JPS61287444A - Double structural carrier - Google Patents

Abstract

PURPOSE:To prevent the deterioration of the performance of the titled carrier by forming both an outside surface being an organic fixed phase having a hydrophilic site and an inside surface of a narrow pore of a porous SiO2 grain which is chemically joined with the organic fixed phase having a hydrophobic site and has a specified grain size. CONSTITUTION:A hydrophobic group is introduced into a porous SiO2 grain and also the inside of the narrow pore of the porous SiO2 grain is substituted with a water-insoluble or water-hard soluble organic solvent. Then a hydrophilic group is selectively introduced into only outside surface of the porous SiO2 grain by performing the hydrolysis treatment and the silane treatment and thereof a double structural carrier having the hydrophobic group in the inside surface of the narrow pore of SiO2 grain is molded. The above-mentioned narrow pore is regulated to 10-200Angstrom mean narrow pore diameter and the mean grain size of the porous SiO2 grain is regulated to 0.1-1,000mu. A low molecular substance can be adsorbed and removed from an aq. soln. wherein coexists a high molecular substance and the low molecular substance by using this double structural carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field of the Invention The present invention relates to a double-structured carrier, and more particularly, an organic stationary phase having hydrophilic sites is chemically bonded to the outer surface of porous silica particles, and The present invention relates to a double-structured carrier that selectively adsorbs only low-molecular substances from an aqueous solution containing both high-molecular substances and low-molecular substances chemically bonded to an organic stationary phase having hydrophobic sites on the inner surface of the pores.

BACKGROUND ART The operation of selectively separating a high molecular weight substance and a low molecular weight substance from a solution containing both high molecular weight substances and low molecular weight substances, respectively, is important particularly in an aqueous solvent system containing a large amount of high molecular weight substances. That is, typical examples include biological fluids such as serum containing low concentrations of low molecular weight substances and large amounts of biopolymer substances such as proteins and nucleic acids, or bacteria.
organs.

Examples include aqueous solutions such as homogenate extracts of plants and the like.

As a method for adsorbing organic substances from an aqueous solvent system, a method using hydrophobic crosslinked polymer particles such as divinylbenzene-styrene is disclosed in U.S. Patent No. 3,531,463; It is not suitable for solutions containing both high-molecular substances and low-molecular substances, since it is also adsorbed by hydrophobic interactions. In addition, the present inventor has already used porous silica particles chemically bonded with hydrophobic groups such as octadecyl groups, and by adsorbing bovine serum albumin to the external surface of the particles, the external hydrophobic sites were removed. An application was filed for a filler with a hydrophilic region (% opening/closing 5
8-226,062). This packing material that adsorbs bovine serum albumin has a pore size that prevents proteins from penetrating into the pores, so bovine serum albumin is adsorbed only on the outer surface through hydrophobic interaction, resulting in core particles. The external surface of the protein is hydrophilic due to the hydrophilic sites of the protein.

Therefore, unlike the above-mentioned carrier, it is a filler that blocks the interaction between water-soluble polymers such as proteins and the filler, while not hindering the penetration of low-molecular substances into the pores. However, since proteins are attached to the outer surface of the particles only by adsorption, this packing material tends to detach proteins when used for a long time, and may adsorb proteins in the sample. Therefore, there are concerns about storage stability, and furthermore, since the adsorbed proteins are huge, the pores are clogged and the adsorption capacity is reduced, resulting in other disadvantages.

Purpose of the Invention The present invention provides, unlike the above-mentioned conventional separation methods and hydrophobic substance adsorption carriers, the external surface of porous silica particles is chemically bonded with a hydrophilic site that does not have hydrophobic interaction with a polymeric substance. It provides a double-structured carrier composed of an organic stationary phase and a chemically bonded organic stationary phase with hydrophobic sites capable of adsorbing low-molecular substances through hydrophobic interaction inside the pores. .

Components of the Invention A method for separating a low-molecular substance from a solution containing both a high-molecular substance and a low-molecular substance is achieved by adsorption onto a carrier using the hydrophobicity or ionicity of the low-molecular substance. Separation by adsorption using hydrophobicity is generally used as an effective method because it can be applied to a wide range of samples. However, in a solution containing a polymeric substance, not only the low molecular weight substance but also the polymeric substance is adsorbed simultaneously due to hydrophobic adsorption. Therefore, in order to adsorb low-molecular substances from solutions containing high-molecular substances,
- If the particles have a site that is inert to high-molecular substances and a hydrophobic site that adsorbs low-molecular substances through hydrophobic interaction, then polymers will not be adsorbed and low-molecular-weight substances will not be adsorbed. As a result of intensive research, we believe that it is effective because it can only adsorb substances.The required parts within the same particle are divided into the inside of the pore and the outside of the pore. It has been found that a structure in which an inert site is chemically bonded to the inside of the pore, and a hydrophobic site for capturing low-molecular substances is useful.

That is, in the present invention, the average pore diameter is 10 to 200'j.

The skeleton is porous silica particles with an average particle diameter of 0.1 to 1000μ, the outer surface of the pores of the particles is composed of a chemically bonded organic stationary phase having sufficient hydrophilic sites, and the inner surface of the pores is composed of a chemically bonded organic stationary phase with sufficient hydrophilic sites. It concerns a double-structured support composed of a chemically bonded organic stationary phase with sufficient hydrophobic sites.

The present invention will be explained in detail below.

In the dual structure support of the present invention, an organic stationary phase having hydrophilic sites is chemically bonded to the external surface of porous silica particles, and an organic stationary phase having hydrophobic sites is chemically bonded to the internal surface of the pores of the particles. It is a double-structure carrier with an average pore diameter of 10 to 200 μm and an average particle diameter of 0.1 to 1000 μm.

In the present invention, the average pore diameter of the porous silica particles is 10-
200x1 preferably in the range of 30-150A. If the pore size is less than 10A, the diffusion of low-molecular substances into the pores will be hindered, and sufficient adsorption capacity will not be obtained.If the pore size exceeds 200X, the permeation of high-molecular substances into the pores will be allowed, resulting in hydrophobicity. This is not preferable because it causes adsorption to sexual sites and a decrease in adsorption ability for low-molecular-weight substances.

In addition, the particle size is Q, 1 to 1000μ, preferably 3 to 1000μ.
It is in the range of 500μ. α less than 1μ, also 1000μ
Exceeding this is undesirable as it loses its practicality. An organic stationary phase having a hydrophilic moiety has a chemical structure having the following general formula: 10H7(CH)t-OT(,OR(JL:1~
6) (1) OH -(0-CH20H2)ln-OCH3 (however, m: 1
~6)(2)-(aH,)ncNH, (however, n: 1~
4) (3)-(0-CH,0H2)p-OH (where p: 1 to 6) A compound having a residue represented by (4). Specifically, compounds with hydroxyl groups in the molecule include polyhydric alcohols such as ethylene glycol, glycerin, and sorbitol; amide groups include lower fatty acid amides such as acetamide chains, propamide chains, and butyramide chains; and ether groups. In the group of compounds with polyethylene oxide or its methyl.

Compounds such as ethers of lower aliphatic compounds such as ethyl, which have hydrogen bonding sites consisting of oxygen atoms or nine-strand atoms, and have low reactivity with proteins, are particularly preferred. Preferred are biltrimethosyphen, ethylene glycol monomethyl ether, diethylene glycol, and the like.

An organic stationary phase having a hydrophobic moiety is defined as having a chemical structure of the following general formula (wherein R8 is an aliphatic hydrocarbon group or aromatic hydrocarbon group having 1 to 36 carbon atoms, R2a and R3 are chloro It is a silanizing agent represented by a group, an alkoxy group or a methyl group (%R, is a chloro group or an alkoxy group). Specifically, organic layers can be easily formed on the surface of porous silica particles by reaction with porous silica particles such as octadecyltrichlorosilane, octyltrichlorosilane, prudichlorosilane, octadecyldimethylchlorosilane, octyldimethylchlorosilane, propyldimethylchlorosilane, and triphenylchlorosilane. Among the examples that can be introduced are those in which R1 is an octadecyl group, horses,
R3 and octadecylchlorosilane in which the cucumber is a chloro group are preferred.

Next, the method for manufacturing the double structure carrier of the present invention will be explained.

A method for introducing different functional groups into the outer and inner surfaces of the pores of a porous silica particle carrier is to fill the inside of the pores of the porous silica particles with an appropriate filler material, and then add hydrophilic sites to the outer surface of the pores. How to do it with Alternatively, after introducing hydrophobic groups into porous silica particles, the insides of the pores are filled with a suitable filling material, and the hydrophobic groups introduced onto the external surfaces of the pores are cut off. Examples include a method of subsequently introducing hydrophilic groups onto the external surface of the particles after removing the filler material. The latter method, which is simple and effective, will be explained below.

Hydrophobic groups can be introduced into porous silica particles by reacting porous silica particles with a silanizing agent having a suitable hydrophobic group. That is, porous silica particles are dispersed in an organic solvent, a silane treatment agent represented by the general formula (5) is added, and the
A hydrophobic group can be introduced by reacting at 0°C for 1 minute or more.

If the reaction temperature is less than 20°C, the reaction time will be longer and 1
If the temperature exceeds 50°C, it is not preferable because it prevents the uniform surface coating reaction of the porous silica particles. Further, if the reaction waiting time is less than 1 minute, it is not preferable because a sufficient surface coating reaction cannot be achieved. Considering the economy and efficiency of the reaction, 30
It is preferable to react for minutes to 24 hours.

Next, the insides of the pores of the porous silica particles having hydrophobic groups are filled with a suitable filling substance to block the outside of the pores, and then only the siloxane bonds on the outside surface are cut using a suitable chemical substance. Filling materials used include aliphatic hydrocarbon compounds such as pentane, hexane and octane, aromatic hydrocarbon compounds such as benzene, toluene and xylene, and hexanol.

Examples include higher alcohols having a carbon chain of 6 or more such as octatool, and halogenated hydrocarbon compounds such as chloroform and carbon tetrachloride, with toluene being particularly preferred.

As a method of filling the inside of the pores of porous silica particles with a filling material, porous silica particles having hydrophobic groups introduced onto their surfaces are dispersed in the above-mentioned organic solvent, and the mixture is stirred or ultrasonicated.
A method that allows sufficient penetration into the pores. Fill a chromato tube etc. with porous silica particles using a dry method or a wet slurry method,
Examples include a method of passing the organic solvent under static pressure or pressure to replace the inside of the pores with the organic solvent, but in particular, a method of replacing the inside of the pores with the organic solvent under pressure using a wet slurry method can achieve the purpose in a short time. This is preferable because it can be achieved.

The porous silica particles whose pores have been substituted with the organic solvent described above are hydrated to break the siloxane bonds (St-O-si) that are the bonds between the hydrophobic groups on the external surface and the silica base material. Treated with degrading reagents. That is, in an acid or alkaline aqueous solution, at 200 to 100°C, preferably at 30°C.
The siloxane bonds are hydrolyzed by treatment at 90°C for a few seconds to 48 hours, preferably 1 minute to 24 hours. Examples of the hydrolysis reagent used here include alkaline aqueous solutions such as potassium hydroxide, sodium hydroxide, and sodium carbonate, and acidic aqueous solutions such as hydrochloric acid, sulfuric acid, and nitric acid. In particular, an alkaline aqueous solution is effective in cleaving siloxane bonds.

These hydrolysis reagents are used to break bonds between chemical bonding groups and silica, but cannot penetrate into the pores because the pores of the particles are filled with water-insoluble or poorly water-soluble organic solvents. As a result, hydrolysis of siloxane bonds proceeds only on the external surface of the particles.

The time spent on hydrolysis must be properly determined; too short a time will prevent sufficient hydrolysis of the external surface of the particles, while over 40 hours will erode the silica skeleton and create porous silica particles. This is not preferable because it results in a decrease in performance as well as a loss in mechanical strength.

Further, if the reaction temperature is less than 20°C, an organic layer remains due to insufficient surface hydrolysis, and if it exceeds 100°C, rapid hydrolysis occurs and the silica skeleton is eroded, which is not preferable.

Porous silica particles that have been subjected to hydrolysis treatment for a certain period of time must be quickly neutralized with acid or alkali. Reagents used for neutralization include dilute aqueous solutions such as hydrochloric acid, sulfuric acid, nitric acid, etc., and aqueous solutions of organic acids such as acetic acid when the hydrolyzing reagent for siloxane bonds is an alkaline aqueous solution, or when the hydrolyzing reagent used is an acidic aqueous solution. If so, potassium hydroxide.

This can be achieved by passing a dilute aqueous solution such as sodium hydroxide or the like or by feeding the liquid under pressure. As the reagent used for neutralization, it is preferable to select, for example, an acid-alkali pair that easily dissolves the by-product salt when it is washed with water, such as sodium acetate.

The porous silica particles obtained as described above have silanol groups produced by a hydrolysis reaction on their outer surfaces, and the inner surfaces of the pores of the particles have a structure that retains the original hydrophobic groups. . The silanol groups newly generated on the external surface of the particles are mixed with a compound having a residue represented by the above-mentioned general formulas (1) to (4) at 20 to 90° in an inert organic solvent.
By treating C9 for 30 minutes to 24 hours, inert hydrophilic groups are introduced into the water-soluble polymeric substance.

In the above method, the above operation can be simplified by using a reversed phase carrier that has a high ability to adsorb hydrophobic substances.

The reversed-phase carrier has hydrophobic chemical bonding groups uniformly bonded to its surface, and therefore has a high ability to adsorb hydrophobic substances. In porous silica particles, the coverage of hydrophobic groups on the outer surface of the pores is extremely small compared to the inner surface of the pores. This fact suggests that the contribution of the hydrophobic groups chemically bonded to the external surface of the particles to the adsorption capacity is small. In other words, the carrier obtained by selectively removing the hydrophobic groups chemically bonded only to the external surface of the particles shows only a small change in its performance and retains the performance as the original reversed-phase carrier. Conceivable.

Therefore, the reversed-phase support obtained by subjecting the reversed-phase support to an appropriate surface treatment and cleaving the hydrophobic groups chemically bonded to the external surface of the particles has newly generated silanol groups on the external surface of the pores. and then the carrier of the above-mentioned general formula (1
) to (4), different functional groups can be introduced to the external surface of the surface-treated carrier by treating with a suitable silane treatment agent represented by (4). That is, as a method for surface treatment of a reversed phase support, for example, after dispersing it in an organic solvent as described above, it is separated on a glass filter, and then the silica base material and the hydrophobic group chemically bonded to the outside of the particles are quickly separated. A method of passing a reagent that hydrolyzes the bond of
One method is to fill a suitable column with a reversed-phase carrier, replace it with an organic solvent, and then send the above-mentioned hydrolyzing reagent under appropriate pressure. It is an effective way to achieve your goals.

Then, it is treated in the same manner with a compound having a residue represented by the general formulas (11 to (4)).

The thus obtained double-structured carrier having hydrophilic sites on the external surface of the particles is inactive against water-soluble polymer substances that cannot penetrate into the pores, while being inactive against low-molecular substances. By capturing the particles using hydrophobic groups inside the pores, it exhibits adsorption performance comparable to that of conventional reversed-phase supports.

As described above, the present invention is a process of introducing a hydrophobic group into porous silica particles to make the inside of the pores of porous silica particles water-insoluble,
Alternatively, hydrophilic groups can be selectively introduced only to the external surface of the particles by a substituting process with a poorly water-soluble organic solvent, a hydrolysis process, and a silane treatment process, and hydrophobic groups can be introduced into the internal surfaces of the pores of the particles. A double structure carrier having a group can be obtained.

Furthermore, without reducing the performance of existing reversed-phase carriers,
Similar dual structure supports can be obtained.

Effects of the Invention The field of application of the double-structured carrier of the present invention is, for example, by adding and mixing the carrier of the present invention into an aqueous solution containing both a high-molecular substance and a low-molecular substance, and removing harmful substances or interfering substances from the aqueous solution. It is possible to adsorb and remove low molecular weight substances and recover the desired high molecular weight substances. For example, blood purification of patients by classifying protein metabolites and harmful substances of low and middle molecular weight in the blood and high molecular weight substances such as proteins, or removal of contaminant low molecular weight substances in the manufacturing process of biopolymer substances such as pharmaceuticals. etc. Further, after removing the high molecular weight substances as interfering substances, the low molecular weight substances captured by the filler of the present invention can be subsequently extracted and recovered with a suitable organic solvent such as methanol, acetonitrile, etc. For example, recovery of pharmaceuticals, biologically related metabolites, neurotransmitters, hormones, hormone-like substances, etc. from biological fluids or homogenate extracts that exist at extremely low levels, or recovery of antibiotics and physiological substances secreted from bacterial cells. Examples include recovery of active substances, etc.

On the other hand, in recent years, especially with the increased sensitivity of liquid chromatography, it has come to be widely used as a useful means for separating and analyzing biological components. Liquid chromatography has been used to analyze biological components because it is quick and suitable for routine work, but analysis of samples containing polymeric substances requires pretreatment of the sample such as protein removal. This complicates the process and also poses problems in the reproducibility of analysis results. Since the carrier of the present invention retains the performance of the original reversed-phase carrier, it can be used as a normal analytical column by being packed in a column for liquid chromatography. That is, the present carrier is outside its pores.

Because the organic stationary phase has hydrophilic moieties and is chemically bonded, it becomes inert to high-molecular substances, allowing high-molecular substances such as proteins to pass through the column, and low-molecular substances can be handled in normal reversed-phase mode. Can be separated and analyzed. As a result,
It enables routine work of specimens, rapid measurement, and minimizes measurement errors, and is particularly useful for analysis of pharmaceuticals and their metabolites in biological fluids.

Furthermore, the support of the present invention can be used as a concentration column for the analysis of low molecular weight substances present in biological fluids at low levels. That is, a large amount of a sample sample containing a trace amount of a target low-molecular substance is passed through the carrier of the present invention packed in a concentration column, and while the high-molecular substance is removed, the low-molecular substance is transferred into the pores of the packing material. In this method, the target low-molecular substance concentrated with an appropriate eluent composition is introduced into the main column and analyzed using a column switching method. For example, it can be used to monitor pharmaceuticals that exhibit medicinal efficacy at low levels, to analyze bioactive peptides that exist in trace amounts in the brain and organs, and to concentrate and separate biologically relevant metabolites. Can be used to analyze specimen samples.

As described above, the carrier of the present invention can be put into an aqueous solution containing both a high molecular weight substance and a low molecular weight substance, or packed into a column for liquid chromatography, and then an analyte sample can be injected into the column, or further packed into the column. By using it as a concentration column, it is possible to selectively separate and recover the target high-molecular substances or low-molecular substances from the target sample solution, and if necessary, it can be subjected to the target analysis method. can.

Examples Example 1 Average pore size is 70 angstroms, average particle size is 10
Disperse 10 g of micron silica gel in 100 d of toluene, add 5 g of octadecyltrichlorosilane, and
The reaction was carried out for 24 hours in an oil bath kept at ℃. The reaction solution was filtered and washed with 100 g of toluene and 100 g of acetone. In order to hydrolyze the remaining chloro groups dispersed in the carrier 70 timeethanol aqueous solution obtained in this way, 6
It was treated at 0°C for 6 hours. After filtering the carrier, methanol 10
0rn! and dried at 100°C for 4 hours. The elemental analysis value of the support obtained in this way is that carbon is 13.5
%, hydrogen was 2.1 h. This reversed phase carrier was
．． 5mt! L, 50 cm long stainless steel column, 4
A 0% ethanol slurry was prepared and filled by a slurry method using ethanol as a filling solvent. After replacing the inside of the column with toluene, an α1N aqueous sodium hydroxide solution was passed through the column at a flow rate of 1 −/min for 5 minutes. A 1 L thiacetic acid aqueous solution was quickly passed through the solution at a flow rate of 1 d/min for 10 minutes.

Furthermore, after passing pure water at a flow rate of 1 ml/min for 10 minutes,
After equilibration with methanol, the elution position of naphthalene was examined by ultraviolet absorption (wavelength 254 nm) using methanol as a bath separation liquid, and it was found to be 2 [lL, 5 minutes (25.4 minutes before treatment). Furthermore, when the amount of methyl red adsorbed on the gel extracted from the column was examined, it was found to be 14.9 mg/9) before 2α3 my/gc treatment.

Disperse 5 ml of this gel in 50 ml of toluene, add 5 ml of γ-glycidoxypropyltrimethoxysilane, and heat to 80°C.
After reacting for 8 hours and separating the filtrate, 50 grams of toluene and 50 grams of acetone were added. After further washing with 50° of methanol, the pH was adjusted to 2.5 with 100° of pure water and depleted chloric acid, and the mixture was treated at 90° C. for 4 hours.

After filtration, the mixture was washed with methanonyl and dried at 100°C for 4 hours. 5 ml of carrier Q thus obtained was added to 1 ml of an aqueous solution containing 0.5 g of bovine serum albumin and 1 μl of theophylline, and stirred for 5 minutes. After filtration, the filtrate was mixed with rJ, 2N IJ acid buffer (pu6.
8) column for high-speed gel filtration chromatography equilibrated with (trade name: T SK gelG2DDO8W).

(inner diameter 7.5 ms, length 60 crn, manufactured by Toyo Soda Kogyo). The chromatogram detected at 280 nm was
Shown in the figure. A is a chromatogram of a solution of bovine serum albumin added with theophylline, B is a chromatogram of a solution treated with the carrier of the present invention, 1 is the absorbance peak of bovine serum albumin, and 2 is the absorbance peak of theophylline. 2 of the protein in the filtrate
When the absorbance at 80 nm was examined, the recovery rate was 99% before adding the carrier of the present invention. On the other hand, theophylline was completely adsorbed onto the carrier.

Example 2 Average pore size is 150 angstroms, average particle size is 1
Silica gel 109 of 5 microns was treated with a silane treatment agent having an octadecyl group according to Example 1. The elemental analysis values of the supports obtained in this way indicate that each carbon
5.9%, and hydrogen was 2.3%. This reversed phase support having octadecyl groups bonded thereto was packed into a stainless steel column according to Example 1, and after being replaced with toluene, 0. Treated with IN sodium hydroxide for 5 minutes. When the elution position of naphthalene was measured using methanol as an eluent, it was found to be 1 & 8 minutes (2 (19 minutes) before treatment).Furthermore, when the amount of methyl red adsorbed was examined after taking it out from the column, it was found to be 11.3 m9.
/9 (1L 11m9/liter before treatment) This carrier was treated with the same γ-glycidoxypropyltrimethoxysilane as in Example 1, and further treated with perchloric acid. In the same manner as in Example 1, this carrier α59 was added to 1 ml of an aqueous solution containing α59 bovine serum albumin and 1 μW theophylline, and the protein recovery rate was determined to be 99%.
Theophylline was adsorbed and was not observed.

Example 3 Average pore size is 60 angstroms, average particle size is 50 angstroms
In the example using 10 g of 0 micron silica gel, the carbon content was 12.2% and the hydrogen content was 2.1%. This carrier was packed into a stainless steel column according to Example 1, and [lI
Treated with N sodium hydroxide for 5 minutes. After neutralization and washing,
When the elution position of naphthalene was measured using methanol as an eluent, it was 19.2 minutes (22.6 minutes before treatment). Furthermore, when the amount of methyl red adsorbed was measured after taking it out from the column, it was found to be 15.2mg/9 (11.1mg/9 before treatment).
g). This carrier 59 was treated with r-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and further treated with perchloric acid. The carrier α thus obtained
5 g of protein-containing aqueous solution 1- in the same manner as in Example 1.
When the protein recovery rate was examined, it was 97%, and theophylline was adsorbed and was not observed.

Example 4 Average pore size is 100 angstroms, average particle size is 1
Disperse 10 g of 0 micron silica gel in 100 d of toluene, add 5 g of octyltrichlorosilane, and
The reaction was carried out at C for 24 hours. The reaction solution was filtered, and the carrier was mixed with 100 mg of toluene, 100 d of acetone, and then 1 d of methanol.
It was washed with 00g and dried at 100°C for 4 hours. The elemental analysis values of the thus obtained reverse phase support to which octyl groups are bonded are 9.5% carbon and 1.5% hydrogen.
It was 4chi. This carrier was packed into a stainless steel column according to Example 1, and then 0. Treated with IN sodium hydroxide for 5 minutes. After neutralization and washing, the elution position of naphthalene was measured using methanol as an eluent and found to be 14.5 minutes (IELI minutes before treatment). Furthermore, when the column was extracted and the amount of methyl red adsorbed was measured, it was found to be 19.
6 rrv)/9 (14.2 minutes before treatment).

This carrier 5 was treated with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and further treated with perchloric acid. In the same manner as in Example 1, 0.5 g of the carrier obtained in this manner was added to an aqueous solution containing protein,
When the protein recovery rate was examined, it was 98%, and theophylline was adsorbed and was not observed.

Example 5 Average pore size is 100 angstroms, average particle size is 1
Disperse 10 g of 0 micron silica gel in 100 g of toluene, add 5 g of propyltrichlorosilane, and
The reaction was carried out at C for 24 hours. After filtering the reaction solution, 100ml of toluene! After washing with 100ml of acetone and 100rnl of methanol, it was dried at 100°C for 4 hours. The elemental analysis values of the thus obtained reverse phase carrier to which propyl groups were bonded were 5.4% carbon and 1.2% hydrogen, respectively. This carrier was packed into a stainless steel column in the same manner as in Example 1, and 0. Treated with IN aqueous sodium hydroxide solution. After neutralization and washing, the elution position of naphthalene was measured using methanol as an eluent, and it was found that 11
2 minutes (158 minutes before treatment).

Furthermore, when it was extracted from the column and the amount of methyl red adsorbed was examined, it was found to be 25.1 ■/g (16.9 da/9 before treatment).
Met. The carrier thus obtained was heated at 100°C for 4 hours.
After drying for an hour, it was treated with γ-glycidoxypropyltrimethoxysilane and further treated with perchloric acid in the same manner as in Example 1. When 5 g of this carrier [L] was added to 1 ml of an aqueous solution containing protein in the same manner as in Example 1 and the protein recovery rate was examined, it was 97.5%, and theophylline was adsorbed and was not observed.

Example 6 Average pore size is 100 angstroms, average particle size is 1
Disperse 10 g of 0 micron silica gel in 100 mt of toluene, add 57 g of phenyltrichlorosilane, and add 90 mt of silica gel.
The reaction was carried out at °C for 24 hours. After filtering the reaction solution, 100ml of toluene was added. After washing with 100 grams of acetone and then 100 grams of methanol, it was dried at 100°C for 4 hours. The elemental analysis values of the thus obtained reverse phase support with phenyl groups bonded are 18fo carbon and 1 hydrogen, respectively.
．． It was 6chi. This carrier was packed into a stainless steel column in the same manner as in Example 1, and treated with a 11N aqueous sodium hydroxide solution for 5 minutes. After neutralization and washing, the elution position of naphthalene was measured using methanol as an eluent, and it was found to be 14.8
minutes (16.5 minutes before treatment).

Furthermore, when it was extracted from the column and the adsorption amount of methyl red was examined, it was found that it was 1z5rv/9 (10.4m9/g before treatment).
)Met. The carrier thus obtained was heated to 100°C.
After drying for 4 hours, it was treated with γ-glycidoxyprobyltrimethoxysilane and then with perchloric acid in the same manner as in Example 1. After drying this carrier at 100°C for 4 hours, this carrier [15F was poured into an aqueous solution 1- containing the same protein as in Example 1, and the protein recovery rate was examined and found to be 96%. Theophylline was adsorbed and was not observed.

Example 7 According to Example 1, octadecyl groups were introduced into 10 g of silica gel with an average pore diameter of 70 angstroms and an average particle diameter of 10 microns, and the gel was further packed into a stainless steel column.
Hydrolyzed. 5 g of this carrier was dispersed in 50 g of toluene, 2.5 g of 4-(methyldichlorosilyl)butyryl chloride was added, and the reaction was carried out at 80°C for 4 hours. Then, 5 rnt of aqueous ammonia was added in 50 g of dioxane. Treated at 50°C for 18 hours. 100° after filtration and washing
It was dried at C for 4 hours. The carrier α5 thus obtained
g is Q as in Example 1, 5 g of bovine serum albumin and 1
When the protein recovery rate was examined by pouring it into an aqueous solution 1- containing μm of theophylline, it was found to be 9.5%, indicating that theophylline was adsorbed and was not observed.

Example 8 The reverse phase carrier into which octadecyl groups were introduced obtained in Example 1 was packed into a stainless steel column according to Example 1, and α1
It was treated with an aqueous solution of IJ (N hydroxide). After treating 5 g of the carrier thus obtained with γ-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, 2.5 g of sorbitol was added in 50 ml of dioxane at 60°C in the presence of boron trifluoride. It reacted for 8 hours. This carrier 0.5
g was added to an aqueous solution containing protein in the same manner as in Example 1, and the protein recovery rate was examined, which was 97 g, and theophylline was adsorbed and was not observed.

Example 9 The reverse phase support into which octadecyl groups were introduced obtained in Example 1 was packed into a stainless steel column according to Example 1, and α1
It was treated with an aqueous solution of IJ (N hydroxide). The carrier thus obtained was treated with r-glycidoxypropyltrimethoxysilane in the same manner as in Example 1, and then 2.5 g of ethylene glycol monomethyl ether was added to 50 ml of dioxane in the presence of boron trifluoride. and 60°C
It reacted for 8 hours. This carrier α5 was poured into an aqueous solution containing protein in the same manner as in Example 1, and the protein recovery rate was examined, and it was found to be 95. In addition, theophylline was adsorbed and was not observed.

Example 10 The reverse phase carrier into which octadecyl groups were introduced obtained in Example 1 was packed into a stainless steel column according to Example 1, and 111
Treated with N aqueous sodium hydroxide solution. The carrier thus obtained was treated with γ-glycidoxyprobyltrimethoxysilane in the same manner as in Example 1, and then 2.59 g of diethylene glycol was added in 50 g of dioxane in the presence of boron trifluoride. , and reacted at 60°C for 8 hours. When 15 g of this carrier was poured into an aqueous solution containing protein in the same manner as in Example 1 and the protein recovery rate was examined, it was 96 g, and theophylline was adsorbed and was not observed.

Example 11 Average pore size is 70 angstroms, average particle size is 10
Micron silica gel 109 was dispersed in 100 g of toluene, 2.5 g of octadecyltrichlorosilane and 2.5 g of propyltrichlorosilane were added, and the mixture was reacted at 90°C for 24 hours. After filtering the reaction solution, it was washed with 10Q of toluene, 100-1 of acetone, and 100mg of methanol, and then treated in a 70% aqueous methanol solution at 80°C for 8 hours.

The carrier thus obtained was dried at 100°C for 4 hours. The elemental analysis value of this carrier is 10.
3%, hydrogen was 1.5%. This carrier was packed into a stainless steel column according to Example 1, and Q, IN hydroxide was applied.
It was treated with an aqueous solution of IJum. The elution position of naphthalene using methanol is 15.4 minutes (1B-1 minute before treatment)
Met. Furthermore, the adsorption amount of methyl red is 17.9 m9
/ri (11.3In9/9 before treatment). The carrier thus obtained was treated with γ-glycidoxyprobyltrimethoxysilane in the same manner as in Example 1, and further treated in an aqueous perchloric acid solution. The carrier thus obtained [1
5 g was poured into an aqueous solution containing protein in the same manner as in Example 1, and the protein recovery rate was determined to be 97%, and theophylline was adsorbed and was not observed.

Example 12 The reversed phase carrier into which octadecyl groups were introduced obtained in Example 1 was hydrolyzed with an aqueous alkali solution according to Example 1, and further treated with γ-glycidoxyprobyltrimethoxysilane. 5 g of the carrier thus obtained was dispersed in 100% dioxane, 2.5 g of glycerin and 2.59 diethylene glycol were added in the presence of boron trifluoride, and 50% of the carrier was dispersed in dioxane.
The reaction was carried out at °C for 8 hours. After filtering and washing with 100 ml of pure water, it was dried at 100° C. for 4 hours. When 15 g of the thus obtained carrier was dispersed in an aqueous solution containing protein according to Example 1 and the protein recovery rate was examined, it was 97 g, and theophylline was adsorbed and was not observed. Ta.

Example 13 0.19 g of the carrier obtained in Example 1 was dispersed in 1 g of serum and stirred for 10 minutes. After filtering off the carrier, the filtrate was subjected to high performance liquid chromatography in the same manner as in Example 1. The results are shown in FIG. C is a chromatogram of theophylline-added standard serum, D is a chromatogram of theophylline-added standard serum treated with the carrier of the present invention, 3 is the absorbance peak of serum protein, and 2 is the absorbance peak of theophylline.
It is. From the absorbance of the protein in the filtrate at 280 nm,
99% of the protein was recovered, while no peak of low molecular weight substances was observed, indicating that it was selectively adsorbed.

Example 14 The carriers [1 and 19 obtained in Examples '2.4 and 8 were each dispersed in 1 tnt of serum and stirred for 10 minutes in the same manner as in Example 9. After filtering off the carrier, the filtrate was subjected to high performance liquid chromatography in the same manner as in Example 1.

The recovery rate of protein based on the absorbance at 280 nm was 9.
On the other hand, no peak of theophylline was observed and it was selectively adsorbed to the carrier.

Example 15 The carrier obtained in Example 1 had an inner diameter of 4.6 ms and a length of 25
The mixture was packed into a stainless steel column with a slurry method. This column was attached to a high performance liquid chromatography machine and 0.1N
Phosphate buffer (pH 5,0) 8 [1 volume and methanol 2
Equilibrate the column with 0 volume of eluent and add 1 µl of the sample containing 5 µl of theophylline and 5 µl of caffeine in 1 d of serum.
00μ was injected and detected with an ultraviolet absorbance meter at 275 mm. The results are shown in FIG. 3. is the absorbance peak of serum protein, 2 is theophylline, and 4 is the absorbance peak of caffeine.

The protein eluted at the exclusion limit position (Vo), while theophylline and caffeine were completely separated by hydrophobic interactions. The recovery rates of theophylline and caffeine determined by the area method were 99 and 98, respectively.

Example 16 The carrier obtained in Example 2 was packed into a column similar to that in Example 15, and the column was installed in a high performance liquid chromatography machine. (LO
The eluent was equilibrated with 65 volumes of 5N phosphate buffer (pH 4,5) and 35 volumes of acetonitrile, and 5 μl of phenytoin was added to 11 nt of serum as shown in Figure 4. 3 is the absorbance peak of serum protein, 5 is the absorbance peak of phenobarbital, and 1.6 is the absorbance peak of phenytoin. The protein eluted at the exclusion limit, and phenytoin and barbital were separated by hydrophobic interactions and were recovered at 985% and 98%, respectively.

Example 17 The carrier obtained in Example 4 was packed into a column similar to that in Example 15, and the column was attached to a high performance liquid chromatography machine. 0.0
Equilibrate with 85 volumes of 5N phosphate buffer (1)H2,5) and 15 volumes of acetonitrile, and remove 1 rnt of serum.
Inside is angiotensin 11μ and angiotensin■
A 100μ sample containing 1μ was injected and detected at 210 nm on an ultraviolet spectrophotometer. The protein elutes at the exclusion limit position, while angiotensin ■ and angiotensin ■ are completely separated due to hydrophobic interactions, each with 9
2, and the recovery rate was 90%.

Example 18 The carrier obtained in Example 6 was packed into a column similar to that in Example 15, and the column was attached to a high performance liquid chromatography machine. 0.1
Sample 1 containing 10 μl of theophylline and 10 μl of caffeine in 1 d of serum, equilibrated with an eluent of 80 volumes of N phosphate buffer (pH 5,0) and 20 volumes of methanol.
00 μt was injected and recorded at 273 nm on an ultraviolet spectrophotometer. The protein was eluted at the exclusion limit position, while theophylline and caffeine were completely separated due to hydrophobic interactions, with recoveries of 99 and 98 hours, respectively.

→Example H-9 - Example 19 The carrier obtained in Example 11 was packed into a column similar to that in Example 15, and the column was mounted on a high-performance liquid chromatography system. 0.
Equilibrated with an eluent of 80 volumes of IN phosphate buffer (PL(5,0) and 20 volumes of methanol, 100 μt of a sample containing 10 μq1 of theophylline and 10 μl of caffeine in 1 volume of serum was injected, and the sample was measured using an ultraviolet absorbance meter. 273 nm
Detected with. The protein was eluted at the exclusion limit position, while theophylline and caffeine were completely separated due to hydrophobic interactions, with recoveries of 98 and 96, respectively.

Example 20 The carrier obtained in Example 1 had an inner diameter of 4 mtn and a length of 5 crn.
A stainless steel column was filled using the slurry method using a sample pretreatment device (product name:
PT-8000 (Toyo Soda Kogyo). The analytical column was equipped with a reversed-phase packing material to which octadecyl groups were bonded (trade name: T S K gel ODS-120T, inner diameter 4.5 mm).
6 mtn, length 25 cm, Toyo Soda Kogyo) using a, o s N IJ acid buffer (pH 2,5).
1 ml of eluent of 70 volume and 30 volume of acetonitrile
The liquid was passed through the tube at a flow rate of /min for equilibration.

The pretreatment column was filled with 0.05 NIJ acid buffer (pH
2, 5) was passed at a flow rate of 1.5 ml/min. Human serum n5mA plus 1μ cupropranolol was injected into the pretreatment column. After 5 minutes, change the flow path,
The substance adsorbed on the pretreatment column was introduced into the analytical column and detected at 215 nm using an ultraviolet absorbance spectrometer. When the recovery rate of propranolol was examined by the area method, it was found to be 9a5%. Further, the solution eluted from the pretreatment column was fractionated and the protein recovery rate was examined by absorbance at 280 nm, and it was found to be 98%. Furthermore, 100 µt of the fraction eluted from the pretreatment column was eluted with 0.2N phosphate buffer (pH 6,8) as an eluent, and then added to a high-performance liquid chromatography packing material (trade name: TSKgel G200).
When the mixture was injected into a 0SW inner diameter 7.5a+s, length 60crn1 (manufactured by Toyo Soda Kogyo Co., Ltd.) and measured at 215 nm using an ultraviolet absorbance meter, no propranolol peak was observed.

The results are shown in FIG. E is a chromatogram of propranolol-added standard serum, F is a chromatogram of propranolol-added standard serum treated with the packing material of the present invention, 3 is the absorbance peak of serum protein, and 7 is the absorbance peak of propranolol.

Example 21 The carrier obtained in Example 2 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. Example 20: Add 0.1 μ9 each of angiotensin ■ and angiotensin ■ to 1.5 ml of human serum.
It was subjected to pretreatment in the same manner as above.

Five minutes after sample injection, the flow path was changed, the pretreatment column adsorbent was introduced into the analytical column, and the 220 nm wavelength of the ultraviolet absorbance meter was measured.
When measured, angiotensin (2) and angiotensin (2) were separated, and the recovery rates by area method were 98% and 97%, respectively.

Furthermore, the eluate from the pretreatment column was fractionated and measured using a gel filtration packing material in the same manner as in Example 20.
The peaks of angiotensin ■ and angiotensin ■ were not observed.

Example 22 The carrier obtained in Example 4 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. 0.1 μl of propranolol was added to 1.5 d of human serum, and the mixture was applied to a pretreatment column in the same manner as in Example 20. After 5 minutes of pretreatment, the flow path was changed and the column was introduced into an analytical column. Propranolol was recovered at 98 points when detected using an ultraviolet absorbance spectrometer.

Furthermore, when the eluate from the pretreatment column was fractionated and measured using a gel filtration column in the same manner as in Example 20, no propranolol peak was observed.

Example 23 The carrier obtained in Example 5 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. 0.1μ of propranolol was added to 0.57ml of human serum, and the mixture was applied to a pretreatment column in the same manner as in Example 20. After passing the solution through the pretreatment column for 5 minutes, the flow path was changed and the solution was introduced into an analytical column, and when detected with an ultraviolet absorbance meter, propranolol was recovered at 97 hours. Furthermore, when the eluate from the pretreatment column was fractionated and measured using the same gel filtration column as in Example 20, no bleed of propranolol was observed.

Example 24 The carrier obtained in Example 7 was packed into a stainless steel column in the same manner as in Example 20, and used as a packing material for sample pretreatment. α1μ of propranolol was added to 0.5d of human serum, and the mixture was applied to a pretreatment column in the same manner as in Example 20. After passing the solution through the pretreatment column for 5 minutes, the flow path was changed and the solution was introduced into an analytical column. Propranolol was recovered at 97.5% when detected using an ultraviolet absorbance spectrometer. Furthermore, when the eluate from the pretreatment column was fractionated and measured using the same gel filtration column as in Example 20, no propranolol peak was observed.

[Brief explanation of drawings]

FIG. 1 is a chromatogram of the theophylline-added bovine serum albumin solution in Example 1, FIG. 2 is a chromatogram of the theophylline-added standard serum in Example 13, and FIG. 3 is the chromatogram of the theophylline-added standard serum in Example 15. The chromatogram of the standard serum, FIG. 4 is the chromatogram of the standard serum added with barbital and phenobarbital in Example 16, and FIG. 5 is the chromatogram of the standard serum added with propranolol in Example 2o. 1. Bovine serum albumin Z Theophylline 3. Serum protein 4. Caffeine & Phenobarbital & Phenytoin 2 Propranolol Patent applicant Toyo Soda Kogyo Co., Ltd. Figure 1 Figure 2 Figure 3 Figure 4

Claims (3)

[Claims] (1) An organic stationary phase with a hydrophilic site on the external surface and a hydrophobic site on the internal surface of the pores, chemically bonded with an average pore diameter of 10 to 200 Å and an average particle diameter of 0.1
Dual structure carrier characterized by being porous silica particles of ~1000μ (2) The organic stationary phase with a hydrophilic moiety has a general formula ▲ mathematical formula, chemical formula, table, etc. ▼ (However, l: 1 to 6
)(1) -(O-CH_2CH_2)_m-OCH_3 (However,
m: 1-6) (2) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(However, n: 1-4
)(3) -(O-CH_2CH_2)_p-OH (however, p: 1
~6) The double structure carrier according to claim (1), which is a compound consisting of at least one type of residue represented by (4) (3) The organic stationary phase having a hydrophobic moiety has a general formula ▲ mathematical formula, chemical formula, table, etc. R_2, R_3: chloro group, alkoxy group, or methyl group; R_4: chloro group or alkoxy group)
Double structure carrier described in )