CN1681852A - Modified polymer particle, packing material and column for chromatography - Google Patents

Abstract

The purpose of this invention is to suppress the separation of carboxyl groups resulting from the hydrolysis of ester bonds, thereby improving the acid and alkali resistance of polymer particles. This hydrolysis occurs when using polymer particles containing ester bonds in the presence of strongly acidic or alkaline solutions. By treating the polymer particles containing ester bonds with acidic or alkaline aqueous solutions, the readily hydrolyzed ester bonds located on and exposed on the particle surface are pre-hydrolyzed, separating the carboxyl groups. The free carboxyl groups are then sealed by amidation, thus solving the aforementioned problem.

Description

Modified polymer particles, packing materials and columns for chromatography

technical field

The present invention relates to a polymer particle with improved acid and alkali resistance, a modified packing material for chromatography, its production and modification methods, and a chromatographic column using the packing material.

Background technique

Acrylate- and methacrylate-based polymer particles have traditionally been used as ion exchange resins or packing materials for various types of chromatography. However, these polymer particles contain ester linkages in the molecule, and use of this polymer in acid or alkaline solutions results in a loss of strength due to hydrolysis of the ester linkages, or use as packing for chromatography due to isolated carboxyl groups. Materials can cause changes in analytical graphics, etc. Therefore, the pH range of the solution that can be used is limited.

Application examples of packing materials for chromatography include chromatography for anion analysis. Anion chromatography includes the inhibitor method (a method that uses an inhibitor) and the non-inhibitor method (a method that does not use an inhibitor). As the eluent, use an alkaline solution with a pH of 10 or more, such as carbonate buffer solution or sodium hydroxide, in the inhibitor method, and a weak acid solution, such as p-hydroxybenzoic acid, o- Phthalic acid and trimesic acid. Therefore, acrylate-based and methacrylate-based filler materials are generally limited to use as filler materials for non-inhibitor methods (see JP-A-2000-221179 (the term "JP-A" used here means "not Examined Published Japanese Patent Application")).

And, a production method of an anion exchanger, an anion exchanger, and a method for measuring anions using the exchanger are known, which are characterized in that an anion exchanger containing an acrylate group and a methacrylate group containing an ester bond is treated with an alkaline solution to make the ester The bond is decomposed, thereby generating a hydroxyl group and/or a carboxyl group (see JP-A-2002-1941117). In this case, however, the treatment of the ester bond-containing polymer is performed with an acidic or basic aqueous solution in order to separate the hydroxyl and/or carboxyl groups and to adjust the anion retention time in analysis by using the separated groups.

Other examples of applications of packing materials for chromatography include reversed phase chromatography. As a packing material for reversed-phase columns, silica-based particles such as octadecanoic acid-treated silica gel are mainly used. However, this filling material has certain problems with respect to chemical stability, and therefore polymer particles are also used. Examples of acrylate-based or methacrylate-based filler materials include ethylene glycol dimethacrylate/alkyl methacrylate copolymers and glyceryl dimethacrylate homopolymers into which stearyl groups have been introduced .

In silica-based particles, unreacted silanol groups still remain in many cases, and therefore, it is impossible to obtain sufficiently high separation performance as reversed-phase columns. The extent of residual silanol groups can be detected by injecting an organic base such as pyridine into a packed column and comparing the peak shapes. It is generally known that peaks tend to tail more as the number of residual silanol groups becomes larger.

In order to solve the above-mentioned problems encountered in the use of silica-based filler materials, it is known to block residual silanol groups with inert groups. Recently, in order to seal at a higher density, for example, a method of reacting two terminal addition agents capable of partially forming a complex in an inert atmosphere in a sealed container has been reported (JP-A-10-72579) and a method of reacting a filling material with a chemical modifier in a supercritical fluid (JP-A-9-49829).

On the other hand, polymer particle packed columns have the advantage of excellent chemical stability compared to silica columns, however, such columns are inferior to silica columns in, for example, (1) batch-to-batch variation in retention volume Large, (2) poor reproducibility of retention volume, (3) enhanced tailing, (4) small number of theoretical plates. Therefore, polymer-based distribution-adsorption columns are very small in terms of product quantities available on the market, and the possibility of using said columns is extremely limited. The above-mentioned problems manifest themselves particularly when completely salt-free eluents are used. Such a problem of polymer particles can be solved by blocking free carboxyl groups of polymer-based filler materials with inert groups (see JP-A-2000-310623). However, also in this case, the resistance to acids and bases is not sufficiently high and limits the pH range of the eluent used.

JP-A-2003-176363 describes a filler material obtained by covering polymer particles with a crosslinkable epoxy compound and introducing a carbon chain through an ether bond, and the filler material has high acid and alkali resistance. However, even in this case, when acrylate-based or methacrylate-based particles are used as the base material gel and a strongly acidic or strongly alkaline solution is used as the eluent, the ester bonds exposed on the surface of the particles are hydrolyzed, New free carboxyl groups are generated, thus limiting the pH range of eluents that can be used for analysis.

Contents of the invention

An object of the present invention is to provide polymer particles with improved resistance to acids and bases, which can be used for liquid chromatographic analysis by using polymer particles containing ester bonds, and which can be used even for strong acids or bases in a neutral solution. More particularly, an object of the present invention is to provide polymer particles with improved acid and alkali resistance, a modified packing material for chromatography, a production method thereof, and a chromatography column using the packing material.

The present inventors have found that when acidic or alkaline aqueous solution is used to treat polymer particles containing ester bonds, so that the ester bonds that are easy to contact with the liquid and easy to hydrolyze are hydrolyzed to separate carboxyl groups in advance, and then the free carboxyl groups are blocked by amidation, It can improve the acid and alkali resistance of polymer particles. The present invention has been accomplished based on the findings.

That is, the present invention relates to, for example, the following subjects.

[1] A modified polymer particle with improved acid and alkali resistance, which is obtained by treating polymer particles containing an ester bond with an acidic or alkaline aqueous solution to partially hydrolyze the ester bond and separate the carboxyl group, and remove the free carboxyl group by amidation. obtained by blocking the carboxyl group.

[2] The modified polymer particle having improved acid and alkali resistance as described in [1], wherein the hydrolyzed ester bond is an ester bond that is easy to contact with a liquid.

[3] The acid and alkali resistance-improved modified polymer particles as described in [1], wherein the amine used for amidation is an amine represented by the formula (1):

NHR ¹ R ² (1)

(wherein ^R1 and ^R2 each independently represent a hydrogen atom, an alkyl group with 18 or less carbon atoms that may be branched or substituted by halogen, or a phenyl group).

[4] The modified polymer particles having improved acid and alkali resistance as described in [3], wherein the amine used for amidation is an amine represented by the formula (2):

NH ₂ R ³ (2)

(wherein, R ³ represents a hydrogen atom, an alkyl group having 18 or less carbon atoms which may be branched or substituted by halogen, or a phenyl group).

[5] A modified polymer particle having improved acid and alkali resistance, wherein when polymer particles are filled in a column having an inner diameter of 4.6 mm and a length of 150 mm and the alkali resistance is evaluated using the column by the following method, by Percent increase in pyridine retention after alkaline eluent of 50% or less:

Evaluation method:

(1) The eluent of acetonitrile/0.1% phosphoric acid aqueous solution=30/70 was passed through for 30 minutes at a flow rate of 0.3ml/min, the column temperature was set at 40°C, and the eluent was washed by using acetonitrile/water=30/70 Deliquification measures pyridine retention at a flow rate of 0.5ml/min,

(2) Make the alkaline eluent of acetonitrile/0.01mol sodium hydroxide aqueous solution=50/50 pass through the same column for 4 hours at a flow rate of 0.5ml/min at a column temperature of 40°C, then make acetonitrile/0.1% phosphoric acid The eluent of aqueous solution=30/70 was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40°C, and the eluent of acetonitrile/water=30/70 was passed at a flow rate of 0.5ml/min Pyridine retention is measured under, and

(3) Compare the pyridine retention of (2) with that of (1).

[6] The modified polymer particles having improved acid and alkali resistance as described in any one of [1] to [5], wherein the average particle size of the polymer particles is 1 to 50 μm.

[7] A polymer-based packing material for chromatography using modified polymer particles with improved acid and alkali resistance, wherein the polymer particles are treated with an acidic or alkaline aqueous solution to The ester bond is partially hydrolyzed and the carboxyl group is separated, and then the free carboxyl group is blocked by amidation.

[8] The polymer-based packing material for chromatography as described in [7], wherein the hydrolyzed ester bond is an ester bond that is easy to contact with a liquid.

[9] The polymer-based packing material for chromatography as described in [7], wherein the amine used for amidation is an amine represented by the formula (1):

NHR ¹ R ² (1)

(wherein ^R1 and ^R2 each independently represent a hydrogen atom, an alkyl group with 18 or less carbon atoms that may be branched or substituted by halogen, or a phenyl group).

[10] The polymer-based packing material for chromatography as described in [9], wherein the amine used for amidation is an amine represented by the formula (2):

NH ₂ R ³ (2)

(wherein, R ³ represents a hydrogen atom, an alkyl group having 18 or less carbon atoms which may be branched or substituted by halogen, or a phenyl group).

[11] A polymer-based packing material for chromatography, which uses modified polymer particles having improved acid and alkali resistance, so that when the polymer particles are filled in a column having an inner diameter of 4.6 mm and a length of 150 mm and passing When the following method uses the column to evaluate the alkali resistance, the percentage increase of pyridine retention after passing through the alkaline eluent is 50% or less:

Evaluation method:

(1) The eluent of acetonitrile/0.1% phosphoric acid aqueous solution=30/70 was passed through for 30 minutes at a flow rate of 0.3ml/min, the column temperature was set at 40°C, and the eluent was washed by using acetonitrile/water=30/70 Deliquification measures pyridine retention at a flow rate of 0.5ml/min,

(2) Make the alkaline eluent of acetonitrile/0.01mol sodium hydroxide aqueous solution=50/50 pass through the same column for 4 hours at a flow rate of 0.5ml/min at a column temperature of 40°C, then make acetonitrile/0.1% phosphoric acid The eluent of aqueous solution=30/70 was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40°C, and the eluent of acetonitrile/water=30/70 was passed at a flow rate of 0.5ml/min Pyridine retention is measured under, and

(3) Compare the pyridine retention of (2) with that of (1).

[12] The polymer-based packing material for chromatography as described in any one of [7] to [11], wherein the average particle size of the polymer particles is 1 to 50 μm.

[13] A method for producing modified polymer particles having improved acid and alkali resistance, which comprises treating polymer particles containing ester bonds with an acidic or alkaline aqueous solution to partially hydrolyze the ester bonds and separate carboxyl groups, and then pass the amide The free carboxyl group is blocked by chemical reaction.

[14] The method for producing modified polymer particles having improved acid and alkali resistance as described in [13], wherein the hydrolyzed ester bond is an ester bond that is easy to contact with a liquid.

[15] The method for producing modified polymer particles having improved acid and alkali resistance as described in [13], wherein the amine used for amidation is an amine represented by the formula (1):

NHR ¹ R ² (1)

(wherein ^R1 and ^R2 each independently represent a hydrogen atom, an alkyl group with 18 or less carbon atoms that may be branched or substituted by halogen, or a phenyl group).

[16] The method for producing modified polymer particles having improved acid and alkali resistance as described in [15], wherein the amine used for amidation is an amine represented by the formula (2):

NH ₂ R ³ (2)

(wherein, R ³ represents a hydrogen atom, an alkyl group having 18 or less carbon atoms which may be branched or substituted by halogen, or a phenyl group).

[17] The method for producing modified polymer particles having improved acid and alkali resistance as described in any one of [13] to [16], wherein when a column having an inner diameter of 4.6 mm and a length of 150 mm is packed into a polymer The percentage increase in pyridine retention after passing through an alkaline eluent is 50% or less when the column is evaluated for alkali resistance by the following method:

Evaluation method:

(1) The eluent of acetonitrile/0.1% phosphoric acid aqueous solution=30/70 was passed through for 30 minutes at a flow rate of 0.3ml/min, the column temperature was set at 40°C, and the eluent was washed by using acetonitrile/water=30/70 Deliquification measures pyridine retention at a flow rate of 0.5ml/min,

(2) Make the alkaline eluent of acetonitrile/0.01mol sodium hydroxide aqueous solution=50/50 pass through the same column for 4 hours at a flow rate of 0.5ml/min at a column temperature of 40°C, then make acetonitrile/0.1% phosphoric acid The eluent of aqueous solution=30/70 was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40°C, and the eluent of acetonitrile/water=30/70 was passed at a flow rate of 0.5ml/min Pyridine retention is measured under, and

(3) Compare the pyridine retention of (2) with that of (1).

[18] The method for producing modified polymer particles having improved acid and alkali resistance as described in any one of [13] to [16], wherein the average particle size of the polymer particles is 1 to 50 μm.

[19] A method of producing a polymer-based packing material for chromatography, which comprises, with the polymer-based packing material packed in a column, by performing hydrolysis using one or both of an acidic or basic aqueous solution Treatment and blocking by amidation produces the polymer-based packing material for chromatography as described in any one of [6] to [12].

[20] A chromatography column using the polymer-based packing material for chromatography with improved acid and alkali resistance described in any one of [6] to [12].

embodiment of the invention

In the present invention, when performing liquid chromatography using polymer particles containing ester bonds, the ester bonds that are easy to come into contact with liquid and are easily hydrolyzed are hydrolyzed in advance to separate carboxyl groups, and the carboxyl groups are blocked by amidation, thereby inhibiting the The hydrolysis of the ester that occurs when used in chromatography improves the acid and alkali resistance of the polymer particles and enables stable analysis.

More specifically, in JP-A-2000-310623, carboxyl groups generated in the polymerization step for producing polymer particles are blocked, whereas in the present invention, easily hydrolyzed ester bonds are actively hydrolyzed, thereby significantly improving Acid and alkali resistance.

The polymer particle modified by the method of the present invention is a polymer particle containing an ester bond in a molecule, and examples thereof include a crosslinkable polymer containing a polyester structure. Furthermore, even in the case of polymer particles in which ester bonds as a problem are contained in a partial structure, when it is considered that ester bonds cause a decrease in acid and alkali resistance, this is a polymer particle modified by the method of the present invention.

Examples of polymer particles containing a polyester structure include ethylene glycol di(meth)acrylate/alkyl(meth)acrylate copolymer, ethylene glycol di(meth)acrylate homopolymer, bis(meth)acrylate base) glyceryl acrylate/alkyl (meth)acrylate copolymer, glyceryl di(meth)acrylate homopolymer and its modified products, ethylene glycol di(meth)acrylate/di(methyl) Glyceryl acrylate copolymer and its modified products, trimethylolpropane tri(meth)acrylate/alkyl(meth)acrylate copolymer, trimethylolpropane tri(meth)acrylate homopolymer , pentaerythritol tri(meth)acrylate/alkyl (meth)acrylate copolymer and its modified products, pentaerythritol tri(meth)acrylate homopolymer and its modified products, vinyl (meth)acrylate Hydrolysis or modification of the oxirane rings of homopolymers and ethylene glycol di(meth)acrylate/glycerol (meth)acrylate copolymers.

Examples of polymer particles containing ester bonds as a problem in partial structures include, in addition to the above-mentioned ester structures, simultaneously containing one or more particles selected from amides, imides, alcohols, ethers, aromatic rings, alkyl chains, etc. Partially structured polymer particles.

The polymer particles contain ester bonds, and when the polymer particles are used in an acidic or alkaline aqueous solution, the ester bonds exposed on the surface of the particles are hydrolyzed. Therefore, the acid and alkali resistance of the polymer particles is low.

The modified polymer particles involved in the present invention include all polymer particles containing the above-mentioned ester bonds, wherein the ester bonds exposed on the surface and easily hydrolyzed are cut off in advance by acidic or alkaline aqueous solution, and the free Carboxy blocked.

The amide bond is not as easy to hydrolyze as the ester bond (see "PROTECTIVE GROUPS in ORGANIC SYNTHESIS" ("PROTECTIVE GROUPS in ORGANIC SYNTHESIS"), third edition, p. And when the generated free carboxyl groups are blocked and converted into amide bonds by amidation, the generation of carboxyl groups can be suppressed and the durability of the polymer can be improved even if the polymer particles are used in an acid or alkali solution.

That is, the ester bond that is easy to come into contact with liquid referred to in the present invention is the ester bond relatively close to the surface of the polymer. By converting the ester bond into an amide bond, the surface of the polymer is protected with an amide bond that is not easily hydrolyzed as described above, and as a result, the ester bond that has not been hydrolyzed by acid or alkali treatment becomes more difficult to combine with Liquid contact, so it is considered that the acid and alkali resistance of the polymer particles is improved.

In the present invention, the polymer particles to be modified may have various particle sizes depending on the use. In the case of use as a packing material for chromatography, preferably the polymer particles have an average particle size of 1-50 μm, more preferably 1.5-30 μm, more preferably 2-10 μm. If the average particle size is less than 1 μm, the column pressure rises too much beyond the strength limit of the particles, which is not preferable, while if the average particle size exceeds 50 μm, the separation ability is low and it is not preferable in practice.

The method of treating with an acidic or basic aqueous solution in order to cleave easily hydrolyzable ester bonds thought to be exposed on the surface of polymer particles in the present invention will be specifically described below.

As the acidic aqueous solution, an aqueous solution of hydrochloric acid, sulfuric acid, or the like can be used. In cases where the polymer particles are less wettable in the solution, an appropriate amount of a water-soluble organic solvent that does not react in a strongly acidic solution, such as dimethyl sulfoxide, may be added prior to use of the aqueous solution. The concentration is not particularly limited as long as the ester bond on the surface of the polymer particles can be hydrolyzed, but the polymer is suspended by appropriately controlling the concentration so that the solution has a pH of 3 or less, preferably 1-2. The reaction temperature is not particularly limited as long as the ester bond on the surface of the polymer particles can be hydrolyzed, but the reaction temperature is suitably set at 20-100°C, preferably 30-80°C, more preferably 40-60°C. The reaction time is not particularly limited as long as the ester bond on the surface of the polymer particles can be hydrolyzed, but the reaction is properly carried out under stirring for 30 minutes to 24 hours, preferably 30 minutes to 10 hours, preferably 1 to 6 hours .

As the alkaline aqueous solution, hydroxides such as aqueous solutions of sodium hydroxide and potassium hydroxide can be used. Where the polymer particles are relatively non-wettable in the solution, an appropriate amount of a water-soluble organic solvent that does not react in a strongly basic solution, such as dimethyl sulfoxide, may be added prior to use of the aqueous solution. The concentration is not particularly limited as long as the ester bond on the surface of the polymer particles can be hydrolyzed, but the polymer is suspended by appropriately controlling the concentration so that the solution has a pH of 10-14, preferably 11-13. The reaction temperature is not particularly limited as long as the ester bond on the surface of the polymer particles can be hydrolyzed, but the reaction temperature is suitably set at 20-100°C, preferably 30-80°C, more preferably 40-60°C. The reaction time is not particularly limited as long as the ester bond on the surface of the polymer particles can be hydrolyzed, but the reaction is properly carried out under stirring for 20 minutes to 24 hours, preferably 30 minutes to 10 hours, preferably 1 to 6 hours .

Ranges of pH, reaction temperature and reaction time in the hydrolysis conditions of ester bond can be adjusted and used individually, but it is preferable to adjust all the conditions in combination.

The amidation method used to achieve blocking in the present invention is described in detail below.

The amine used for amidation is basically not limited, as long as the free carboxyl group can be blocked, but considering the reactivity under amidation, the limitations introduced due to steric hindrance, and the stability after conversion into an amide, it is preferred to use The amine is an amine represented by formula (1):

NHR ¹ R ² (1)

(wherein ^R1 and ^R2 each independently represent a hydrogen atom, an alkyl group with 18 or less carbon atoms that may be branched or substituted by halogen, or a phenyl group).

More preferably the amine is a primary amine represented by formula (2):

NH ₂ R ³ (2)

(wherein, R ³ represents a hydrogen atom, an alkyl group having 18 or less carbon atoms which may be branched or substituted by halogen, or a phenyl group).

The advantage of smaller substituent group volume is that the introduction efficiency is improved due to small steric hindrance and the hydrophobicity of the polymer particles before modification does not change much. However, in the case where the number of free carboxyl groups to be blocked is very small, even if a relatively large inert group is introduced, the change in hydrophobicity is small. Thus, substituents that are bulky to a certain extent can be used. More specifically, in the case of an alkylamine, the alkyl group preferably has a carbon number of 18 or less, more preferably 12 or less, more preferably 6 or less, and particularly preferably 4 or less.

Examples include ammonia, methylamine, ethylamine, 1-propylamine, isopropylamine, 1-butylamine, isobutylamine, t-butylamine, 1-hexylamine, cyclohexylamine, 1-octylamine, dimethylamine, diethylamine and di(1-propyl)amine.

For amidation in the present invention, methods generally used in peptide synthesis and the like can be used. However, there must be no adverse effect on moieties other than the free carboxyl groups of the polymer particles to be modified. In addition, amidation is preferably performed by using a combination of reagents described below in consideration of practical aspects such as introduction efficiency, ease of handling, and cost.

(A) (i) reagents for producing mixed acid anhydrides, such as ethyl chloroformate; tertiary amines, such as triethylamine; and organic solvents, such as chloroform, and (ii) amines represented by formula (1).

(B) Amines represented by formula (2), such as 1-propylamine; dehydration condensation agents, such as N,N'-diisopropylcarbodiimide; and organic solvents, such as toluene and N,N-dimethyl Formamide.

The method for modifying polymer particles containing ester bonds of the present invention is characterized in that the polymer particles are treated with the above-mentioned acidic or alkaline aqueous solution, and brought into contact with the solution or suspension containing the amidating agent.

That is, the readily hydrolyzable ester bonds thought to be exposed on the surface of the polymer particles are first hydrolyzed by treating the polymer particles with an acidic or alkaline aqueous solution, and then the isolated Carboxyl groups are converted to amide groups.

The column may or may not be prepacked with polymer particles to be modified. More specifically, one or two steps of hydrolysis treatment with an acidic or basic aqueous solution and blocking treatment by amidation may be performed with polymer particles packed into a column. In order to process the polymer in a packed state, a method of adding a solution or suspension containing a reagent to the column is used. The form concerning the addition amount, temperature, rate and time is appropriately selected according to the need to complete the reaction.

With conventional LC columns packed with polymer particles containing ester bonds that are readily in contact with liquids, the elution time and peak shape of organic bases such as pyridine sometimes change between before and after passage through strongly acidic or strongly basic eluents very big. On the other hand, the use of the liquid chromatography column filled with the polymer particles of the present invention significantly suppresses the change in elution time and peak shape of organic bases such as pyridine between before and after passing through a strongly acidic or strongly basic eluent .

It is believed that the problems encountered when measuring with conventional liquid chromatography columns filled with polymer particles containing ester linkages that are readily contacted by liquids are due to the generation of free carboxyl groups that are generated by passing through strongly acidic or strongly basic eluents Produced by hydrolysis of ester linkages exposed on the surface of polymer particles. As the free carboxyl group, it is considered that a proton type (R-COOH) carboxyl group and a metal type (R-COOM) carboxyl group exist at the same time. Organic bases such as pyridine (organic bases are hereinafter referred to as "pyridine") are mainly strongly adsorbed to the former. This is because the pH is locally low in the vicinity of the protic carboxylic acid (R-COOH) and the balance of pyridine and pyridinium salt ions tends toward pyridinium salt ions, and as a result, it is easy to generate ionic bonds. This phenomenon is more pronounced if a completely salt-free eluent is used. Because if a small amount of metal is mixed in the eluent, the metal is trapped and concentrated in the column when it passes near the proton-type carboxylic acid (R-COOH), and as a result, the metal-type carboxylic acid (R-COOM) increases and makes pyridine less less adsorption. In order to accurately evaluate the alkali resistance, it is necessary to convert the metal-type carboxylic acid (R-COOM) into the proton-type carboxylic acid (R-COOH). For this purpose, an acid solution containing phosphoric acid can be passed through the column prior to the analysis of pyridine.

Taking this into consideration, the effect of modification can be clearly detected when pyridine analysis is performed before and after passing a strongly acidic or strongly basic eluent through the subject column to be evaluated.

More particularly, when the ester bond is hydrolyzed and a free carboxyl group is generated, the retention volume of pyridine increases and the peak shape tails, therefore, it can be obtained by comparing the polymer particles after modification according to the present invention with the polymer particles before modification The effect of the present invention was checked.

By this method, the influence on acid and alkali resistance of polymer particles obtained by modification including hydrolysis treatment with an acidic or alkaline aqueous solution and blocking treatment by amidation can be confirmed.

Example

The present invention is described in more detail below by referring to Examples, however, the present invention is not limited to these Examples. In Example 1, the production method of a polymer-based packing material suitable for a reversed-phase liquid chromatography column is described from the first preparation of the base material gel until the final introduction of the carbon chain, but steps 3 and 4 are mainly related to the present invention. part. In this way, the method of the invention can be utilized by introducing it into the course of a series of steps in the production of polymer particles.

(Example 1)

<Step 1: Synthesis of base material cross-linked polymer particles>

In a mixed solution containing 2,000 g of glyceryl dimethacrylate and 900 g of 1-hexanol, 30 g of 2,2'-azobisisobutyronitrile was dissolved to prepare an oil phase. 180 g of polyvinyl alcohol (KURARAYPOVAL PVA-224, manufactured by Kuraray Co., Ltd.) was dissolved in 3 liters of water, and 7 liters of water were added thereto, and then 240 g of sodium chloride obtained by dissolving 240 g of sodium chloride in 2 liters of water was added. A solution and a solution obtained by dissolving and mixing 15 g of sodium lauryl sulfate in 485 ml of water were prepared to prepare an aqueous phase. The obtained oil phase and water phase were mixed in a 20 L volume stainless steel container and dispersed in a high-speed disperser (homogenizer) by adjusting the number of revolutions and dispersion time so that the maximum particle size of oil droplets was 3 μm.

While stirring at 150 rpm, the obtained dispersion was reacted at 70° C. for 4 hours. The resulting cross-linked polymer particles were centrifuged (10 minutes at 2,000 rpm), and after removing the supernatant, the precipitate was dispersed in 12 liters of hot water at 70° C. (by using an ultrasonic cleaner), and then heated at 70 Stir at °C for 3 hours. The resulting solution was suction-filtered, and the gel on the funnel was washed with 60 liters of hot water at 70° C., then washed with 18 liters of acetone, air-dried by dispersing it in a stainless steel tank, and further decompressed at 60° C. Dry for 24 hours. The obtained polymer particles were classified with an air classifier to obtain 716 g of crosslinked polymer particles having a weight average particle size of 3 μm (hereinafter, the polymer particles are referred to as “base material gel”).

<Step 2: Wash thoroughly>

Pure water (500 ml) was added to 50 g of the base material gel obtained in Step 1, and heated and stirred at 60° C. for 5 hours. Thereafter, the particles were collected by filtration, sequentially washed with 2,000 ml of hot water at 70°C, and then washed with 300 ml of methanol, then air-dried by dispersing the particles in a stainless steel tank, and further dried at 60°C under reduced pressure for 24 hours, 49 g of a thoroughly washed base material gel were obtained.

<Step 3: Hydrolysis treatment with alkaline aqueous solution>

The thoroughly washed base material gel (10 g) obtained in step 2 was dispersed in 50 ml of 0.1 N aqueous sodium hydroxide solution, and stirred at 50° C. for 2 hours. The reaction liquid was suction filtered, and successively adopted 100ml of 0.01N hydrochloric acid solution, adopted 500ml of water and adopted 100ml of acetone to clean the gel on the funnel, then air-dried the gel by dispersing it in a stainless steel tank, and further dried it at 60 It was dried under reduced pressure at °C for 3 hours to obtain 10 g of a hydrolyzed gel.

<Step 4: Blocking by amidation>

The hydrolyzed gel (10 g) obtained in step 3 was dispersed in 50 ml of chloroform at 25°C. While stirring the dispersion in a water bath at 25°C, 2.3 g of triethylamine were added, 5 minutes later, 2.5 g of ethyl chloroformate, and 30 minutes later, 1.6 g of 1-propylamine. Then, they were allowed to react for 3 hours. The reaction mixture was filtered with suction, and the gel on the funnel was washed successively with 100 ml of chloroform, 500 ml of water and 100 ml of acetone, then air-dried by dispersing the gel in a stainless steel tank, and further at 60 ° C under reduced pressure. Drying under pressure for 3 hours yielded 10 g of amidated and blocked gel.

<Step 5: Surface crosslinking and epoxy ring opening reaction>

The amidated and blocked gel (10 g) obtained in step 4 and 1 g of ethylene glycol diglycidyl ether were added to 50 ml of toluene, and they were stirred in a water bath at 40° C. at the same time. 0.5 g of boron trifluoride diethyl ether complex was added, and they were allowed to react for 3 hours. Suction filter the reaction mixture, and use 100ml of acetone, then use 500ml of water to wash the gel on the funnel, then transfer the gel to a container, and add 50ml of 0.1N hydrochloric acid therein, and react at 50°C 1 hour. The reaction mixture was suction-filtered, and the gel on the funnel was washed with 500 ml of water and then with 100 ml of acetone, then air-dried by dispersing the gel in a stainless steel tank, and further dried at 60° C. under reduced pressure for 3 Hours, 11.5 g of a surface-crosslinked gel was obtained.

<Step 6: Reaction of C18 Formation>

The surface-crosslinked gel (10 g) obtained in Step 5 and 3 g of stearyl glycidyl ether were dispersed in 100 ml of toluene while stirring in a water bath at 40°C. 0.5 g of boron trifluoride diethyl ether complex was added, and they were allowed to react for 3 hours. The reaction mixture was suction filtered, and successively adopted 100ml of toluene, adopted 300ml of tetrahydrofuran, adopted 500ml of water and adopted 100ml of acetone to clean the gel on the funnel, then air-dried the gel by dispersing it in a stainless steel tank, and further It was dried under reduced pressure at 60° C. for 3 hours to obtain 10.5 g of a carbon chain-introduced gel.

<Filling of modified gel>

The carbon chain-introduced gel obtained in step 6 was packed into a stainless steel column of 4.6 mm (inner diameter) x 150 mm (length) by the slurry method to produce a reverse chromatography column (hereinafter referred to as "column A").

(comparative example 1)

A column was produced by removing steps 3 and 4 in the column production method of Example 1 (hereinafter referred to as "column R").

(comparative example 2)

A column was produced by removing step 4 in the column production method of Example 1 (hereinafter referred to as "column C").

(1. Alkali resistance comparison)

Example 1 (column A) and Comparative Example 1 (column B) were evaluated for alkali resistance by the following method.

In order to clearly show the effect of blocking by amidation, the results of the pyridine/phenol test for the comparative example (column C) are also shown.

1-1. Alkali resistance test method

1-1-1. Pyridine/phenol test in the initial state

The acidic eluent was passed through the column to be tested under the following conditions. The above steps were performed to favor the protic form (RCOOH) of free carboxyl groups, if present.

·Eluent: CH ₃ CN/0.1% phosphoric acid aqueous solution=30/70(v/v)

·Flow rate: 0.3ml/min

·Column temperature: 25℃

Eluent passage time: 30 minutes

Subsequently, the conditions were changed to the following conditions, and after liquid replacement for 30 minutes or longer, pyridine and phenol were analyzed.

·Eluent: CH ₃ CN/H ₂ O=30/70 (v/v)

·Flow rate: 1.00ml/min

·Column temperature: 40℃

1-1-2. Through strong alkaline eluent

After completion of the pyridine/phenol test in the initial state, the column to be tested was passed through, and the strongly basic eluent was passed under the following conditions. By such manipulations, hydrolysis of the ester bond, if present, readily accessible to liquids yields free carboxyl groups.

·Eluent: CH ₃ CN/0.01N-NaOH (pH: 12) = 50/50 (v/v)

·Flow rate: 0.50ml/min

·Column temperature: 40℃

Eluent passing time: 4 hours

1-1-3. Pyridine/phenol test after passing through strong alkaline eluent

After passing through the strong basic eluent, the pyridine/phenol test was performed by the same method as in the above-mentioned 1-1-1 using the column to be tested.

1-2. Alkali resistance evaluation results

Table 1 shows the evaluation results of column A and column B. In the table, the retention k' is calculated from the no-retention time _t0 and the composite retention time _tr according to the following formula:

k'＝(t _r -t ₀ )/t ₀

Calculate the tailing coefficient T from the peak width W _0.05h at 5% of the peak height and the peak width f on the rising side of the peak according to the following formula:

T=W _0.05h /f

Table 1 Pyridine retention k' Phenol retention k' Pyridine tailing coefficient initial state After passing through a strong alkaline eluent initial state After passing through a strong alkaline eluent initial state After passing through a strong alkaline eluent Column A 1.5 1.6 3.6 3.6 0.75 0.75 Column B 1.6 2.5 3.6 3.6 1.5 4.3 Column C 4.7 - 3.6 - 5.8 -

In columns A and B, the phenol retention k' was unchanged in the initial state and after passing through the strong basic eluent, but in column B the pyridine retention increased by 56% and the tailing coefficient also increased . This suggests that the ester bond hydrolyzes and generates free carboxyl groups during passage through the strongly basic eluent. In contrast, in column A, the pyridine peak hardly changed, which remarkably shows the effect of the present invention.

Furthermore, by comparing the pyridine retention and tailing coefficient of column A in the initial state with those of column C, it was confirmed that the carboxyl group generated by the alkali treatment in the production method of the packing material was blocked by amidation.

(2. Comparison of acid resistance)

Example 1 (Column A) and Comparative Example 1 (Column B) were compared for acid resistance by the following method.

2-1. Acid resistance evaluation method

2-1-1. Pyridine/phenol test in the initial state

The pyridine/phenol test was performed by the same method as in 1-1-1 above.

2-1-2. Through strong acid eluent

After completion of the pyridine/phenol test in the initial state, a strongly acidic eluent was passed through the column to be tested under the following conditions. By such manipulations, hydrolysis of the liquid-accessible ester linkages, if present, yields free carboxyl groups.

Eluent: MeOH/0.1% trifluoroacetic acid aqueous solution (pH: 1.2) = 10/90 (v/v)

·Flow rate: 1.00ml/min

·Column temperature: 40℃

Eluent passing time: 70 hours

2-1-3. Pyridine/phenol test after passing through strong acid eluent

After passing through the strongly acidic eluent, the pyridine/phenol test was performed by the same method as in the above-mentioned 1-1-1 using the test subject column.

2-2. Acid resistance evaluation results

Table 2 shows the evaluation results of column A and column B. In the table, the retention k' is calculated from the no-retention time _t0 and the composite retention time _tr according to the following formula:

k'＝(t _r -t ₀ )/t ₀

Calculate the tailing coefficient T from the peak width W _0.05h at 5% of the peak height and the peak width f on the rising side of the peak according to the following formula:

T=W _0.05h /f

Table 2 Pyridine retention k' Phenol retention k' Pyridine tailing coefficient initial state After passing through a strongly acidic eluent initial state After passing through a strongly acidic eluent initial state After passing through a strongly acidic eluent Column A 1.4 1.6 3.6 3.6 0.75 0.75 Column B 1.6 2.6 3.6 3.6 1.5 3.6

In columns A and B, the phenol retention k' was not changed in the initial state and after passing through the strongly acidic eluent, but in column B, the pyridine retention increased by 63%, and the tailing coefficient also increased. This suggests that the ester bond hydrolyzes and generates free carboxyl groups during passage through the strongly acidic eluent. In contrast, in column A, the pyridine peak hardly changed, which remarkably shows the effect of the present invention.

The polymer particles modified by the method of the present invention were packed to produce a column for chromatography, and organic bases such as pyridine were analyzed before and after passing through a strongly acidic or strongly basic eluent, and compared, as a result, significantly suppressed The increase in retention and the tailing of peaks can be observed, and the effect of the present invention can be confirmed.

Industrial applicability

In the modified polymer particles of the present invention, the ester linkages present near the surface and readily in contact with liquids are hydrolyzed and the resulting carboxyl groups are converted into amide groups, so that, after treatment, the ester bonds present on the surface are less susceptible to hydrolysis. amide bond, and the remaining ester bond becomes difficult to contact with liquid, as a result, it is considered that the acid and alkali resistance is improved.

The polymer particle of the present invention can be used as a packing material for chromatography, and the column filled with the polymer particle shows high acid and alkali resistance, therefore, it is used in a wide range of fields such as pharmaceutical preparations or agricultural chemical preparations It is useful in the field of separation and analysis of food additives, their intermediates, natural or synthetic polymers, their additives and environmental pollutants.

Claims (20)

1. A modified polymer particle improved in acid-base resistance obtained by treating a polymer particle containing an ester bond with an acidic or basic aqueous solution to partially hydrolyze the ester bond and separate the carboxyl group, and blocking the free carboxyl group by amidation.

2. The modified polymer particle improved in acid-alkali resistance according to claim 1, wherein the ester bond that has been hydrolyzed is an ester bond that is easily contacted with a liquid.

3. The modified polymer particle improved in acid-alkali resistance of claim 1, wherein the amine used for amidation is an amine represented by formula (1):

NHR¹R²(1)

wherein R is¹And R²Each independently represents a hydrogen atom, an alkyl group having a carbon number of 18 or less which may be branched or may be substituted with a halogen, or a phenyl group.

4. The modified polymer particle improved in acid-alkali resistance of claim 3, wherein the amine used for amidation is an amine represented by formula (2):

NH₂R³(2)

wherein R is³Represents a hydrogen atom, an alkyl group having a carbon number of 18 or less which may be branched or may be substituted with a halogen, or a phenyl group.

5. A modified polymer particle improved in acid-alkali resistance, wherein when the polymer particle is packed in a column having an inner diameter of 4.6mm and a length of 150mm and the alkali resistance is evaluated using the column by the following method, the percentage of increase in pyridine retentivity after passing through an alkali eluent is 50% or less:

the evaluation method comprises the following steps:

(1) an eluent of acetonitrile/0.1% aqueous phosphoric acid solution (30/70) was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40 ℃, and the pyridine retentivity was measured by using an eluent of acetonitrile/water (30/70) at a flow rate of 0.5ml/min,

(2) an alkaline eluent of acetonitrile/0.01 mol of aqueous sodium hydroxide solution of 50/50 was passed through the same column at a column temperature of 40 ℃ for 4 hours at a flow rate of 0.5ml/min, then an eluent of acetonitrile/0.1% aqueous phosphoric acid solution of 30/70 was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40 ℃, and pyridine retentivity was measured by using an eluent of acetonitrile/water of 30/70 at a flow rate of 0.5ml/min, and

(3) the pyridine retention of (2) was compared to the retention of (1).

6. The modified polymer particles with improved acid-base resistance of any one of claims 1 to 5, wherein the average particle size of the polymer particles is 1 to 50 μm.

7. A polymer-based packing material for chromatography, which uses modified polymer particles improved in acid-base resistance, wherein the polymer particles are obtained by treating polymer particles containing ester bonds with an acidic or basic aqueous solution to partially hydrolyze the ester bonds and separate carboxyl groups, and then blocking the free carboxyl groups by amidation.

8. The polymer-based packing material for chromatography according to claim 7, wherein the ester bond that has been hydrolyzed is an ester bond that is easily contacted with a liquid.

9. The polymer-based packing material for chromatography according to claim 7, wherein the amine used for amidation is an amine represented by formula (1):

NHR¹R²(1)

wherein R is¹And R²Each independently represents a hydrogen atom, an alkyl group having a carbon number of 18 or less which may be branched or may be substituted with a halogen, or a phenyl group.

10. The polymer-based packing material for chromatography according to claim 9, wherein the amine used for amidation is an amine represented by formula (2):

NH₂R³(2)

wherein R is³Represents a hydrogen atom, an alkyl group having a carbon number of 18 or less which may be branched or may be substituted with a halogen, or a phenyl group.

11. A polymer-based packing material for chromatography, which uses modified polymer particles improved in acid-alkali resistance, so that when the polymer particles are packed in a column having an inner diameter of 4.6mm and a length of 150mm and the alkali resistance is evaluated using the column by the following method, the percentage of increase in pyridine retention after passing an alkali eluent is 50% or less:

the evaluation method comprises the following steps:

(1) an eluent of acetonitrile/0.1% aqueous phosphoric acid solution (30/70) was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40 ℃, and the pyridine retentivity was measured by using an eluent of acetonitrile/water (30/70) at a flow rate of 0.5ml/min,

(2) an alkaline eluent of acetonitrile/0.01 mol of aqueous sodium hydroxide solution of 50/50 was passed through the same column at a column temperature of 40 ℃ for 4 hours at a flow rate of 0.5ml/min, then an eluent of acetonitrile/0.1% aqueous phosphoric acid solution of 30/70 was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40 ℃, and pyridine retentivity was measured by using an eluent of acetonitrile/water of 30/70 at a flow rate of 0.5ml/min, and

(3) the pyridine retention of (2) was compared to the retention of (1).

12. A polymer-based packing material for chromatography as claimed in any one of claims 7 to 11, wherein the average particlesize of the polymer particles is in the range of 1 to 50 μm.

13. A method for producing a modified polymer particle improved in acid-alkali resistance, which comprises treating a polymer particle containing an ester bond with an acidic or alkaline aqueous solution to partially hydrolyze the ester bond and separate the carboxyl group, and then blocking the free carboxyl group by amidation.

14. The method for producing modified polymer particles improved in acid-alkali resistance according to claim 13, wherein the ester bond that has been hydrolyzed is an ester bond that is easily contacted with a liquid.

15. The method for producing modified polymer particles improved in acid-alkali resistance according to claim 13, wherein the amine used for amidation is an amine represented by formula (1):

NHR¹R²(1)

wherein R is¹And R²Each independently represents a hydrogen atom, an alkyl group having a carbon number of 18 or less which may be branched or may be substituted with a halogen, or a phenyl group.

16. The method for producing modified polymer particles improved in acid-alkali resistance according to claim 15, wherein the amine used for amidation is an amine represented by formula (2):

NH₂R³(2)

wherein R is³Represents a hydrogen atom, a carbon number of 18 or less which may be branched or substituted by halogenAn alkyl group, or a phenyl group.

17. The method for producing a modified polymer particle improved in acid-alkali resistance according to any one of claims 13 to 16, wherein when the polymer particle is packed in a column having an inner diameter of 4.6mm and a length of 150mm and the alkali resistance is evaluated using the column by the following method, the percentage of increase in pyridine retention after passing through an alkali eluent is 50% or less:

the evaluation method comprises the following steps:

(1) an eluent of acetonitrile/0.1% aqueous phosphoric acid solution (30/70) was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40 ℃, and the pyridine retentivity was measured by using an eluent of acetonitrile/water (30/70) at a flow rate of 0.5ml/min,

(2) an alkaline eluent of acetonitrile/0.01 mol of aqueous sodium hydroxide solution of 50/50 was passed through the same column at a column temperature of 40 ℃ for 4 hours at a flow rate of 0.5ml/min, then an eluent of acetonitrile/0.1% aqueous phosphoric acid solution of 30/70 was passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature was set at 40 ℃, and pyridine retentivity was measured by using an eluent of acetonitrile/water of 30/70 at a flow rate of 0.5ml/min, and

(3) the pyridine retention of (2) was compared to the retention of (1).

18. The method for producing modified polymer particles improved in acid-alkali resistance according to any one of claims 13 to 16, wherein the average particle size of the polymer particles is from 1 to 50 μm.

19. A method for producing a polymer-based packing material for chromatography, comprising producing the polymer-based packing material for chromatography described in any one of 6 to 12 by performing hydrolysis treatment with one or both of acidic or basic aqueous solutions and blocking treatment by amidation in the case of packing the polymer-based packing material in a column.

20A column for chromatography using the polymer-based packing material for chromatography having improved acid-base resistance as described in any one of 6 to 12.