calcium salt with calcium carbonate in a reaction medium containing an organic solvent (Japanese Unexamined Patent Publication No. 59-190807)
107912) etc. can be adopted. HAp produced by these methods has little difference in adsorption capacity between production lots, can be produced by simple operations, and is a powder with excellent physical properties. This thing is
It can be used not only for HPLC but also for ordinary column chromatography. According to the above method, HAp, which is an aggregate of hexagonal columnar or cylindrical crystals with a specific surface area of 50 m ² /g or less and which can have a packing density of 0.5 g/cm ³ or more, is produced, which is packed in a column. Compressive strength is 10g/cm ²
The above can be done. This aggregate has a nearly spherical shape and has excellent practical pressure strength for HPLC. Preferred physical properties of HAp used in the present invention are as follows. Ca/P ratio (gram atom ratio) 1.30-1.90 X-ray diffraction Strong diffraction peak. Specific surface area: 50 m ² /g or less Crystal shape: Aggregate of hexagonal columnar crystals Compressive strength when packed in column: 10 Kg/cm ² or more (effects of the invention) Conventional HAp columns for HPLC have almost reached their limit in separation ability, and HAp The packing density of HPLC of the present invention was 0.3 g/cm ³ at the highest.
The column has a packing density of HAp of 0.5 g/cm ³ or more, and exhibits further improved separation performance under development conditions unique to HPLC, especially at high development back pressures. Furthermore, whereas conventional columns are inferior in pressure resistance and durability, in the column of the present invention, HAp has crystal aggregates that are close to spherical, and it is possible to use crystalline materials that are good in X-rays. The crystals are not easily destroyed and can be easily packed at a density of 0.5 g/ ^cm
satisfies the practical filling pressure of 10Kg/ ^cm2 , and
There is no problem at all even if high pressure of 500Kg/cm ² or more is applied. Furthermore, unlike conventional products, the HAp column of the present invention does not require the complicated operation of orienting the crystals while filling the HPLC column, so it has the advantage that the column can be easily filled. Furthermore, as mentioned above, the improved pressure resistance means that the column has a longer lifespan and can be used over 200 times.
Has excellent durability. Another advantage is that the flow rate can be controlled by adjusting the pressure, so a smaller amount of developing liquid is required compared to conventional products. Furthermore, this HAp column can be used not only for analytical HPLC purposes, but also as a preparative HPLC column for preparative separation purposes, making it possible to separate and purify the required sample with high purity in a short period of time. Since it is possible, its value is also great. (Examples) Hereinafter, the present invention will be explained in detail with reference to production examples, examples, and comparative examples. Production example 1 Monobasic calcium phosphate monohydrate (Ca(H ₂ PO ₄ ) ₂ H ₂ O) 75.6 g, calcium carbonate (CaCO ₃ ) 40.0 g, water 100 g and n-hexane
Pour 350g into a 1-volume flask, raise the temperature while stirring, and heat for about 2 hours below the reflux temperature (63℃), then evaporate the water and n-hexane in the reaction system and distill the n-hexane and water. n of capacity corresponding to
- Heating was performed while adding hexane to the system. As the operation progresses, the internal temperature rises and reaches 68℃.
Heating was stopped when the temperature exceeded 100 mL, and after cooling, the product was filtered from the solvent and dried to obtain a white powder. The X-ray diffraction pattern of this powder is shown in FIG. As is clear from Figure 9, the V-ray diffraction diagram has main peaks at diffraction angles 2θ = 31.7, 32.2, and 32.8,
This coincided with the characteristic diffraction peak of HAp described in ASTM Card 9-432. The grain shape is spherical or similar as shown in FIG. 11, and is an aggregation of fine hexagonal columnar crystals. The physical property analysis results are shown in Table 2. Production Examples 2 to 4 Anhydrous dicalcium phosphate, dicalcium phosphate dihydrate, calcium hydroxide, calcium carbonate, water, and organic solvent were prepared under the conditions shown in Table 1. For conditions other than Table 1, , HAp was obtained by the same operation as in Example 1. Its physical properties are shown in Table 2.

【table】

[Table] Production example 5 Conventional HAp synthesis method (A. Tiselius et al. Arch.Biochem.
Biophys., 65, 132 (1956)). Add 0.5 mol/CaCl ₂ aqueous solution 1 to 3 volume flask
and 0.5 mol/Na ₂ HPO ₄ aqueous solution 1 per minute.
The mixture was added dropwise at the same time at a rate of 120 drops, and the reaction was carried out while stirring slowly 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 two portions of distilled water each time. Next, distilled water from Step 2 was added to this precipitate, and 50 ml of 40% by weight NaOH was added thereto, followed by boiling for 1 hour.
After cooling, the supernatant liquid was removed again by decantation, and the mixture was again washed four times with distilled water from step 2. Then PH
6.8 0.01mol/sodium phosphate buffer solution
Add and heat until just before boiling. This operation was repeated twice, with boiling times of 5 minutes and 15 minutes, respectively. Next, two 0.001 mol/sodium phosphate buffer solutions with a pH of 6.8 were added and the mixture was boiled twice (for 15 minutes). The thus obtained HAp was placed in a 0.001 mol/sodium phosphate buffer solution with a pH of 6.8 and stored in a refrigerator. As a result, HAp with the physical properties shown in Table 2 was obtained. Further, an X-ray diffraction diagram is shown in FIG. 10, and an electron micrograph is shown in FIG. 12. Example 1 HAp obtained in Production Example 1 was transferred to a metal column for HPLC with an inner diameter of 8 mm and a length of 10 cm using a routine method.
The column was packed at a pressure of 10 Kg/cm ² , and after filling was completed, the column was attached to an HPLC device. The packing density was 0.52 g/cm ³ . An electron micrograph of HAp obtained in Production Example 1 is shown in FIG. As can be seen from this, this HAp has a roughly spherical aggregate of hexagonal columnar crystals.
In addition, to evaluate the separation ability of the column, tryptophan (0.16% by weight), bovine serum albumin (4.6% by weight), lysotium (2.0% by weight), and cytochrome C
(1.0% by weight) was used as a standard sample for development. Deployed with 10m of phosphate buffer
The measurement was carried out by the linear density gradient method using M (PH6.8) and 350mM (PH6.8), and 10.0μ of the above standard sample was injected. Also, the flow rate is 7.5Kg/min so that it is 1ml/min.
The development pressure was set at ^cm2 . Protein was detected by measuring absorbance at 280 nm. A chromatogram obtained with this column is shown in FIG. As is clear from FIG. 1, main peaks appear at four locations, indicating that the four components of the standard sample are clearly separated. It was confirmed that each peak corresponds to tryptophan, bovine serum albumin, lysotium, and cytochrome C from the left side. Furthermore, cytochrome C has
There are two types of isomers, and the peaks are separated into two. Example 2 80Kg/inner diameter 8mm, length 10cm HPLC column
Filled with HAp obtained from Production Example 3 at a pressure of cm ² ,
The subsequent operations were performed using HPLC in the same manner as in Example 1.
The operation of the column was carried out. Packing density is 0.60g/ ^cm3
It was hot. The results are shown in FIG. As can be seen from FIG. 2, the same results as the chromatogram obtained in Example 1 were shown. Example 3 Using the column used in Example 2, standard sample separation was performed 200 times. The chromatogram obtained at the 200th time is shown in Figure 3. As can be seen from Figure 3, the column of the present invention exhibits good separation performance even after repeated use 200 times, and has excellent durability. Example 4 500 ml in an HPLC column with an inner diameter of 8 mm and a length of 10 cm.
The HAp obtained in Production Example 4 was packed at a pressure of Kg/cm ² to a density of 1.03 g/cm ³ , and the subsequent operations were performed in the same manner as in Example 1 to operate the HPLC column. The results are shown in FIG. From FIG. 4, it can be seen that even though the column was filled with HAp at high pressure, the chromatograms showed the same results as those obtained in Examples 1 and 2, indicating that the column had excellent pressure resistance. Example 5 A preparative column with an inner diameter of 20 mm and a length of 25 cm was loaded with 80 kg/
HAp obtained according to Preparation Example 2 at a pressure of cm ² with a density of 0.61
cm ³ and operated in the same manner as in Example 1.
Manipulated HPLC columns. However, the standard sample was injected at 70μ. The chromatogram thus obtained is shown in FIG. From the results shown in FIG. 5, it was found that the column of the present invention gave the same results as conventional HPLC and could be sufficiently used for preparative separation. Example 6 A HPLC column with an inner diameter of 8 mm and a length of 10 cm was
The density of HAp obtained according to Preparation Example 4 at a pressure of Kg/cm ² was
Filled to 0.58g/cm ³ and the subsequent operations were as in Example 1.
I went in the same way. However, the pressure was adjusted to 50 Kg/cm ² so that the pressure was 3.4 ml/min during development. The chromatogram thus obtained is shown in FIG. As can be seen from FIG. 6, each of the four peaks was clearly separated even though the separation was performed in a short time. Example 7 Using a column packed under the same conditions as Example 1, LDH
(lactate dehydrogenase) was separated. The development was carried out under the same conditions as for the standard sample, and the sample was
8μ was injected. Protein was detected by measuring absorbance at 280 nm, and enzyme activity was measured by measuring absorbance at 340 nm. The chromatogram thus obtained is shown in FIG. As can be seen from Figure 7, the peak indicating protein elution and the peak indicating enzyme activity coincide on the right side, indicating that LDH is separated here. Furthermore, since the impure protein contained in the sample appears on the left side, it can be seen that LDH has been separated and purified to a high degree of purity. Comparative Example 1 HAp obtained according to Production Example 5 was prepared with an inner diameter of 8 mm and a length of
Packed into a 10cm column, and the subsequent operations were as in Example 1.
The standard sample was separated using the same procedure. In addition,
The packing density at this time was 0.28 g/cm ³ . Further, in order to set the flow rate to 1 ml/min, the pressure was adjusted to 1 Kg/cm ² , but the pressure was too low for HPLC, making the operation difficult. The chromatogram thus obtained is shown in FIG. As can be seen from the comparison between Figure 8 and Figure 1,
The sample filled with the conventional product showed a broad peak overall. Comparative Example 2 HAp from Production Example 5 was packed into a column with an inner diameter of 8 mm and a length of 10 cm at a packing pressure of 15 Kg/cm ² . However, the crystals inside were destroyed and clogged, making it impossible to deploy.

[Brief explanation of the drawing]

1 to 7 show chromatograms obtained in Examples 1 to 7, respectively.
FIG. 8 shows a chromatogram obtained in Comparative Example 1. 9 and 10 show X-ray diffraction patterns of HAp obtained in Production Example 1 and Production Example 5, respectively. FIGS. 11 and 12 are photographs in place of drawings, and are electron micrographs showing the crystal structures of HAp (hydroxyapatite) obtained in Production Examples 1 and 5, respectively.

Claims (1)

[Scope of Claims] 1. A column for high performance liquid chromatography, characterized in that it is packed with hydroxyapatite having a specific surface area of 50 m ² /g or less at a high density of 0.5 g/cm ³ or more. 2. The column for high performance liquid chromatography according to claim 1, wherein the hydroxyapatite is a columnar crystal. 3. The column for high performance liquid chromatography according to claim 2, wherein the aggregate of columnar crystals is approximately spherical.