JPH0572546B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to a packing material for high performance liquid chromatography and a method for producing the same.

[Background of the invention]

In recent years, high performance liquid chromatography (hereinafter referred to as
HLC (HLC) is used in all chemical fields as an important separation and purification technique because of its short measurement time and high resolution.
This HLC was particularly used in the field of analytical chemistry, but recently it has been used for molecular weight separation of synthetic polymers, separation of various natural proteins, separation of blood proteins in living organisms, separation of hormones, and even in the field of biochemistry. It is now being used for purposes such as separating enzymes, nucleic acids, and pharmaceuticals, and its scope of application is rapidly expanding.

As mentioned above, high performance liquid chromatography is a useful analytical technique, and in order to further speed up separation and improve performance, improvements to HLC equipment have been made, for example: 1. Method of filling a column with a packing material, 2. High pressure liquid feeding section, 3. Separation packed column, 4.
Detection unit, 5. This is done from various viewpoints such as selection of elution conditions.

Among the above improvements in separation packed columns are generally based on improvements in the packing material. The properties required for HLC fillers include the following properties.

(1) Pore size must be controlled.

Among HCLs, in gel permeation chromatography (hereinafter referred to as GPC) that utilizes the molecular sieve effect, the solute can enter the particle pores depending on the relative relationship between the pore size of the stationary phase particle and the size of the solute molecule. As a result, a time lag occurs in elution time, and molecules elute sequentially from large molecules to small molecules. In particular, when separating a wide range of compounds from low molecular weight compounds such as amino acids and oligomers to high molecular weight compounds such as synthetic polymer polypeptides, the pores of the packing material need to be controlled over a wide range and arbitrarily. be. However, when separating a mixture of compounds with similar molecular weights, a filler with a sharp pore size distribution is naturally required.

Therefore, control of pore size over a wide range and sharpness of pore size distribution are important quality characteristics of fillers for GPC.

(2) The particle size is small and the particles are spherical.

In HLC, the number of theoretical plates N based on the following formula is generally used as a parameter indicating peak broadening, and it can be said that the larger N is, the better the separation performance of the column is.

N=16 (Ve/W) ² Ve: peak elution volume W: peak width By reducing the particle size, the diffusion equilibrium time of the solute in the stationary phase becomes shorter, making it possible to reduce W. At the same time, if the particle size distribution is sharpened, N becomes larger. This is because if the particle size distribution is wide, a separation zone will occur between large and small particles in the column. Also, in order to increase Ve,
It is necessary to have a large pore volume and total porosity.

Furthermore, it is also important to make the particle shape spherical. This is because the spheroidization of the particle shape reduces the interparticle voids, increasing the holding capacity, making the flow of the mobile phase uniform, and preventing the occurrence of vortices and abnormal flow paths.

(3) Must be a stable hard gel.

When reducing the particle size of the filler to improve the performance of HLC, pressure loss increases, and high pressure must necessarily be applied to obtain a constant flow rate.
Therefore, the filler must have high mechanical strength so as not to be deformed or damaged even under high pressure, and must also be chemically free from swelling, shrinkage, and denaturation.

As fillers for HCL, hard types such as silica gel and porous glass particles, semi-hard types such as polystyrene particles, and soft types such as polyacrylamide particles and polysaccharide gel have been commercially available and used.

Among these, soft gels are not suitable for HLC due to swelling, shrinkage, and pressure deformation. Semi-rigid types are commonly used as organic solvent-based fillers, but they cannot be applied to aqueous systems unless special surface modification is performed, and there are restrictions on the solvents that can be used. On the other hand, the disadvantages of silica gel are that the pore size control range is relatively narrow (approximately 500A° or less), making it difficult to obtain large pores, and because the pore distribution is poor (wide), it is difficult to separate substances with similar molecular weights. It cannot be said that it is suitable for GPC use.

Porous glass particles are fillers that overcome the shortcomings of silica gel and have excellent pore properties. A general method for making porous glass particles is described in U.S. Patent No.
No. 2106744, No. 2221709, No. 3549524, No.
In the specifications such as No. 3758284, and “Membrane Journal” 4 (4)
221-227 (1979) and "Gel Permeation Chromatography of Giant Particles" 3-18 (1980). However, the manufacturing methods described in these publications produce porous glasses with fine particles suitable for HLC (particle diameters of approx.
(30 μm or less, especially about 10 μm or less) is difficult to manufacture.

[Object of the Invention] An object of the present invention is to provide a porous glass filler suitable for use in a high performance liquid chromatography device.

It is also an object of the present invention to provide a porous glass filler suitable for use in high performance liquid chromatography devices, particularly in adsorption and gel permeation chromatography devices.

[Summary of the invention]

The present invention provides porous glass particles of sodium borosilicate consisting of a three-component system of SiO ₂・B ₂ O ₃・Na ₂ O,
The average diameter of the porous glass particles is 0.1 to 10 μm, the average pore size is 80 to 3000 angstroms, and the pore size distribution (cumulative 10, 90% point standard) is ±15
% and a specific surface area of 10 to 250 m ² /g.

The porous glass filler for high performance liquid chromatography described above is made by heat-treating phase-splitting soda borosilicate glass, which has a three-component system of SiO ₂ , B ₂ O ₃ , and Na ₂ O, to separate the phases. Porous glass particles obtained by eluting and removing at least a portion of the acid-soluble phase by acid elution treatment are subjected to a fine pulverization operation and a classification operation to obtain particles with an average diameter of 0.1 to 10 μm and an average pore size of 80 to 3000.
It can be advantageously produced by a method for obtaining porous glass particles having a pore size distribution (accumulated 10, 90% point reference) within ±15% and a specific surface area of 10 to 250 m ² /g.

[Detailed description of the invention]

The porous glass used in the present invention includes SiO ₂ , B ₂ O ₃ ,
It is a three-component borosilicate glass consisting of Na ₂ O.
It is manufactured by utilizing the phase separation phenomenon in a specific composition range. The method for producing porous glass particles from the above three-component borosilicate glass is described, for example, in the publications listed above and is well known. Therefore, typical examples of the manufacturing method will be shown below.

A raw material mixed batch of SiO ₂ , B ₂ O ₃ , and Na ₂ O is placed in a platinum crucible and melted at 1300 to 1450°C. After the melt is rapidly cooled to room temperature, it is subjected to phase separation heat treatment at 500-700°C. By this process, inside the melt
Separation of the SiO ₂ rich phase and the B ₂ O ₃ .Na ₂ O rich phase takes place. During this separation, each separated phase exhibits an entangled structure in which they are entangled with each other, and the size of each separated phase can be arbitrarily controlled by the temperature and time of the phase separation heat treatment. After heat treatment, the cooled material is crushed into particles with a diameter of approximately 50 μm or more using a crusher. Then,
In order to elute the acid-soluble B ₂ O ₃ · Na ₂ O phase and make it porous, an inorganic acid such as hydrochloric acid is used under heating (e.g.
Acid treatment at approximately 90℃). By this acid treatment
The B ₂ O ₃ .Na ₂ O phase is eluted, thereby forming a SiO ₂ -rich skeletal phase with many pores. Colloidal silica remains in these pores, which remain and partially block the pores, so an alkaline aqueous solution (e.g.
Colloidal silica is eluted using 1/2N-NaOH aqueous solution). Thereafter, water washing, pickling, and water washing are repeated to clean the inside of the pores and obtain porous glass.

However, no example has been reported to date of producing fine-particle porous glass having a particle diameter of 30 μm or less using the above method. One reason for this may be that such an attempt has simply not been made, but another reason is that it is extremely difficult to produce fine-particle porous glass with particle diameters of 30 μm or less using only the above method. One thing can be mentioned. That is, in the above method, an alkaline aqueous solution is applied to elute colloidal silica, but at this time, fine particles of 30 μm or less are also dissolved at the same time.

According to the studies of the present inventors, by subjecting the porous glass produced as described above to fine pulverization and classification, the average particle diameter of the porous glass, which is suitable as a filler for HLC, is approximately 0.1 to 1. It has been found that fine particle porous glass with a diameter of 10 μm can be obtained. less than,
The pulverization operation and classification operation performed on porous glass will be explained by giving an example.

The porous glass produced as described above is first pulverized using an impact type pin mill, for example. It is desirable to use a pin mill lined with anti-wear ceramic made of zirconia for the powder contacting part. This is to prevent metal powder from being mixed in. Using a metal pin mill causes significant wear,
It is difficult to separate fine metal powder mixed into porous glass from the porous glass even through a strong magnetic separation device.

Next, it is roughly classified using a centrifugal air classifier. Although it is difficult to obtain the particle size distribution required for an HLC filler by air classification alone, it is desirable to perform this coarse classification in order to improve the efficiency of water sieve classification in the later process.

Next, water sieve classification is performed. This water sieve classification is based on the usual Stokes sedimentation principle, but requires water sieving several times to more than ten times. The most preferable dispersion medium is water, but if necessary, thickeners such as glycerin and polyethylene glycols can be added to control the sedimentation rate, and sodium hexametaphosphate, sodium pyrophosphate, sodium dodecyl sulfate, etc. The dispersibility of particles can also be improved by adding a surfactant. Finally, it is washed with water and dried to complete the fine-particle porous glass.

Examples of physical and chemical properties of micronized porous glass obtained by the above-mentioned pulverization and classification operations are shown below.

(1) Pore characteristics 1) Average pore size range: pore diameter = 80 to 3000 Å (any pore size within this range can be manufactured) 2) Pore size distribution (cumulative 10, 90% point standard): ≦±15 % 3) Pore volume: about 0.8 ml/g 4) Specific surface area: about 10 to 250 m ² /g The above pore characteristics can be measured using a known porosimeter using mercury porosimetry. Regarding 2) pore size distribution (cumulative 10, 90% point standard), "MEMBRANE" 4
(4) 221-227 (1979), and 11 of “Gel Permeation Chromatography of Giant Particles - Particle Size Distribution of Biological Particles” (Kitami Shobo, published in 1980).
It can be obtained from the cumulative distribution curve of the pore size measured by mercury intrusion method, as described on page 1. To explain this in detail, we use a porosimeter based on mercury intrusion to determine the pore diameter (Angstrom).
Create a cumulative distribution curve of pore diameter showing the relationship with "relative cumulative specific pore volume (%)", and calculate the relative cumulative specific pore volume 10
"φ ₅₀ %" (average pore diameter) of the pore diameters corresponding to the % and 90% points, i.e. "φ ₁₀ %" and "φ ₉₀ %"
The relative percentages "-PD" and "+DD" with reference to "-PD" are calculated using the following formula.

−PD (%) = (φ ₁₀ % − φ ₅₀ %) / φ ₅₀ % × 100 + PD (%) = (φ ₉₀ % − φ ₅₀ % / φ ₅₀ % × 100 In other words, pore size distribution (cumulative 10, 90% point reference) is the value expressed by the above-mentioned -PD value (%) and +PD value (%).

(2) Particle size distribution 1) Average particle size: particle diameter = 0.1 to 30 μm 2) Particle size distribution (cumulative 10, 90% point standard): ≦±35% (3) Other physicochemical properties 1) Chemical composition: SiO ₂ 92%, B ₂ O ₃ 7% Na ₂ O <1wt% 2) True specific gravity = 2.2 3) High specific gravity = 0.50 4) Silanol group = approximately 1.5 μmol/m ² (based on the measured value of methyl Red adsorption in toluene) 5) High temperature resistance: Can be used up to 600℃ 6) Linear thermal expansion coefficient = 8 x 10 ^-7 cm/cm・℃ (up to 600℃) The above fine particulate porous glass has a about
It has adsorption activity because it retains 1.5 μmol/m ² of silanol groups, and therefore high performance liquid chromatography can be performed in adsorption mode. It can also be used in GPC mode that utilizes the hole size.

The column can be filled with the filler of the present invention by a conventional wet slurry filling method.

Examples of the present invention are described below.

[Example 1] Using silica sand, boric acid and sodium nitrate as raw materials, the composition in terms of oxides is SiO ₂ = 60.3%, B ₂ O ₃
= 30.0% and Na ₂ O = 9.7% (both weight %), and were uniformly mixed in a ball mill to obtain 80 kg of a raw material mixture. This raw material mixture was placed in a platinum crucible and melted in an electric furnace at 1350°C with stirring for 10 hours. Then, it was rapidly cooled to room temperature and subjected to phase separation heat treatment at 550°C for 96 hours. Thereafter, it was coarsely ground using a cross beater mill, adjusted to a particle size of 80 to 400 mesh using a sieve, and acid-treated with 3N hydrochloric acid at 90°C for 24 hours. After washing with water, colloidal silica removal treatment was performed at 20 to 25°C for 2 hours using 1/2N sodium hydroxide solution.

In order to remove the alkaline solution, it was thoroughly washed with water, and then acid washed with 1N hydrochloric acid at room temperature for 2 hours. The glass was washed with water until the pH reached 7, and finally dried at 100°C to produce a coarse-particle porous glass.

The characteristics of this sample are average pore diameter 170Å, specific pore volume 0.81ml/g, pore size distribution +11/-15%, and specific surface area.
The area was 162 m ² /g, and the silanol group was 227 μmol/g. Pore properties were measured by mercury porosimetry, specific surface area by BET method, and silanol groups by methyl red adsorption method.

After finely pulverizing the above coarse particle porous glass with an impact pin mill, it was classified using an air classifier with a classification point of 4.0μm and
The particles were classified at 6.0 μm, and the particles of 4.0 to 6.0 μm were classified using a water sieve.

Water sieve classification: sedimentation height 10cm, sedimentation time 1.5 and 2.0
This was repeated 10 times for an hour. The dispersion medium was pure water. Finally, vacuum drying was performed at 100°C to obtain fine particulate porous glass. The particle size characteristics of the obtained microparticulate porous glass are as follows: average particle size (D=50)=
The particle size distribution (D90/D50) was 1.30, and the particle size distribution (D50/D10) was 1.31.

Adsorption mode high performance liquid chromatography was carried out using the above-mentioned particulate porous glass filler under the following conditions.

Sample: Nitrobenzene/o-cresol/p-
Cresol mixture (1:1:1, weight ratio) Column size: Diameter 4.6 mm x length 150 mm Eluent: Methylene chloride (50% water saturation) Flow rate: 1.0 ml/min The chart obtained is shown in Figure 1. . In FIG. 1, peak numbers 1, 2, and 3 mean the peaks of nitrobenzene (1), o-cresol (2), and p-cresol (3), respectively.

As is clear from FIG. 1, mixtures of compounds with similar molecular weights and chemical structures can be separated with high accuracy by adsorption mode high performance liquid chromatography using the fine particulate porous glass packing material of the present invention. confirmed.

[Example 2] Using the particulate porous glass filler obtained in Example 1, GPC mode high performance liquid chromatography was performed under the following conditions.

Sample: DMP adenine/blue dextran mixture (2:1, weight ratio) Column size: Diameter 4.6 mm x length 150 mm Eluent: 0.1 M sodium phosphate buffer (PH
7.0) + 6M guanidine hydrochloride Flow rate: 1.0ml/min The obtained chart is shown in Figure 2. In FIG. 2, peak numbers A and B mean the peaks of DMP adenine (A) and blue dextran (B), respectively.

As is clear from FIG. 2, by GPC mode high performance liquid chromatography using the fine particulate porous glass filler of the present invention, similar molecular weight,
It was confirmed that mixtures of compounds with chemical structures can be separated with high precision.

[Brief explanation of the drawing]

FIG. 1 is a graph showing an example of an elution curve obtained by an adsorption mode high performance liquid chromatography operation performed using the packing material of the present invention. Second
The figure is a graph showing an example of an elution curve obtained by GPC mode high performance liquid chromatography operation using the packing material of the present invention.

Claims (1)

[Claims] 1. Porous glass particles made of sodium borosilicate consisting of a ternary system of SiO ₂ .B ₂ O ₃ .Na ₂ O, wherein the porous glass particles have an average diameter of 0.1 to 10 μm, A high performance liquid chromatograph having an average pore diameter of 80 to 3000 angstroms, a pore diameter distribution (cumulative 10, 90% point reference) within ±15%, and a specific surface area of 10 to 250 m ² /g. Porous glass filler for e. 2 After heat-treating the ternary phase-splitting borosilicate soda glass of SiO ₂・B ₂ O ₃・Na ₂ O to cause phase separation,
Porous glass particles obtained by eluting and removing at least a portion of the acid-soluble phase by acid elution treatment are subjected to a fine pulverization operation and a classification operation to obtain porous glass particles with an average diameter of 0.1 to 10 μm, an average pore diameter of 80 to 3000 angstroms, Pore size distribution (cumulative 10, 90% point reference) within ±15%,
and a method for producing a porous glass filler for high performance liquid chromatography, characterized by obtaining porous glass particles having a specific surface area of 10 to 250 m ² /g.