JPS6359463B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a packing material for high performance liquid chromatography that has ion exchange action as a separation mechanism. It has been known to use an ion exchanger made by copolymerizing methacrylic acid and divinylbenzene as a packing material for liquid chromatography, but when this is used to separate biological substances such as proteins, enzymes, and amino acids, These components are adsorbed by the highly hydrophobic divinylbenzene part of the packing material, resulting in a long time for analysis, or the target product does not flow out, making analysis impossible. Furthermore, if the copolymerized amount of divinylbenzene is reduced, the strength as a filler will be lost, making it unsuitable as a filler for liquid chromatography. The purpose of the present invention is to provide a packing material suitable for separating biological substances such as proteins, enzymes, and amino acids using ion exchange liquid chromatography. , 5 to 90% by weight of a polymerizable monomer (A) having one polymerizable double bond and one or more carboxyl groups in the molecule and having a hydrophobic parameter of 2.3 or less,
A cross-linked polymerizable monomer having two or more polymerizable double bonds in the molecule and a hydrophobic parameter of 0 or less
(B) 10 to 95% by weight, a monomer having one polymerizable double bond in the molecule and having a hydrophobic parameter of 2.3 or less and different from the above polymerizable monomer (A) (C) 0 to 85% by weight A packing material for high performance liquid chromatography is characterized in that it is composed of an ion exchanger obtained by copolymerizing a monomer mixture consisting of: As mentioned above, the packing material for high performance liquid chromatography of the present invention is composed of an ion exchanger in which the portion in contact with the mobile phase is copolymerized with a specific monomer. In order to use the above-mentioned copolymerized ion exchanger as a filler, for example, the copolymer particles obtained by copolymerization may be made to have the same particle size and used as they are as a filler; In this case, a pulverized copolymer with uniform particle size may be used, or the surface of an appropriate core material such as a glass bulb may be coated with the above copolymer by an appropriate method. However, the copolymer constituting the filler of the present invention is
5 to 90% by weight of a polymerizable monomer (A) having one polymerizable double bond and one or more carboxyl group in the molecule and having a hydrophobicity parameter of 2.3 or less;
10 to 95 cross-linked polymerizable monomers (B) having 1 or more polymerizable double bonds and a hydrophobic parameter of 0 or less
A monomer mixture containing 0 to 85% by weight of a monomer (C) different from the above polymerizable monomer (A) having one polymerizable double bond in the molecule and a hydrophobic parameter of 2.3 or less by weight. It is a copolymerized product, and conventional polymerization methods such as suspension polymerization, emulsion polymerization, solution polymerization, and bulk polymerization can be used for this copolymerization. Suspension polymerization is preferred because it can be used as an agent. Also, during suspension polymerization,
If a small amount of an organic solvent that dissolves the monomer but does not dissolve the polymer is added, the resulting copolymer particles become porous, and a filler with an increased contact area with the mobile phase can be obtained. Here, the hydrophobic parameter is the logarithm of the solubility ratio of a certain compound in an aqueous octyl alcohol system, and is a value specific to that compound. Although this parameter can be determined by experiment, the contribution of each fragment in the molecule (hydrophobic fragment constant) can be determined by calculation, and the hydrophobic parameter value of the compound is calculated as the sum of these. can. For information on how to calculate hydrophobicity parameter values, see
“The Hydrophobic Fragmental” by RFRekker
"Constant" (Elsevier Scientific Publishing)
Co., published in 1977), Chapter 3, and the parameter values in the present invention are based on this calculation method. That is, in this calculation method, the hydrophobic fragment constant (f value) is calculated for each fragment constituting the compound, such as methyl group, carboxyl group, etc.
is determined, and for a particular compound, the hydrophobicity parameter of the compound can be determined by summing the above constants for each fragment of the compound. For example, according to Table 2 on page 48 of Chapter 3, the f value of each fragment is as shown in the following table.

[Table] And, for example, acrylic acid (CH ₂ = CH-
Regarding the hydrophobic parameters of COOH), CH ₂ , CH,
Since there is one COOH, using the constants in Table 1, the hydrophobic parameter can be calculated as follows: 0.528 x 1 + 0.261 x 1 + (-0.987) x 1 ≒ -0.20. Next, the polymerizable monomers (A) preferably used in the present invention include acrylic acid (hydrophobic parameter = 2.0, below the parameter values are shown in parentheses), methacrylic acid (0.39), crotonic acid (0.23), maleic acid ( -1.46) etc. Moreover, the crosslinking polymerizable monomer (B) used in the present invention
Examples of the Alkylene glycol diacrylates (or methacrylates) such as polyethylene glycol diacrylate (or methacrylate) and polypropylene glycol diacrylate (or methacrylate) can also be used. In addition, the monomer (C) different from the monomer (A) can be used in the present invention up to a quantitative range of 85% by weight if necessary.
as NN-dimethylmethacrylamide (-
0.88), 2-hydroxyethyl methacrylate (-0.41), glycidyl methacrylate (-
0.48) and the like are preferably used, and other substances such as NN-dimethylacrylamide, 2-hydroxyethyl acrylate, and glycidyl acrylate can also be used. In the present invention, as mentioned above, the polymerizable monomer
A monomer mixture consisting of (A) 5 to 90% by weight and a crosslinking polymerizable monomer (B) 10 to 95% by weight, or a monomer mixture in which the above monomer (C) is added up to 85% by weight as necessary, is copolymerized. However, the obtained copolymer is a polymerizable monomer having a carboxyl group.
The presence of (A) in the molecule makes it an ion exchanger that itself exhibits ion exchange action. And in the monomer mixture, 1 having a carboxyl group
If the amount of functional monomer (A) is too small, the ion exchange ability will decrease, so 5% by weight or more of monomer (A) is required. If the amount of the reactive monomer (B) is too small, the density of crosslinks in the copolymer will decrease, causing problems in the mechanical strength to withstand high pressure, especially when used as a packing material for high performance liquid chromatography. Therefore, the monomer (B) must be present in an amount of 10% by weight or more. In addition, the monomer (C) having a polymerizable dibond other than the polymerizable monomer (A) may be mixed within a range in which the proportion of the polymerizable monomer (A) and the crosslinking polymerizable monomer (B) does not become less than the required amount. It can be included in the monomer. All of the monomers contained in the monomer mixture produced by copolymerizing the filler of the present invention have hydrophobic parameters of 2.3 or less, and furthermore, the crosslinking polymerizable monomer (B) has a hydrophobicity parameter of 0 or less. However, this means that the hydrophobic parameter is
This invention is based on the knowledge of the present invention that a packing material made of an ion exchanger copolymerized with 2.3 or less monomers exhibits excellent separation ability in the analysis of biological substances or hydrophilic substances such as proteins, enzymes, and amino acids. For example, divinylbenzene, which has traditionally been used as a crosslinking polymerizable monomer to produce ion exchangers, has a high hydrophobicity parameter of 3.8, so an ion exchanger containing this as a copolymerization component is used as a filler. However, the effects of the present invention cannot be achieved even if the present invention is used. In addition, the hydrophobic parameters of the polymerizable monomer (A) and the monomer (C) added as necessary are also 0 as in the crosslinking polymerizable monomer (B).
It is preferable that the value be below or on the negative side in order to prevent adsorption of hydrophilic substances to the packing material during analysis of the substances. The packing material of the present invention may be packed into a column by a conventional method and used for analysis by high performance liquid chromatography, and exhibits analytical behavior based on an ion exchange mechanism. Since the part of the packing material of the present invention that comes into contact with the mobile phase is made of a copolymer of the monomer mixture having the specific composition, it is possible to analyze biological substances such as proteins, enzymes, and amino acids, which were difficult to analyze in the past, by liquid chromatography. Accurate analysis and analysis using graphs
It has excellent separation properties, and the mechanical strength of the filler itself is also excellent, making it suitable as a filler for high performance liquid chromatography. Examples of the present invention will be described below. Example 1 400 ml of 4% by weight aqueous polyvinyl alcohol solution, 40 g of tetraethylene glycol dimethacrylate, 10 g of tetramethylolmethane triacrylate, and 50 g of methacrylic acid were placed in two separable flasks equipped with a cooler, stirrer, thermometer, and dropping funnel. , 40 g of toluene and 1.5 g of benzoyl peroxide were supplied. Next, the temperature was raised to 80° C. while stirring at a stirring speed of 400 rpm, reaction was carried out for 10 hours, and the mixture was cooled. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a spherical polymer having a particle size of 5 to 20 microns. The particles of 8 to 12 microns obtained by removing fine particles and coarse particles were dispersed in 800 ml of ion-exchanged water, and the ion-exchanged water was poured into a stainless steel column (diameter 7.9 mm, length 30 cm) using a high-pressure constant flow pump. It was filled by pumping at a rate of 1.6 ml/min. The obtained packed column was connected to a high performance liquid chromatograph (trade name: Shimadzu DuPont High Performance Liquid Chromatograph Model 830), and the following analytical operations were performed. 10μ of standard serum was injected using a microsyringe. As an eluent, disodium phosphate 0.1N30
%, monopotassium phosphate 0.1N70%, PH = 6.4 is used as solution A, and 3% of solution is added to solution A as solution B.
Using a solution containing NaCl, elution was started with 100% solution A, and solution B was increased at a rate of 10% every 5 minutes. The chromatogram thus obtained was as shown in FIG. In this chromatogram, ultraviolet light with a wavelength of 280 nm was used as a detector. Example 2 36 g of tetraethylene glycol dimethacrylate, 10 g of tetramethylolmethane triacrylate, and 5 g of methacrylic acid were used as monomer components, and 40 g of isoamyl alcohol was used as an organic solvent.
A packing material and a packed column were prepared in the same manner as in Example 1, except that a standard serum was analyzed under the same conditions, and a chromatogram almost the same as that shown in FIG. 1 was obtained. Example 3 Nonaethylene glycol dimethacrylate (-
3.60) Prepare a packing material and a packed column in the same manner as in Example 1 using 40 g of acrylic acid, 10 g of acrylic acid, and 50 g of isoamyl alcohol as an organic solvent,
Amino acid analysis was performed. Lysine as an amino acid,
Using a mixture of histidine and arginine, the effluent was reacted with ninhydrin at 80°C for 30 seconds and analyzed by absorption at 590 nm. A 0.35N aqueous sodium citrate solution at pH 5.3 was used as the mobile phase. The obtained chromatogram is shown in FIG. Example 4 Tetraethylene glycol dimethacrylate 40
g, 50 g of methacrylic acid and 1.5 g of benzoyl peroxide were placed in a 500 ml separable flask, bulk polymerized at 80°C for 5 hours with stirring, cooled, crushed, and fine particles and coarse particles were removed using a sieve. Particles of around micron size are dispersed in water to form a slurry, and a stainless steel column (diameter 7.9 mm,
1.6 cm of water by high pressure constant flow pump to length 30cm)
It was filled by pressure feeding at a rate of ml/min. The obtained packed column was connected to a high performance liquid chromatograph (product name: Shimadzu DuPont High Performance Liquid Chromatograph Model 830) and the same amino acid mixture as in Example 3 was analyzed, and the same good results as in Example 3 were obtained. Obtained. Comparative Example 1 A packing material and a packed column were prepared in the same manner as in Example 1, except that 50 g of neopentyl glycol dimethacrylate (hydrophobicity parameter = 2.40) and 45 g of acrylic acid were used as monomers, and n-pentanol was used as an organic solvent. When a standard serum was prepared and analyzed under the same conditions, the target substance was adsorbed to the packing material and could not flow out of the column. Comparative Example 2 A packing material and a packed column were prepared in the same manner as in Example 1, except that 10 g of divinylbenzene (hydrophobicity parameter = 3.8) and 40 g of methacrylic acid were used as monomers, and 40 g of toluene was used as an organic solvent, and the same conditions were used. When the standard serum was analyzed, the target did not flow out of the column.

[Brief explanation of drawings]

Figure 1 shows Example 1, Figure 2 shows Example 3.
The chromatogram obtained is shown below. 1... Ignoglobulin G aggregate, 2... Ignoglobulin A, 3... Ignoglobulin G, 4
...Albumin, 5...Transferrin, 6...
...Lysine, 7...Histidine, 8...Arginine.

Claims (1)

[Claims] 1 The part in contact with the mobile phase has one polymerizable double bond and one or more carboxyl groups in the molecule,
Polymerizable monomers with a hydrophobicity parameter of 2.3 or less
(A) 5 to 90% by weight, a cross-linked polymerizable monomer having two or more polymerizable double bonds in the molecule and a hydrophobic parameter of 0 or less (B) 10 to 95% by weight, 1 in the molecule It has a polymerizable double bond and a hydrophobic parameter of
A high performance liquid chromatograph comprising an ion exchanger copolymerized with a monomer mixture of 0 to 85% by weight of a monomer (C) different from the polymerizable monomer (A) to 2.3 or less. filler for use.