JPH0312256B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a packing material for liquid chromatography.

In recent years, liquid chromatography has shown remarkable technological progress, which is largely due to the development of packing materials that can withstand high pressures of several hundred kg/cm ² . Conventional packing materials for liquid chromatography include:
Porous silica, which has the mechanical strength to withstand high pressure, has been introduced with octadecyl groups to give it separation properties.

However, with the development of clinical testing technology, technology to rapidly and highly accurately separate proteins from biological samples such as blood and urine has been explored, but silica fillers with octadecyl groups have been found to be effective. The disadvantage was that it was not possible to perform a precise separation.

For this reason, fillers with excellent performance in separating components such as proteins in biological samples have been sought, and for example, synthetic polymer fillers such as methacrylic acid-divinylbenzene copolymer have been used. However, such synthetic polymer fillers have low pressure resistance and about
Since it can withstand only a pressure of about 30 kg/cm ² , it is not possible to sufficiently increase the speed of liquid chromatography, and the filler itself is easily destroyed, leaving problems with durability.

The present invention was made with the aim of eliminating the above-mentioned drawbacks of conventional packing materials for liquid chromatography, and its gist is to provide hydrophilic polymers containing vinyl monomers as a polymerization component. Hollow, substantially spherical particles formed by coalescence, wherein the ratio (L/D) of particle diameter (D) to wall thickness (L) at any location of the hollow, substantially spherical particles is in the range of 0.06 to 0.45. Among the existing packing materials for liquid chromatography.

Next, the packing material for liquid chromatography of the present invention will be explained in more detail.

The packing material for liquid chromatography of the present invention is made of a hydrophilic polymer containing a vinyl monomer as a polymerization component. The reason why polymers are made to have hydrophilic properties is to give them separation ability from aqueous eluents.For this reason, the vinyl monomers constituting the polymers are those with carboxyl groups, such as acrylics. The acid, methacrylic acid may also be used or the following general formula (However, in the formula, R ₁ and R ₂ are hydrogen atoms or methyl groups,
n is an integer from 2 to 18). Divinyl monomers are suitable for use.

For example, when the divinyl monomer represented by the above general formula is used, when R ₁ and R ₂ are methyl groups and n = 14, it is tetradecyl glycol dimethacrylate, and when n = 2, it is tetradecyl glycol dimethacrylate. is diethylene glycol dimethacrylate,
These polymers have moderate hydrophilicity,
A polymer with good separation performance for proteins, etc. in blood and urine can be obtained.

Therefore, in the present invention, a copolymer of the vinyl monomer and a polyfunctional monomer such as divinylbenzene, divinyltoluene, diallyl phthalate, diallyl maleate, triallyl isocyanurate, etc. is used. Thus, the degree of hydrophilicity can be adjusted.

The degree of hydrophilicity is preferably such that the solubility parameter of the polymer is 8.4 or more.

Solubility parameters (SD values) are density (P),
It is a value determined from the molecular weight (M) and cohesive energy constant (G) using the following formula.

SP=ρΣG/M When the solubility parameter of the polymer is smaller than 8.4, the polymer has poor wettability with water;
This is because it becomes unsuitable for separation from an aqueous eluent.

The packing material for liquid chromatography in the present invention consists of hollow, substantially spherical particles. Since the packing material is made of hollow, substantially spherical particles, when the column is filled and subjected to liquid chromatography, a high flow rate can be obtained at low pressure, making it possible to perform high-speed separation.

Even if the packing material is approximately spherical particles, if they are not hollow, when the column is filled and subjected to liquid chromatography, the column pressure will increase significantly if the same flow rate is to be obtained, and the column pressure will increase significantly. If it is held to the same extent as when it is hollow, the separation performance is likely to be impaired.

Further, in the liquid chromatography packing material of the present invention, the ratio (L/D) of particle size (D) to wall thickness (L) at any location is in the range of 0.06 to 0.45.
Here, the term "arbitrary location" refers to any location arbitrarily selected, and for approximately spherical particles, the (L/D) value is smaller than 0.06 or larger than 0.45. This means that there are no locations where the value is large.

When the ratio of particle size (D) to wall thickness (L) (L/D) is smaller than 0.06, the packing material tends to form dents, and when subjected to liquid chromatography after filling the column, the dents may be formed. As a result, the fractionation peak becomes broader. Also, the ratio of particle size (D) to wall thickness (L) (L/D) is
When it is larger than 0.45, when trying to obtain the same flow rate when the column is packed and subjected to liquid chromatography, the column pressure will increase significantly, and if the column pressure is lowered, the separation performance will be impaired.

The packing material for liquid chromatography of the present invention can be obtained by aqueous suspension polymerization of the above-mentioned monomers. The polymerization reaction is carried out in the presence of a diluent with poor compatibility. For example, when methacrylic acid or diethylene glycol dimethacrylate is used as a monomer, a mixture of toluene/n-octanol can be used as a diluent.

In the aqueous suspension polymerization, for example, a radical generating catalyst is dissolved in a mixture of the monomers and a diluent, and the solution is added to an aqueous phase in which a suspension polymerization stabilizer such as polyvinyl alcohol or calcium phosphate is dispersed, and the mixture is stirred for 50 to 50 min.
This is done by heating to 100°C.

The above-mentioned radical generating catalyst is a catalyst that generates radicals as a reaction initiator, and examples thereof include organic peroxides such as benzoyl peroxide and cumene oxide, inorganic peroxides such as hydrogen peroxide and ammonium persulfate, and azobisisomer. Azo compounds such as butyronitrile and azobisisobutyramide are used.

The polymer is washed with water, dried, and then classified to have a uniform particle size.

The packing material for liquid chromatography of the present invention has excellent performance in separating and quantifying trace amounts of components such as proteins from biological samples such as blood and urine, and can be used at low column pressure and high speed during liquid chromatography. It becomes something that can be fractionated.

Example 1 In a 2-capacity separable flask, 400 ml of 4% by weight polyvinyl alcohol aqueous solution, 60 g of diethylene glycol dimethacrylate, and 40 g of methacrylic acid were added.
g, 40 g of toluene/n-octanol mixture (mixing ratio 2/1) and 1.5 g of benzoyl peroxide.
A mixed solution consisting of 50 g was added thereto, and the temperature was raised to 80° C. while stirring at a stirring speed of 400 revolutions/min, and a polymerization reaction was carried out for 10 hours. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a substantially spherical methacrylic acid-diethylene glycol dimethacrylate copolymer with a particle diameter of 7 to 15 μm.

Scanning electron micrographs of these approximately spherical copolymer particles are shown in FIGS. 1 and 2. Also, particle diameter (D) and wall thickness (L) at arbitrary locations of approximately spherical copolymer particles.
The ratio (L/D) was about 0.14.

The roughly spherical copolymer particles obtained in this way were classified to have almost the same particle size. 15 ml was dispersed in 120 ml of water, and a stainless steel column (7.6 mm in diameter, 25 mm in length) was dispersed in 120 ml of water.
cm) was filled with water by pumping water at a rate of 25 ml/min using a high-pressure constant flow pump.

The column obtained in this way was connected to a high-performance liquid chromatography device, and a UV-visible spectrophotometer (measurement wavelength 415 nm) was used as a detector and as an eluent.
As a result of separation analysis using normal human hemolysate as a sample using 50mM phosphate buffer PH6.5 and 200nM phosphate buffer PH6.3, a liquid chromatogram with the pattern shown in Figure 8 was obtained. .

Separate each peak part of the detection pattern,
As a result of identification, P ₁ was hemoglobin A ₁ a+b, P ₂ was hemoglobin A ₁ c, and P ₃ was other hemoglobin, and at a flow rate of 1.0 ml/min, the column pressure was 30 Kg/cm ² . In addition, the above separation and analysis were repeated 500 times, but
No phenomena such as pressure increase occurred.

Example 2 In a 2-capacity separable flask, 100 g of tetradecyl ethylene glycol dimethacrylate and a toluene/n-octanol mixture (mixing ratio 1/
1) 40 g of the mixed solution was added, and the temperature was raised to 80° C. while stirring at a stirring speed of 400 rpm, and a polymerization reaction was carried out for 10 hours. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain approximately spherical polymer particles. Next, classification was performed.

A scanning electron microscope photograph of this approximately spherical polymer particle is shown in FIG. Further, the ratio (L/D) between the particle size (D) and the wall thickness (L) at any location of this approximately spherical polymer was about 0.08.

These approximately spherical polymer particles were packed into a stainless steel column (inner diameter 7.9 mm, length 50 cm) in the same manner as in Example 1, gel vermiaction chromatography was performed using dextran as a sample, and a differential spectrometer was used. As a result of separation analysis, dextran of each molecular weight was eluted in descending order of molecular weight, and the exclusion limit was 200,000.

Comparative Example 1 Polymerization, washing, and classification were carried out in the same manner as in Example 1 except that the amount of toluene/n-octanol mixture (mixing ratio 2/1) was changed to 60 g to obtain a copolymer. However, as shown in Figures 4 and 5, a part of the wall surface was deformed in a concave manner, and as shown in Figure 5, the thickness of the fractured surface was significantly uneven in some places. It was.

The thus obtained copolymer particles were packed into a stainless steel column in the same manner as in Example 1, and measured using the same measuring device and under the same elution conditions as in Example 1. As a result, a liquid chromatogram with a pattern as shown in Figure 8 was obtained. grams were obtained. When this copolymer particle was used, the separation performance was poorer than that in Example 1, and the column pressure at a flow rate of 1.0 ml/min was 28 Kg/cm ²
It was hot.

Comparative Example 2 Polymerization, washing, and classification were carried out in the same manner as in Example 1 except that the amount of toluene/n-octanol mixture (mixing ratio 2/1) was changed to 20 g to obtain a copolymer. Obtained. Scanning electron micrographs of this copolymer are shown in Figures 6 and 7.
As shown in the figure, the particles were not hollow particles as shown in the fractured surface of FIG.

Further, although a stainless steel column was filled in the same manner as in Example 1, the pressure rose rapidly, causing deformation and breakage of the packing material.

[Brief explanation of the drawing]

FIG. 1 is a scanning electron micrograph of a hollow approximately spherical particle in Example 1, FIG. 2 is a scanning electron micrograph showing an enlarged fracture surface of the same, and FIG. It is a scanning electron micrograph showing an enlarged view of a fractured surface of a substantially spherical particle. FIG. 4 is a scanning electron micrograph of particles in Comparative Example 1, FIG. 5 is an enlarged scanning electron micrograph of the same, FIG. 6 is a scanning electron micrograph of particles in Comparative Example 2, and FIG. is a scanning electron micrograph showing an enlarged view of the fracture surface of the same as above. FIG. 8 is a liquid chromatogram in Example 1, and FIG. 9 is a liquid chromatogram in Comparative Example 1.

Claims (1)

[Scope of Claims] 1 Hollow approximately spherical particles made of a hydrophilic polymer containing a vinyl monomer as a polymerization component, the particle diameter (D) at any location of the hollow approximately spherical particles Filler 2 for liquid chromatography, having a ratio (L/D) of 0.06 to 0.45, characterized in that the vinyl monomer contains a carboxyl group. A packing material 3 for liquid chromatography according to claim 1, wherein the vinyl monomer has the following general formula: (However, in the formula, R ₁ and R ₂ are hydrogen atoms or methyl groups,
n is an integer from 2 to 18). Packing material for liquid chromatography according to claim 1