SE457797B - PROCEDURES FOR PREPARING POROEST SUBSTRATE INCLUDING PEARLS THAT HAVE METHYLOL GROUPS OF CO-POLYMS BASED BY THE CIRCUIT BOARD AND APPLICATION OF THE SUBSTRATE BY SCIENCE CHROMATOGRAPHY - Google Patents

Description

Due to the viscosity of the adhesive polymer and some of which caused undesired reactions. On the other hand, a relatively pure crosslinked polymer of styrene can be obtained if a phosphate which is sparingly soluble in water, such as calcium phosphate and magnesium phosphate, is used as the suspending agent, since the phosphate can be easily removed by washing the resulting minimal beads with acid or the like. . However, the resulting polymer can not be satisfactorily dispersed in water, various buffer solutions, serum, urine and the like, depending on the hydrophobic surface thereof.

An object of the present invention is to provide a method for providing a substrate for the analysis of low molecular weight hydrophilic substances, the outer surface of the substrate being highly hydrophilic while the inner surface of the pores therein is less hydrophilic than the outer surface.

A further object of the invention is to provide a use of such a substrate in liquid chromatography.

These objects are achieved with a method according to claim 1 and a use according to claim 9.

The process according to the invention thus means that a mixture of a monomer from the styrene group and a crosslinking agent, which is copolymerizable with the monomer, is subjected to suspension polymerization in water in the presence of a pore-regulating agent and a water-soluble polymer, drying the resulting crosslinked copolymer , which is then provided with methylol groups.

The invention is described in detail below.

The porous crosslinked copolymer is prepared by suspension polymerization of a monomer from the styrene group together with a crosslinking agent which is copolymerizable with the monomer in a solution of a water-soluble polymer in the presence of a pore-regulating agent. By monomer from the styrene group is meant herein styrene, α-methylstyrene, chloromethylstyrene and a mixture thereof. As the crosslinking agent, copolymerizable with the monomer, one can use divinylbenzene, trivinylbenzene, triallyl isocyanurate, a dimethacrylate of a polyhydroxy alcohol, a diacrylate of a polyhydroxyalcohol or diallyl phthalate. Of these agents, divinylbenzene is the most preferred because the methylol groups can be readily added in this case. The amount of crosslinking agent is preferably in the range of 30-70% by weight of the total amount of monomer and crosslinking agent; The most preferred copolymer of the invention is a styrene-divinylbenzene copolymer.

The monomer and the crosslinking agent are radically polymerized in the presence of a high concentration water-soluble polymer, according to the invention. Examples of water-soluble polymers are polyethylene oxide, polyvinyl alcohol, saponified polyvinyl acetates with different degrees of saponification, polyvinylpyrrolidone, methylcellulose and the like.

The amount of water-soluble polymer is preferably in the range of 5-60 parts by weight and in particular 10 to 40 parts by weight per 100 parts by weight of the mixture of the monomer and the crosslinking agent in order for the resulting copolymer to have good hydrophilicity. Less than 5 parts by weight of water-soluble polymer does not provide sufficient hydrophilicity, and with more than 60 parts by weight of water-soluble polymer, Imniej completely completes spherical minimal beads and in addition this causes some difficulty in introducing the methylol groups into the resulting beads as the viscosity of the system increases. To the aqueous solution of the water-soluble polymer may be added a small amount of phosphate suspending agent, such as hydroxyapatite and the like, or an anionic surfactant.

The diameter of the copolymer can be controlled according to the invention, depending on the type and amount of the water-soluble polymer, the ratio between the amounts of monomer and water, the stirring speed, the amount of surfactant and the like. The preferred substrate according to the invention consists of spherical beads with a diameter in the range 1-30 μ.

The pore regulating agent is added to modify the pore size of the substrate, and it may consist of an inert organic solvent which is soluble in the monomer. Examples of pore regulators are aromatic hydrocarbons such as benzene, toluene, xylene and the like, chlorinated hydrocarbons such as trichlorethylene, chloroform, carbon tetrachloride and the like, aliphatic hydrocarbons such as n-hexane, n-heptane, n-octane, n- dodecane and the like, or a mixture thereof. According to the invention, the pore size can be easily moderated by using different types and / or amounts of the pore regulating agent. Generally, 20-300 parts by weight of pore regulating agent per 100 parts by weight of a mixture of monomer and crosslinking agent are preferably used.

The pore size of the substrate may vary depending on the area of use. For analysis of low molecular weight hydrophilic substances without adsorption of high molecular weight protein, the particular molecular weight of the substrate according to the invention is preferably lower than 30,000, i.e. the molecular weight of serum protein.

If, on the other hand, it is desired to adsorb protein having a molecular weight of 30,000 or more during analysis of the low molecular weight hydrophilic substances, then the substrate molecular weight should preferably be at least 30,000. The polymerization initiator may be a conventional initiator used in radical suspension polymerization of vinyl monomers, for example an organic peroxide such as benzoyl peroxide and butyl perbenzoate, an azo compound such as azobisisobutyronitrile and the like. Preferably benzoyl peroxide is used because the water-soluble polymer can be easily grafted.

When the porous crosslinked copolymer produced, with the water-soluble polymer adhering or grafted onto the surface thereof, is used as a substrate, it is difficult for a hydrophilic substance to diffuse into the pores because the hydrophilicity is not yet sufficient, although the copolymer may be dispersed. in water. Furthermore, the copolymer is highly viscous due to the water-soluble polymer present on its surface, and the pressure rises upon elution resulting in low flow rate of the eluent and poor reproduction at the elution, when the substrate is packed in a column. According to the invention, therefore, methylol groups are introduced into the copolymer to eliminate this disadvantage. A known way of introducing methylol groups is, for example, chloromethylation of the copolymer followed by hydrolysis. An example of the chloromethylation method is a process using formaldehyde or a derivative thereof and hydrochloric acid and zinc chloride as catalyst, or a process using chloro-. .m-e ~ F-> fl m, -, ~. «, .. i ,, ~, ~ ..,. ,, 4_.,. di. ..,. . _ Wï ^ "r.jïif fi« vz '.' W, '^' * '13; .W? ¿Í-s ', ~ «; iïl_;' [fl ^ '^. "“ «' ~ ''» '- “«:' \ .'- = “i" '~ «^ f'. 457 797 In the chloromethylation reaction, the water-soluble polymer can also react on the surface of the copolymer, and the copolymer acquires a stronger color than when no water-soluble polymer is present, which results in the substrate having a brown surface. When, for example, chloromethyl-methyl ether is used, 2-20 parts by weight of chloromethyl-methyl ether per part by weight of crosslinked copolymer are preferably added before use. The most preferred weight ratio of chloromethyl-methyl ether to crosslinked copolymer is about 5: 1 due to homogeneous stirring and loss of clear methyl methyl ethers due to evaporation during the course of the reaction. The amount of catalyst is generally in the range of 0.5 to 30 parts by weight per 100 parts by weight of chloromethyl methyl ether, and it is suitably selected according to the desired degree of chloromethylation. . The reaction is usually carried out at a temperature in the range 0 DEG-58 DEG C. (boiling point of chloromethyl methyl ether).

The degree of chloromethylation of the copolymer is preferably 0.2 chloromethyl groups or more per aromatic moiety.

After the chloromethylation, the chloromethyl groups are converted to methylol groups (-CH 2 OH) by hydrolysis under alkaline conditions, at room temperature or higher temperature. In the H 2 O-NaOH system, the reaction rate is low, but addition of methanol accelerates the reaction- Alkali concentration; the amount of methanol, the temperature and the ion. the reaction time can be selected according to the desired introduction of methylol groups.

The degree of hydrolysis of the chloromethyl groups is preferably 50% or more. The number of methylol groups introduced into the substrate is thus preferably 0.1-0.5 group per aromatic moiety.

The prepared substrate has an outer surface which is highly hydrophilic, and consequently it can be easily dispersed in an aqueous medium. The inner surface of the pores in the substrate is also hydrophilic, but to a lesser extent than the outer surface, and consequently hydrophilic substances with add molecular weight can enter the pores unhindered. The inner surface of the pores is only provided with methylol groups and has no adhesive water-soluble polymer, and it is more hydrophobic than the outer surface.

The viscosity of the substrate according to the invention is so low that a high flow rate can be obtained through a column packed with the substrate by flash-liquid chromatography. Furthermore, the representation in fast-liquid chromatography is good and low molecular weight hydrophilic substances are well fractionated.

The substrate with a special molecular weight lower than 30,000 and a diameter of 1-30 μm is the substrate which can be preferably used in the analysis of low molecular weight hydrophilic substances, and especially those present in a sample which also contains high molecular weight substances. , e.g., serum protein, since it cannot adsorb such high molecular weight substances, and the substrate can thus be used in the continuous analysis of such a sample. On the other hand, a substrate having a special molecular weight of at least 30,000 can be used in various fields, for example as a substrate for separating low molecular weight hydrophilic substances, as a precolumn for protein removal, in an antigen-antibody reaction or the like, since this can completely adsorb high molecular weight substances, such as hydrophilic proteins. In this yall, the diameter of the substrate may be in the range of 1-300 n, although this is preferably in the range of 1-30 n when it is desired to use the substrate in a column for the analysis of low molecular weight hydrophilic substances. molecular weight and co-exist with high molecular weight protein, eg serum protein with a molecular weight of 30,000, there are two methods. (1) Use of a substrate having a molecular weight of less than 30,000. In this case, the hydrophilic substances are adsorbed on the surface of the substrate and the serum protein having a molecular weight of 30,000 can not diffuse into the pores of the substrate nor can it be adsorbed on the hydrophilic surface of the substrate. Accordingly, the hydrophilic substance and the serum protein can be separated and analyzed. (2) Use of a substrate having a molecular weight of at least 30,000. In this case, the serum protein may diffuse into the pores and due to the hydrophilicity within the pores, the protein is adsorbed there and can not be rinsed off under the conditions for rinsing away hydrophilic substances. adsorbed on the surface. As a result, the hydrophilic substances and the protein are separated and can be analyzed. (3) By "exclusive" molecular weight of the substrate is meant that due to the size of the pores of the substrate, a substance having a molecular weight higher than the exclusive molecular weight cannot diffuse into the pores. Accordingly, in the present invention, both substrates having an exclusive molecular weight of less than 30,000 and having an exclusive molecular weight of at least 30,000 can be used to analyze hydrophilic substances which coexist with protein having a molecular weight of 30,000. A substrate according to the invention however, having an exclusive molecular weight of less than 30,000 is preferably used for the analysis of low molecular weight hydrophilic substances coexisting with, for example, serum protein, because the protein cannot be adsorbed both on the surface and inside the pores of the substrate and the substrate can be used for continuous analysis. time.

The invention will now be described in more detail with reference to the following non-limiting examples.

Example 1 In an autoclave, a solution of 12.5 g of methylcellulose in 625 g of water containing 0.125 g of sodium lauryl sulfate was introduced, and after the addition of 21.8 g of styrene, 18.2 g of divinylbenzene and 60 g of toluene as pore regulators to the autoclave, the polymerization by adding benzoyl peroxide as initiator, after which it was allowed to proceed at 60 ° C for 17 hours and with vigorous stirring.

The resulting polymer was washed thoroughly with water and then with acetone. The washed polymer was dried at 40 ° C at reduced pressure. The dried polymer had a diameter of 3-20 .mu.m. when sieving the polymer, beads with a diameter of 10-20 p were collected.

The specific molecular weight of the polymer was about 7000, which was determined using polystyrene of known molecular weight and tetrahydrofuran as eluent.

To 35 ml of chloromethyl methyl ether was added 5 g of beads and 1.5 ml of anhydrous tin tetrachloride, and the mixture was heated under a reflux condenser for 6 hours at about 50-60 ° C. The color of the beads darkened to blackish brown during the course of the reaction. After completion of the reaction, the beads were washed several times with methanol containing hydrochloric acid and then with acetone until the color turned yellow. The chloromethylation degree of the beads was about 0.62 chloromethyl groups per aromatic part, ~ and this degree was determined by comparing the infrared absorption peaks at 1600 cm -1 from the aromatic moiety and at 1260 cm -1 from the chloromethyl group with a calibration curve obtained by mixtures. of cumene and p-isopropylbenzyl chloride in different mixing ratios. The chloromethylated beads were heated in a 10% aqueous solution of sodium hydroxide at 60 ° C for 9 hours for hydrolysis. In the hydrolyzed beads, the strength of the infrared absorption peak at 1260 cm -1 originating from the chloromethyl group was reduced, but an absorption peak occurred at 1090-1100 cm -1 originating from the C-O group. From the extent to which the strength of the peak at 1260 cm 3 was reduced, it could be found that about 55% of the chloromethyl groups were converted to methylol groups, ie the introduction of methylol groups into the beads was about 0.34 per aromatic moiety.

The beads produced by the invention could be extremely easily dispersed in the vat and various buffer solutions without any appreciable coagulation or agglomeration.

After packing a stainless steel tube, with a diameter of 4 mm and a length of S00 mm, with the substrate according to the invention, a mixture of p-aminobenzoic acid, creatinine and uric acid was subjected to chromatography using the column and an aqueous 1/20 M phosphoric acid buffer solution as eluent. The conditions during the chromatography were the following liquid detector at 250 nm, elution rate 1 ml / min and a pressure of 20 kg / cm 2. The elution time was 10.4 minutes for p-aminobenzoic acid, 7.5 minutes for creatinine and 5.5 minutes for uric acid.

Furthermore, a mixture of bovine serum albumin and the three components mentioned above was subjected to chromatography on the same column.

The albumin was eluted first and the four components were separated - completely from each other (see Fig. 1 which shows the elution curve).

Upon repeated chromatography, only a slight adsorption of the albumin was observed the first and second time, and then not at all. Consequently, almost the same result was obtained thereafter.

It was thus found that the substrate prepared by the process of the invention was particularly suitable for use in the analysis of low molecular weight water-soluble substances, and that the high molecular weight non-adsorbed protein. f hä, Vä, 55:; ...) \: \ .._ :. t, - ,, =; __. ¶., _. ~ _¿.: Twh_ Ai fl ïy 'Wv: .i,. _. After packing the same stainless steel tube with the polymer prepared in Example 1 but before the chloromethylation, the same substances as in Example 1 were subjected to chromatography. The pressure showed a value higher than 80 kg / cm 2 at a flow rate of 1 ml / min, which confirms that the methylcellulose which adheres to the inner surface of the pores in the polymer has a great effect on the viscosity. The four components of the sample were hardly separated from each other and eluted very rapidly.

Comparative Example 2 - V5 g of the polymer prepared in Example 1 was chloromethylated in the same manner as in Example 1, except that the reaction time was 24 hours instead of 6 hours in Example 1, to obtain beads with a degree of chloromethylation of about 64%. The beads were hydrolyzed in a solution of 25 g of sodium hydroxide in 100 g of methanol at 60 ° C for 15 hours to give a substrate having (on average) about 0.58 methylol groups per aromatic moiety.

After packing the same stainless steel tube as in Example 1 with the substrate, a mixture of bovine serum albumin, p-aminobenzoic acid, creatinine and uric acid was subjected to chromatography using the column with a flow rate of the same eluent of 1 ml / min, eluting times the following: p-aminobenzoic acid 6.4 min, creatinine 5.9 min and uric acid 4.8 min.

It was thus found that the elution time was shorter than in Example 1 and p-aminobenzoic acid and creatinine could not be separated from each other. The elution curve for this chromatography experiment is shown in Figure 2. The results show that there was a greater interaction between the low molecular weight hydrophilic substances and the inner surface of the pores in the substrate according to the invention than in the substrate of this comparative example 2.

Example 2 In the same manner as in Example 1, 21.8 g of styrene and 18.2 g of divinylbenzene were polymerized in the presence of 5656.4 g of toluene and 3.6 g of n-V / dodecane as pore regulating agents, to prepare a styrene-.divinylbenzene copolymer having a specific molecular weight of about 16,000. The vapor polymer was subjected to chloromethylation under the same conditions as, ".fs-" ~, "w ~ _f» f- "aw-af ', except that the reaction time was 2 hours instead of 6 hours as in Example 1, to give a chloromethylated copolymer having a degree of chloromethylation of about 0.42 chloromethyl groups per aromatic moiety. By exposing the copolymer to hydrolysis in a 10% aqueous solution of sodium hydroxide at 60 ° C for about 5 hours, a substrate containing methylol groups in an (average) amount of about 0.2 per aromatic moiety was obtained. obtained by packing the same stainless steel pipe as in Example 1 with the prepared substrate according to the invention, showed a pressure of 25 kg / cm 2 at a flow rate of 1 ml / min for an aqueous 1/20 M phosphoric acid buffer solution.

The same amount, 20 μl, of a mixture of uric acid, creatinine and p-aminobenzoic acid was subjected to chromatography using the column. The three components were completely separated. Experiments with a mixture of bovine serum albumin and the three components mentioned above showed that albumin was not adsorbed on the column at all.

Example 39 Methylcellulose was dissolved in 125 g of water and 0.025 g of sodium lauryl sulfate was added. The mixture was then introduced into a 300 ml ampoule, to the ampoule were added 3.27 g of styrene, 2.73 g of divinylbenzene and 6.75 g of toluene and 2.25 g of n-dodecane as pore regulators, and then the mixture was stirred at 60 ° C for 17 hours for reaction, using benzoyl peroxide as initiator. The resulting beads were washed with water and acetone, then dried at room temperature under reduced pressure. The spherical diameters of the spheres were essentially the same and about 20u.

The specific molecular weight of the beads was about 100,000, which value was determined by eluting polystyrene with different predetermined molecular weights using a column packed with the beads.

S'g beads were introduced into a reaction vessel, then 35 ml of chloromethyl methyl ether and 1.5 ml of anhydrous tin tetrachloride were added.

The mixture was heated under reflux at 50-60 ° C for 6 hours to chloromethylate the beads. The beads became increasingly dark brown during the course of the reaction. After the reaction, the beads were washed repeatedly with methanol containing hydrochloric acid and finally with acetone.

The final color of the pearls was amber. The new absorption peaks appeared at 1260 and 670 cm -1 in the beads' infrared absorption spectrum after the chloromethylation. The degree of chloromethylation was about 0.5 chloromethyl groups per aromatic moiety. The chloromethylated beads were added to a solution of 25 g of sodium hyaroxia 1 to 9 menanols, and the solution was kept at room temperature for 15 hours for reaction. The new IR absorption peaks appeared at 1090 cm -1 due to the introduction of -CH 2 OH groups. The number of methylol groups was 0.3 per aromatic moiety. The beads obtained were very easy to disperse in water, various buffer solutions, urine and the like, without aggregation. The beads were packed in a stainless steel column, with a diameter of 4 mm and a length of 500 mm, for fast liquid chromatography.

A solution of bovine serum albumin in a 1/20 M phosphoric acid buffer was subjected to flash liquid chromatography using the phosphoric acid buffer solution as an eluent at a flow rate of 1 ml / min. No albumin eluted. In addition, no albumin was eluted even after 50-fold repeated 50 μl of 10% bovine serum albumin. A 20 μl sample of a solution of uric acid, creatinine, p-aminobenzoic acid and albumin in 1/20 M phosphoric acid buffer solution was passed through the column. Uric acid, creatinine and p-aminobenzoic acid were completely separated from each other, while no albumin was eluted. See Fig. 3 which shows a curve of elution peaks at a speed of 1 cm / 3 min according to the diagram and measured at a wavelength of 250 nm. The pressure was 25 kg / cm 2 at a flow rate of 1 ml / min.

Example 4 A styrene-divinylbenzene copolymer was prepared in the same manner as in Example 1, except that 45 g of toluene and 15 g of n-dodecane were used as pore regulators instead of 60 g of toluene. The specific molecular weight of the polymer produced was about 100,000. Methylol groups were then introduced into the polymer in the same manner as in Example 1 in an amount of about 0.4 methylol groups per day. Then, the obtained substrate was packed in the same column as in Example 1 and the albumin was chromatographed. However, the albumin was adsorbed on the column and did not elute.

Comparative Example 3 A styrene-divinylbenzene copolymer was prepared in the same manner as in Example 3, using 1.3.9 g of calcium phosphate and 4.07 mg of sodium n-dodecylbenzene sulfonate instead of methylcellulose and sodium lauryl sulfate as in Example 3. After washing with water, the copolymer was sieved to give beads with a diameter of 10-20u. However, the distribution of the bead diameters was wide. The specific molecular weight was about 100,000.

The beads produced were washed thoroughly with hydrochloric acid to remove the calcium phosphate. The beads did not disperse at all in water but agglutinated.

After drying the beads, chloromethylation was carried out as in Example 3. The beads were only slightly colored to a final light yellow color. The chloromethyl groups were converted to methylol groups as in Example 3. The obtained beads were less hydrophilic than those according to Example 3 and the water or urine dispersions were not satisfactory. In a column packed with the beads, the albumin was not completely adsorbed and a very wide albumin peak was obtained. The reason for this was considered to be that the beads were less hydrophilic than those of Example 3, and the protein could not penetrate into the pores.

Example 5 The specific molecular weight 300,000 was prepared in the same manner as in Example 3, using 4.5 g of toluene and 4.5 g of n-dodecane as pore regulators. The beads were treated in the same manner as in Example 3 to give a substrate having methylol groups. The resulting substrate was readily dispersed in serum and urine. In a column packed with the substrate, albumin was not eluted, not even after 50 ul of 10% solution of bovine serum albumin was passed 50 times through the column.

Porous minimal beads of styrene-divinylbenzene copolymer with

Claims (9)

Patent claims. »_ A process for producing porous substrate comprising beads having methylol groups of copolymer based on crosslinked styrene, wherein the outer surface of the beads is hydrophilic and the porous surface of the substrate is hydrophobic or less hydrophilic than the outer surface, known from a For suspension polymerization in water, at least one styrene or monomer selected from the group consisting of styrene, β-methylstyrene and chloromethylstyrene, and a crosslinking agent selected from the group consisting of divinylbenzene, trivinylbenzene, triallyl isocyanurate, a dimethacrylate of a polyhydroxy a diacrylate of a polyhydroxy alcohol and diallyl phthalate in the presence of methylcellulose, polyethylene oxide, polyvinyl alcohol, saponified polyvinyl acetate or polyvinylpyrrolidone as a water-soluble polymer and a pornographic agent, the amount of water-soluble polymer being 5-60 parts by weight 20-300 parts by weight per 100 parts by weight of the mixture of monomer and crosslinking m noble, b) then drying takes place, c) the beads produced are subjected to chloromethylation by using 2-20 parts by weight of chloromethyl-methyl ether per part by weight of crosslinked copolymer and anhydrous SnCl 4 as catalyst in an amount of 0.5- 30 parts by weight per 100 parts by weight of chloromethyl methyl ether at a temperature of 0-58 ° C, and d) the chloromethylated beads are subjected to hydrolysis under alkaline conditions, the degree of hydrolysis of the methyl chloride groups being 50% or more and the number of methylol groups introduced into the substrate, is 0.1 to 0.5 per aromatic moiety. 2. A process according to claim 1, characterized in that the pore regulating agent is selected from the group consisting of aromatic hydrocarbons, aliphatic hydrocarbons, chlorinated hydrocarbons, and a mixture thereof. ÜÉ 3. A process according to claim 1, characterized in that the copolymer is a styrene-divinylbenzene copolymer. A method according to any one of claims 1-4, characterized in that the substrate comprises spherical beads with a diameter in the range 1-30; 5. A process according to any one of claims 1-5, characterized in that it has a specific molecular weight lower than 30,000. Process according to any one of claims 1-5, characterized in that it has a specific molecular weight of at least 30,000 and is capable of adsorbing that protein. 457 797 M 7. A process according to claim 1, characterized in that the water-soluble polymer is methylcycle. _ yï _ 8. A process according to claim 1, characterized in that the amount of crosslinking agent is 30-70% by weight of the total amount of monomer and ɧ: verbfnaanae mixture while f A Use of the substrate prepared by the process according to claim 1 in liquid chromatography. m.