JP2000074894A - Polymer packings for liquid chromatography, and preparation thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymer packings for liquid chromatography having a superior separation function, and preparation thereof. SOLUTION: A particle size monodisperse polymer packings having a styrene backbone makes an inside of a micropore existing in a pore of the polymer packings hydrophilic. This preparation of the packings is carried out by polymerizing glycerol dimethacrylate of a crosslinker and 2-ethylhexyl metheacrylate of a monomer pursuant to two stage swelling polymerization method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a packing material for liquid chromatography, particularly to a polymer packing material for liquid chromatography obtained by a two-stage swelling polymerization method, and an improvement of a method for producing the same.

[0002]

2. Description of the Related Art High performance liquid chromatography (HPL)
Fillers for C) can be broadly classified into those based on inorganic carriers and those based on organic carriers. However, in practice, fillers based on silica gel, which is an inorganic carrier, are frequently used, and in reversed-phase liquid chromatography, which accounts for more than 60% of all separation modes of HPLC, alkyl-silylated silica gel with a chemically modified silica gel carrier surface. Is mainly used. These silica gel-based fillers exhibit excellent separation performance and good mechanical strength, but on the other hand, have low chemical stability, and are also affected by silanol groups remaining on the surface or metal impurities of the silica gel base. It has disadvantages such as the appearance of an undesirable secondary holding effect.

On the other hand, organic polymer fillers are chemically stable and have been used as fillers for size exclusion chromatography and ion exchange chromatography as well as fillers for silica gel. In addition, it can be used in reverse phase chromatography even under separation conditions where silica gel packing cannot be used.In recent years, understanding of the separation characteristics has been advanced, and examples of separation showing separation characteristics superior to silica gel have come to be seen. ing.

[0004] Polymer fillers are broadly classified into those prepared from natural polymers via crosslinking and those based on crosslinked polymers obtained by polymerization of vinyl monomers. Typical examples of the former include fillers synthesized from polysaccharide derivatives such as agarose, dextran, and mannan, but generally have low pressure resistance and cannot be used as a filler for HPLC in some cases. On the other hand, synthetic polymer fillers include polystyrene divinylbenzene and its derivatives, polymethacrylate gels, and polyacrylamide gels. Among them, polystyrene divinylbenzene and polymethacrylate gels are used as reversed-phase chromatography packing materials, are chemically stable, can be used in a wider pH range than silica gel packings, and have metal impurities. No adverse effects due to Synthetic polymer fillers are often synthesized by mixing a monomer and a crosslinking agent with a diluent, polymerizing the mixture, and increasing the porosity. Small pores are formed when a good solvent is used for the polymer to be synthesized, and larger pores are formed when a poor solvent is used, and the pore diameter can be controlled by combining this diluent with a monomer. Therefore, a spherical porous polymer filler has been synthesized by combining it with a suspension polymerization method which is easy to operate.

[0005]

However, this polymer filler has a structural problem related to the distribution of the pores of the polymer particles. That is, the polymer filler has a double pore structure having micropores (usually 2 nm or less) called micropores derived from the crosslinked structure of the polymer, apart from so-called pores (mesopores), unlike silica gel fillers. ing. Therefore, in general, the range in which solute molecules can penetrate into a polymer filler may be increased.
When these are packed in a chromatography column and used for separation of a sample, they exhibit significantly different separation characteristics from the case where a silica gel filler is used. Since it is difficult to control the influence of the separation ability due to the presence of such micropores, the influence has appeared as a decrease in the separation ability for the analysis sample.

[0006] In actual HPLC analysis, the adverse effect of the micropores is more remarkable as the analytical sample is strongly retained in the packing material and the larger the three-dimensional structure, the wider the peak width and the number of theoretical plates in the chromatogram. Lower. The effect of the structure of the target substance is that when the analysis sample passes through the micropore, molecules that are sufficiently smaller than this micropore space are not significantly affected by retention, but large molecules are affected by hydrophobic interaction in this space. This is because retention occurs.

[0007] The adverse effect of the fine pores on chromatography can be reduced by changing the state of polymerization and crosslinking of the polymer. That is, if the linear portion of the polymer formed by polymerization is made the same length and the distance between the cross-linking points is lengthened, the micropore space becomes large and the micropores have approximately the same size, and the deterioration of the filler performance is suppressed. It is possible. It is possible to increase the distance between the crosslinking points by reducing the percentage of crosslinking,
Simply reducing the percentage of cross-linking results in particles that are weak in terms of strength and cannot be used as a filler for HPLC. Therefore, it is necessary to prepare the filler by controlling the method and conditions of polymerization and crosslinking. For example, as one of the control methods, there is a method using oxidation-reduction polymerization in which polymerization is performed at a low temperature as a method for polymerizing monomers. Oxidation-reduction polymerization is generally used for preparing particles, and can increase the space of micropores as compared with thermal polymerization, and can suppress a decrease in the number of theoretical plates in chromatography of a polymer filler. Although this method is extremely useful in controlling micropores, the chemical properties within the micropores have not changed, and there has been some limitations in improving the performance of the filler.

Therefore, the problem of micropores is still solved,
No polymer filler utilizing this property has been obtained.
An object of the present invention is to solve the problem of micropores and to provide a polymer packing for liquid chromatography having excellent separation performance utilizing its properties and a method for producing the same.

[0009]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies. As a result, the chemical properties in the micropores of the polymer filler are modified to obtain a monodispersed particle size system. It was found that a polymer packing material for liquid chromatography capable of improving the separation performance of a chromatography column was obtained. We have also developed an appropriate manufacturing method.

That is, the chemical properties of the micropores are largely determined by the type of the monomer or the cross-linking agent constituting the polymer. Further, as described above, in the packing material for reversed phase chromatography, the decrease in the separation performance in the micropores is due to the hydrophobic interaction between the sample and the inside of the micropores. Therefore, it was decided to weaken the hydrophobic interaction between the sample and the micropores by increasing the hydrophilicity in the micropores while maintaining the holding power as a reversed phase chromatography packing material.

However, merely making the micropores hydrophilic does not result in the polymer filler according to the present invention exhibiting its properties sufficiently. The present inventors have further found that the cause is a problem related to the particle size distribution of the polymer filler. This is largely due to the method for producing the polymer filler. The suspension polymerization method is mainly used for the synthesis of polymer fillers, but the particle size distribution of the particles obtained by this method is extremely wide. Performance cannot be obtained. The large range of the particle size distribution causes a decrease in the number of theoretical plates. Particles that are considerably smaller than the target particle size may cause an increase in column pressure during analysis, and may damage the polymer filler, which is said to have low mechanical strength. On the other hand, the smaller the particle size, the higher the separation performance, so that mixing of particles larger than the target particle size results in lower column performance.

Therefore, the obtained polymer particles are usually classified and used only as a filler having an appropriate particle size as a filler for chromatography by a classification operation. However, the classification operation requires a large facility and is difficult in terms of operation, and there is a practical limit to the classification, and a certain degree of distribution is inevitably generated. In addition, when the classification accuracy is increased, only a small part of the polymer particles obtained first is used, so that the yield as a filler is reduced and the cost is increased.

Therefore, the present inventors have adopted a known two-stage swelling polymerization method in order to obtain a monodisperse polymer filler having a particle diameter. As a specific production method, hydrophilic glycerin dimethacrylate is used as a cross-linking agent, hydrophobic 2-ethylhexyl methacrylate is used as a monomer, and cross-linking polymerization is performed using a two-step swelling polymerization method. An agent was prepared.

That is, the polymer filler according to the present invention is characterized in that in a monodisperse polymer filler having a particle diameter of styrene as a skeleton, the inside of the fine pores existing in the pores of the polymer filler is made hydrophilic. It is characterized by. The polymer filler of the present invention preferably contains a unit having the following structure.

[0015]

Embedded image The polymer filler of the present invention preferably further contains a unit having the following structure.

[0016]

Embedded image (R represents a C1-18 hydrocarbon group)

In the polymer filler of the present invention, R is preferably a 2-ethylhexyl group. Further, the polymer filler according to the present invention is a polymer filler obtained by a two-step swelling polymerization method, wherein the inside of the fine pores present in the pores of the polymer is made hydrophilic. filler. The polymer filler obtained by the two-stage swelling polymerization method preferably contains a unit having the following structure.

[0018]

Embedded image

The polymer filler obtained by the two-stage swelling polymerization method preferably further contains a unit having the following structure.

[0020]

Embedded image (R represents a hydrocarbon group having 1 to 18 carbon atoms)

Further, in the polymer filler according to the present invention, it is preferable that R is a 2-ethylhexyl group. Further, the method for producing a polymer filler according to the present invention is characterized in that it is obtained by polymerizing glycerin dimethacrylate as a crosslinking agent and 2-ethylhexyl methacrylate as a monomer by a two-stage swelling polymerization method.

[0022]

Embodiments of the present invention will be described below in detail. The present invention provides a polymer filler in which the inside of fine pores present in the pores of the filler particles is made hydrophilic in a filler having a monodisperse particle diameter. It is shown in FIG. In ordinary polymer fillers, micropores (micropores: usually 2 nm or less) derived from the crosslinked structure of the polymer exist in addition to the pores (mesopores), but in the present invention, the hydrophilic group (-O in FIG. 1) is used.
H) can be made hydrophilic and its adverse effects can be reduced.

As a specific cross-linking agent for imparting hydrophilicity to the micropores used in preparing the polymer filler according to the present invention, glycerin dimethacrylate can be mentioned.
Glycerin dimethacrylate used in the polymer filler of the present invention can be synthesized, for example, from an addition reaction of glycidyl methacrylate and methacrylic acid. Glycerin dimethacrylate is mixed and polymerized with a hydrophobic monomer copolymerizable therewith. By this ratio, the balance between hydrophilicity and hydrophobicity of the micropores of the polymer can be controlled.

Examples of the hydrophobic monomer include methacrylates having 5 to 22 carbon atoms such as methyl methacrylate, ethyl methacrylate, butyl methacrylate, 2-ethylhexyl methacrylate and propyl methacrylate.

Among these monomers, methyl methacrylate, butyl methacrylate and 2-ethylhexyl methacrylate are preferred, and 2-ethylhexyl methacrylate is particularly preferred. Further, when glycerin dimethacrylate and methyl methacrylate are copolymerized, the molar ratio thereof is preferably 1: 9 to 9: 1,
When glycerin dimethacrylate and 2-ethylhexyl methacrylate are copolymerized, their molar ratio is 1:
It is preferably 9 to 9: 1.

Therefore, in the polymer filler according to the present invention, the one using glycerin dimethacrylate as a cross-linking agent has the structure

Embedded image Has the unit of Therefore, in the polymer filler having this structural unit, the micropores are made hydrophilic by the hydroxyl group, which is a hydrophilic group.

In the case where methacrylates having 5 to 22 carbon atoms are used as the monomer,

Embedded image (R represents a hydrocarbon group having 1 to 18 carbon atoms). The hydrocarbon group R can have up to 18 carbon atoms.

The polymer filler for chromatography of the present invention having a monodispersed particle size can be produced by using a known two-stage swelling polymerization method using a crosslinking agent such as glycerin dimethacrylate and a monomer.

In the two-step swelling polymerization method, seed particles having a very uniform particle diameter of about 1 μm obtained by soap-free seed polymerization are swollen with a swelling aid, a monomer and a cross-linking agent in a two-step process. And then polymerizing to obtain polymer particles having a uniform particle size. In this case, the uniformity of the particle size is not impaired in the swelling step, and highly crosslinked particles can be synthesized.If the seed particles are swollen with a diluent together with a monomer, the particles should be porous. Can also. The monodispersity of the particles obtained by this method is very good according to the particle distribution of the seed particles, without any classification work,
A filler having a monodispersed particle size can be easily prepared. This has much higher particle size uniformity than that obtained by repeating a classification operation after preparation by a suspension polymerization method generally used conventionally.

In the above two-stage swelling polymerization method, styrene is used as seed particles serving as nuclei, and a skeleton is formed in the polymer filler. Also, dibutyl phthalate as a swelling aid, cyclohexanol, toluene as a diluent,
Examples of the polymerization initiator include, but are not limited to, benzoyl peroxide, azobisisobutyronitrile and the like.

The chromatography column packed with the packing material of the present invention has a high particle size uniformity, so that the pressure can be suppressed. Therefore, it can be used without problem even for a polymer filler having low mechanical strength. If the column pressure can be suppressed, a packing material having a small particle diameter, which can achieve higher performance, can be easily used, and the separation performance can be improved and the speed can be increased.

[0032]

The present invention will now be described in more detail with reference to Examples.
Will be described.Example Polymer packing for liquid chromatography  (Crosslinking agent) Glycerin dimethacrylate (monomer) 2-ethylhexyl methacrylate (molar ratio) Crosslinking agent: monomer = 5: 5 (Production method) Two-stage swelling polymerization method

1 obtained by soap-free seed polymerization
First-stage swelling was performed on styrene seed particles having a very uniform particle diameter of about μm with dibutyl phthalate as a swelling aid. Next, a mixed solution consisting of 2-ethylhexyl methacrylate, glycerin dimethacrylate, cyclohexanol and azobisisobutyronitrile was added, followed by stirring and swelling in the second stage. And 70 ° C
And polymerized for 24 hours.

After completion of the reaction, the polymerization reactor is cooled to room temperature,
The product was separated. The product was sufficiently washed with water, and cyclohexanol was removed using acetone, and then dried to obtain a porous spherical gel having fine styrene as a skeleton. FIG. 2 shows the result of performing liquid chromatography using the polymer filler thus obtained. The sample was benzene,
A mixture of five monocyclic compounds of naphthalene, anthracene, pyrene, and triphenylene was used. As is clear from the figure, the peaks of the five simple ring compounds are sharp, indicating that the number of theoretical plates (N) is high and the resolving power is extremely excellent.

[0035]Comparative Example Polymer for Liquid Chromatography
ー filler  (Crosslinking agent) Glycerin dimethacrylate (monomer) 2-ethylhexyl methacrylate (molar ratio) Crosslinking agent: monomer = 5: 5 (polymerization method) Suspension polymerization

An aqueous solution in which polyvinyl alcohol was dissolved was charged into a polymerization reactor. Then, the purity 9
A mixed solution composed of 5% glycerin dimethacrylate, cyclohexanol and azobisisobutyronitrile was added, and the mixture was heated to 70 ° C. with stirring to perform a polymerization reaction for 24 hours.

After the completion of the reaction, the polymerization reactor was cooled to room temperature, and the product was separated. After sufficiently washing the product with water and removing cyclohexanol using acetone, the product is dried to obtain a fine porous spherical gel, and classified to a particle size of 15 to 20.
The filler having a size of μm was used as a filler of Comparative Example.

FIG. 3 shows the result of liquid chromatography using the polymer filler thus obtained. As a sample, a mixture of five kinds of monocyclic compounds was used as in the example. As is clear from the figure, the elution peak of benzene having a small molecular weight is relatively sharp, but as the molecular weight increases, the peak broadens and the resolution decreases.

Therefore, comparing the examples with the comparative examples, it can be seen that even if the filler is polymerized using the same crosslinking agent and monomer, the particle diameter of the filler is not very uniform.
It can be seen that the effect of making the micropores hydrophilic cannot be obtained.
Further, it is understood that the method for producing a polymer filler according to the present invention is preferably polymerized by a two-stage swelling polymerization method.

[0040]

The polymer filler for liquid chromatography according to the present invention is a polymer filler having a monodisperse particle diameter having a styrene skeleton, wherein the inside of the fine pores existing in the pores of the polymer filler is hydrophilic. As a result, excellent separation ability can be obtained. Further, the polymer filler for liquid chromatography according to the present invention, in the polymer filler obtained by the two-step swelling polymerization method, since the inside of the fine pores present in the pores of the polymer is made hydrophilic, Excellent resolution can be obtained. Further, a method for producing a polymer filler obtained by polymerizing glycerin dimethacrylate as a crosslinking agent and 2-ethylhexyl methacrylate as a monomer by a two-stage swelling polymerization method is provided.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a polymer filler according to the present invention.

FIG. 2 is an elution pattern obtained as a result of partitioning the column with the polymer filler obtained in the example and using a mixture of homocyclic compounds as a sample in a reversed-phase system.

FIG. 3 is an elution pattern obtained by packing a polymer filler obtained in a comparative example into a column, using a mixture of homocyclic compounds as a sample, and performing partition chromatography in a reversed-phase system.

Continued on the front page (72) Inventor Hiroshi Otsu 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture Inside Shiseido Daiichi Research Center (72) Inventor Tomohiko Kimura 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture F-term in Shiseido Daiichi Research Center (reference) 4J002 BC021 BC031 BG052

Claims (9)

[Claims] 1. A polymer filler having a particle size monodisperse polymer having a styrene skeleton, wherein the inside of micropores existing in the pores of the polymer filler is made hydrophilic. 2. The polymer filler according to claim 1, comprising a unit having the following structure. Embedded image 3. The polymer filler according to claim 2, further comprising a unit having the following structure. Embedded image (R represents a hydrocarbon group having 1 to 18 carbon atoms) 4. The polymer filler according to claim 3, wherein R is a 2-ethylhexyl group. 5. A polymer filler obtained by a two-stage swelling polymerization method, wherein the inside of micropores existing in the pores of the polymer is made hydrophilic. 6. The polymer filler according to claim 5, comprising a unit having the following structure. Embedded image 7. The polymer filler according to claim 6, further comprising a unit having the following structure. Embedded image (R represents a hydrocarbon group having 1 to 18 carbon atoms) 8. The polymer filler according to claim 7, wherein R is a 2-ethylhexyl group. 9. A method for producing a polymer filler, which is obtained by polymerizing glycerin dimethacrylate as a crosslinking agent and 2-ethylhexyl methacrylate as a monomer by a two-stage swelling polymerization method.