JPWO2019117063A1 - Porous fine particles, their production methods, chromatographic fillers and chromatographic columns - Google Patents

Abstract

An object of the present invention is to provide porous fine particles having excellent separation characteristics and an increased retention capacity, a method for producing the same, a packing material for chromatography using the same, and a column for chromatography. The porous fine particles of the present invention are porous fine particles whose surface is modified with a chemical modifier, and the inside of the pores existing in the porous fine particles is chemically modified.

Description

The present invention relates to porous fine particles, a method for producing the same, a packing material for chromatography using the same, and a column for chromatography. More specifically, the porous fine particles having excellent separation characteristics and an increased retention capacity thereof. The present invention relates to a production method, a packing material for chromatography using the same, and a column for chromatography.

High performance liquid chromatography (HPLC) is known as a technique for separating a mixture of chemical substances and exhibiting excellent performance in both analysis and purification. In HPLC, a filler (also referred to as a stationary phase or a carrier) is filled in a cylinder called a column, and a solvent (also referred to as a mobile phase) and a sample are injected into the filler to separate the components in the sample. Most of the commonly used fillers (stationary phases) are porous fine particles such as silica gel, but there are also those using organic polymer materials, and the types are diverse. In addition, there are many variations in which the surface is chemically modified. On the other hand, the mobile phase can be variously selected from liquids such as water and organic solvents. HPLC is used in a very wide range of technical fields because of its excellent separation characteristics of compounds, ease of operation, and wide range of target compounds.

In recent years, in the purification of organic electronics materials and drug substances, the purity required for the materials is extremely strict, so it is indispensable to further improve the separation ability of chromatography depending on the amount of the compound mixed with the target product. Become.

In the purification technology field where extremely high purity is required, such as the organic electronics materials and pharmaceutical drug substances, a filler (stationary phase) that does not cause separation failure even when a large amount of components are injected while reducing the amount of solvent used. ) Is required to apply the excellent separation properties of chromatography to purification operations on an industrial scale.

On the other hand, supercritical fluid chromatography (SFC) is known as an advanced technique of HPLC. A feature of SFC is that a supercritical fluid is used as the main solvent (mobile phase), and in most cases, a carbon dioxide supercritical fluid is used. In this system, the solvent (mobile phase) that has passed through the column is no longer in a supercritical state. Therefore, the supercritical carbon dioxide used as the main solvent returns to gas, and the load of solvent drying can be reduced.

Supercritical fluids have intermediate properties between gas and liquid, and diffuse much faster than ordinary solvents. In addition, it is known that the viscosity is low and the filler is densely packed, so that the separation ability is generally superior to that of HPLC. On the other hand, it has the same problems as HPLC in terms of application to the industrial scale, and causes poor separation due to an increase in the injection amount. Therefore, developing a filler (stationary phase) that does not cause separation failure even when a large amount of components are injected as in the case of HPLC applies the excellent separation characteristics of SFC to purification operations on an industrial scale. It is required to do so.

In line with the above requirements, a technique for improving separation characteristics by increasing the modification rate when chemically modifying a packing material (stationary phase) for chromatography with an organic structure has been disclosed, and chromatography in a supercritical fluid has been disclosed. A production method for chemically modifying silica gel for chromatography is disclosed (see, for example, Patent Document 1 and Patent Document 2). However, although the modification rate is superior to that of the liquid phase method, the modification rate is not sufficient, and the above-mentioned supercritical fluid chromatography

-(SFC) did not have sufficient resolution.

Japanese Unexamined Patent Publication No. 62-194457 Japanese Patent No. 2818857

The present invention has been made in view of the above problems and situations, and the problem to be solved thereof is a porous fine particle having excellent separation characteristics and an increased retention capacity, a method for producing the same, and a chromatography using the same. To provide a filler and a column for chromatography.

In the process of investigating the cause of the above problem in order to solve the above-mentioned problem, the present inventor is a porous fine particle whose particle surface is modified by a chemical modifier, and the inside of a hole existing in the porous fine particle. It has been found that the porous fine particles chemically modified to the above can provide porous fine particles having excellent separation characteristics and an increased retention capacity.

That is, the above problem according to the present invention is solved by the following means.

1. 1. Porous fine particles whose surface is modified with a chemical modifier, and the inside of the pores existing in the porous fine particles is chemically modified.

2. 2. The porous fine particles according to item 1, wherein the chemical modifying agent has a modifying group containing a 5-membered or 6-membered aromatic heterocycle or each condensed ring as a structure.

3. 3. The porous fine particles according to item 1 or 2, wherein the porous fine particles are fine particles containing a metal oxide.

4. The porous fine particles according to any one of items 1 to 3, wherein the porous fine particles are fine particles containing silicon dioxide.

5. A packing material for chromatography, which comprises the porous fine particles according to any one of items 1 to 4.

6. A column for chromatography, which comprises the porous fine particles according to any one of items 1 to 4.

7. A method for producing porous fine particles according to items 1 to 4, wherein the porous fine particles are produced.
A step of replacing the system containing the porous fine particles with a dry gas after depressurizing,
A step of reacting the porous fine particles with the chemical modifier in a supercritical fluid to modify the particles,
A method for producing porous fine particles, which comprises at least.

According to the above means of the present invention, it is possible to provide porous fine particles having excellent separation characteristics and an increased retention capacity, a method for producing the same, a packing material for chromatography using the same, and a column for chromatography.

Although the mechanism of expression or mechanism of action of the effect of the present invention has not been clarified, it is inferred as follows.

Recrystallization, filtration, distillation, sublimation, chromatography and the like are known as methods for separating a mixture of chemical substances. Recrystallization, filtration, distillation, sublimation, etc. are excellent in terms of cost and are practically used for industrial scale purification. Such an operation is not always suitable for separating the mixture, but the state on the sample side is set to a state in which impurities are less mixed from the beginning, or only those that are very easy to separate are mixed. Purification can be achieved by leaving it in a state where it does not exist. However, in the purification of organic electronics materials and drug substances, the purity requirements for the materials are very strict, and there is a problem that the required purity is not met in recrystallization, filtration, distillation, sublimation and the like.

On the other hand, the chromatography method can separate a sample with high purity without exposing it to high temperature conditions, and is widely used in the analysis and purification of a wide range of organic compounds such as pharmaceuticals, foods, and electronic device materials. ing. In the chromatography method, adsorption and desorption are repeated due to the interaction between the filler (stationary phase) filled in the column and the compound, and the number of theoretical plates is higher than that of recrystallization, filtration, distillation, sublimation, etc. Separation is possible. Therefore, the ability of the compound to be separated by the chromatography method is greatly affected by the performance of the packing material (stationary phase). Therefore, improving the performance of the filler (stationary phase) contributes to the analysis and purification of organic compounds in general.

The above-mentioned high performance liquid chromatography (HPLC) is mentioned as a technique for separating a mixture of chemical substances and exhibiting excellent performance in both analysis and purification.

HPLC is excellent in both analysis and purification and is very important on the laboratory scale, but its use on the industrial scale is particularly limited.

There are two major problems with this. One is the load of the drying process, which is derived from the fact that after separating the separated components, the large amount of solvent used must be volatilized to concentrate the target components. The other is the problem of separation accuracy on a large scale, and in general, HPLC has a problem that the components cannot be separated well when trying to separate a large amount of the mixture with respect to the amount of the filling material of the stationary phase. If this is simply solved by increasing the size of the equipment, a larger amount of solvent is consumed for operation, which leads to an increase in drying load and cost.

Further, in the purification of organic electronics materials and drug substances, the purity requirements for the materials are very strict, so that the separation and purification by HPLC has a large load as described above, and is not suitable for the industrial scale.

Therefore, in purification operations that require extremely high purity, such as organic electronics materials and pharmaceutical drug substances, the amount of solvent used should be reduced, and a filler (fixed phase) that does not cause separation failure even when a large amount of components are injected. ) Is required to apply the excellent separation properties of chromatography to purification operations on an industrial scale.

In recent years, supercritical fluid chromatography (SFC) has been known as an advanced technique of HPLC. A feature of SFC is that a supercritical fluid is used as the main solvent (mobile phase), and in most cases, a carbon dioxide supercritical fluid is used. In this system, the mobile phase that has passed through the column is no longer in a supercritical state. Therefore, the supercritical carbon dioxide used as the main solvent returns to gas, and the load of solvent drying can be reduced.

Supercritical fluids have intermediate properties between gas and liquid, and have a much higher diffusion rate than ordinary solvents.

Is fast. Further, it is known that the viscosity is low and the filler (stationary phase) is densely packed, so that the separation ability is generally superior to that of HPLC. On the other hand, it has the same problems as HPLC in terms of application to the industrial scale, and causes poor separation due to an increase in the injection amount. Therefore, developing a filler (stationary phase) that does not cause separation failure even when a large amount of components are injected as in the case of HPLC applies the excellent separation characteristics of SFC to purification operations on an industrial scale. Is required to do.

The porous fine particles of the present invention are porous fine particles that are densely chemically modified to the deep part of the pores of the porous fine particles such as silica gel, and are used, for example, as a filler (stationary phase) not only for HPLC but also for SFC. This gives excellent separation characteristics and increases the holding capacity.

In the present invention, the porous fine particles are exposed to reduced pressure conditions before being chemically modified, and then the inside of the system is filled (purged) with a dry gas so that water or the like is not adsorbed inside the pores of the porous fine particles. Create a state. After that, by subjecting it to a chemical modification reaction under supercritical conditions, the diffusivity of the supercritical fluid as a medium is fully utilized, the transportability of the reaction reagent is improved, and the porous fine particles are densely packed to the deep pores. Chemically modified porous fine particles can be provided.

Here, it will be described again that it is held in the above-mentioned column. In column chromatography, the adsorption of the filler (stationary phase) on the compound is called retention. In column chromatography, the compound moves toward the outlet of the column while repeating adsorption to the packing material (stationary phase) and elution to the solvent (mobile phase). While adsorbed on the packing material (stationary phase), the compound does not advance inside the column, but proceeds to the tip of the column when it elutes into the solvent (mobile phase). Therefore, a compound having a strong interaction, that is, a compound that has been adsorbed on the filler (stationary phase) for a long time, takes longer to pass through the column.

In FIG. 1, the time t ₁ until the compound passes through the column is called the hold time, and the time until the compound that does not interact with the filler (stationary phase) passes through the column is called the hold-up time. At this time, t ₁ ′, which is obtained by subtracting the hold-up time from the holding time, is called the space-corrected holding time, and this compound was adsorbed by the packing material (stationary phase) of the column and did not proceed in the column. It will be a net time. Further, the ratio k of the space-corrected holding time and the hold-up time is called a holding coefficient, and is an index showing how easily the compound is held in the filler (stationary phase).

Further, the meaning of the retention coefficient k will be described. Regarding the retention of the compound by the stationary phase, it can be expressed as shown in FIG. 2 assuming that the single compound is A and the retaining site of the filler (stationary phase) is B. This is just a chemical equilibrium, and the equilibrium constant K of this equilibrium can be expressed as [A + B] / [A] + [B]. Considering in correspondence with FIG. 1, t ₀ represents the time during which the compound and the filler (stationary phase) exist in the relationship of [A] + [B], and t ₁ ′ (that is, in FIG. 1, t ₁ ′ = t _{1 −} t _{0. )} Represents the time existing in the relation of [A + B]. Therefore, it can be replaced with k = t ₁ ′ / t ₀ = [A + B] / [A] + [B], which is the same as the equilibrium constant K. That is, in HPLC, the retention coefficient k gives the strength (affinity) of the interaction between the filler (stationary phase) and the compound in the form of an equilibrium constant.

In the actual measurement system, both the compound amount [A] and the retention site amount [B] of the packing material (stationary phase) are finite, and both can be settled at the ratio of the equilibrium constant for correct separation. It is necessary to have a large amount. For example, in the case of a compound having an equilibrium constant of 10000 and a filler (stationary phase), the equilibrium relationship can be correctly realized if there are 10001 and 10001 retaining sites of the compound. However, if 100,000 compounds were present for the retention site 10001, 10,000 retention sites were bound to the compound and still 900.

A state occurs in which 00 surplus compounds try to be adsorbed on the remaining one retention site, and equilibrium is not established. As a result, the balance of suction and desorption is lost, leading to reading, tailing, and broadening of the peak width, and as a result, it is presumed that separation is hindered.

Therefore, it is considered that the shape loss such as peak tailing, reading, or broadening due to overload can be solved by preparing more adsorption sites on the filler (stationary phase).

The present inventors have focused on chemical modification in a supercritical fluid as a method for imparting more modifying groups serving as adsorption sites on a filler (stationary phase). Further, as a result of the examination, as a method for introducing more modifying groups than the method disclosed in the prior art, it is effective to replace the water content in the porous fine particles with a dry gas after depressurizing the system containing the porous fine particles. I found that.

There are various possible reasons for this to improve the modification rate, but it is considered as follows.

First, a small amount of water remaining inside the pores of the porous fine particles is completely removed under reduced pressure conditions, and then the pores are replaced with a dry gas to bring the inside of the pores into a completely dry state without water. Subsequently, it is presumed that the chemical modification could be efficiently performed inside the pores by rapidly diffusing the supercritical fluid and transporting the reaction reagent by the exposure to the supercritical fluid. ..

Schematic diagram showing the time it takes for a compound to pass through a column Conceptual diagram showing how the transferred compound is adsorbed on the filler (stationary phase) Schematic diagram showing the symmetry coefficient Schematic of an apparatus using a packed column in supercritical or subcritical chromatography

The porous fine particles of the present invention are porous fine particles whose surface is modified with a chemical modifier, and the inside of the pores existing in the porous fine particles is chemically modified. This feature is a technical feature common to or corresponding to each embodiment.

As an embodiment of the present invention, from the viewpoint of exhibiting the effect of the present invention, it is preferable that the chemical modifying agent has a modifying group containing a 5-membered or 6-membered aromatic heterocycle or a condensed ring thereof as a structure. ..

Further, from the viewpoint of exhibiting the effect of the present invention, it is preferable that the porous fine particles contain a metal oxide and are fine particles containing silicon dioxide.

The chromatographic filler and the chromatographic column of the present invention are characterized by containing the porous fine particles of the present invention, which provide excellent separation characteristics and increase the holding capacity.

The method for producing porous fine particles of the present invention includes a step of replacing the system containing the porous fine particles with a dry gas after depressurizing, and modifying the porous fine particles by reacting them with the chemical modifier in a supercritical fluid. It is characterized by including at least a step.

Hereinafter, the present invention, its constituent elements, and modes and modes for carrying out the present invention will be described in detail. In the present application, "~" is used to mean that the numerical values described before and after the value are included as the lower limit value and the upper limit value.

<< Outline of the porous fine particles of the present invention >>
The porous fine particles of the present invention are porous fine particles whose surface is modified with a chemical modifier, and the inside of the pores existing in the porous fine particles is chemically modified.

The "porous fine particles" in the present invention are defined as fine particles having a large number of pores inside the fine particles and capable of adsorbing gas molecules and chemical substances in a solution by the fine particles surface and the pores. "Inside the pore" refers to the surface portion of the pore.

The "chemical modification" as used in the present invention means adsorbing a compound on the surfaces (outer surface and inner surface) of porous fine particles.

Here, the adsorption may be chemical adsorption (adsorption with a chemical bond) or physical adsorption (adsorption by physical interaction), or a combination of these. In the case of chemical adsorption, the chemical bond includes a hydrogen bond, a covalent bond, a coordination bond, an ionic bond, and the like, and further, any combination of two or more of these can be used. In the case of physical adsorption, the physical interaction includes an ion-ion interaction, a dipole interaction, Van der Waals, etc., and a combination of two or more of these. May be good.

[1] Porous fine particles The porous fine particles of the present invention are characterized in that the surface of the particles is modified with a chemical modifier, and the inside of the pores existing in the porous fine particles is chemically modified. And.

The fact that the porous fine particles of the present invention are chemically modified to the inside of the pores means that, for example, after the porous fine particles sample is dried under reduced pressure, SEM / EDS (Energy Dispersive X-ray Spectoroscopy: Energy Dispersive X-ray) It can be estimated by performing elemental analysis using an analyzer), a carbon / hydrogen / nitrogen simultaneous quantifier CHN elemental analyzer (CHN coder), etc., and measuring and comparing the carbon concentration.

The method for producing porous fine particles of the present invention includes a step of replacing the system containing the porous fine particles with a dry gas after depressurizing in order to chemically modify the inside of the pores, and the above-mentioned porous fine particles in a supercritical fluid. It is characterized by including at least a step of reacting with a chemical modifier to perform modification.

This is because the water and the like slightly remaining inside the pores of the porous fine particles are removed under reduced pressure conditions and then replaced with a dry gas, so that the inside of the pores becomes a completely water-free dry state. Subsequently, it is presumed that the chemical modification could be efficiently performed inside the pores by rapidly diffusing the supercritical fluid and transporting the reaction reagent by the exposure to the supercritical fluid. ..

The porous fine particles, which are the starting materials of the porous fine particles of the present invention, are not particularly limited as long as they are porous fine particles, but are preferably metal oxides, and examples thereof include silicon dioxide (silica gel), alumina, and zeolite. Typical examples are inorganic metal oxides. Further, a polymer material may be used, or a composite material thereof may be used as a carrier.

Further, a material obtained by chemically modifying the porous fine particles and chemically bonding a modifying group to the surface thereof, or a product obtained by further performing an end capping treatment on the chemically modified porous fine particles can also be used.

As an example of preferable porous fine particles, the silica gel generally used as a packing material for chromatography is illustrated, and a preferable specific surface area is about 1 to 1500 m ² / g.

The particle size is in the range of 1 to 1000 μm, more preferably in the range of 1 to 50 μm, and even more preferably in the range of 1 to 10 μm. The pore diameter is in the range of 1 to 100 nm, the depth is in the range of 20 to 1000 nm, the more preferred depth range is 50 to 500 nm, and the most preferred depth range is 50 to 150 nm. Further, when the pitch of the pores is defined as the distance between the centers of the pores, the preferable pitch is in the range of 2 to 500 nm, the more preferable pitch range is 2 to 100 nm, and the most preferable pitch range is 2 to 50 nm. Is.

The present invention is characterized in that the water content in the pores of the porous fine particles is replaced with a dry gas to the deep part thereof. Moisture and oxygen contained in the porous fine particles are completely removed by keeping them under reduced pressure conditions. Vacuum is preferable as the depressurizing condition.

Next, by being replaced by a dry gas, the inside of the pores becomes completely dry without moisture. As the dry gas, those preferably inactive with respect to the reaction reagent, the stationary phase carrier, the solvent used as the reaction medium or the supercritical fluid, and nitrogen, carbon dioxide, argon and the like are preferably used. It is not limited. Of these, nitrogen is preferable from the viewpoint of handleability.

Subsequently, by being exposed to the supercritical fluid, the diffusion of the supercritical fluid and the rapid transport of the reaction reagent by the diffusion proceed, so that the chemical modification can be efficiently performed even inside the pores. The supercritical fluid used is not particularly limited as long as it takes a supercritical state, but a fluid having poor reactivity is preferable due to the property of chemically modifying an organic substance. For example, carbon dioxide, ethane, propane, butane, benzene, diethyl ether and the like can be mentioned, and carbon dioxide is particularly preferable because the pressure and temperature at the critical point are low. Each of these supercritical fluids may be used alone, or may be used as a modifier in a mixed state of various solvents.

The supercritical fluid that can be used in the present invention can be used if the critical temperature is 400 ° C. or lower, but 300 ° C. or lower, more preferably 200 ° C. or lower, and most preferably 100 ° C. or lower to avoid side reactions. It is preferable that the critical pressure is 300 kg / cm ² or less, more preferably 200 kg / cm ² or less, and most preferably 100 kg / cm ² or less. Examples of supercritical fluids suitable for the present invention that meet these conditions include carbon dioxide, dinitrogen monoxide, ethane, propane, butane, benzene, diethyl ether, and the like, and in particular carbon dioxide (critical temperature 31.3 ° C.). , Critical pressure 72.9 atm (75.3 kg / cm ² ) is preferred. Each of these supercritical fluids can be used alone or as a modifier such as methanol or toluene to improve the solubility of the chemical modifier. It can also be used as a mixed fluid with.

[2] Chemical modifier The structure of the modifying group in the chemical modifier according to the present invention is not particularly limited, but a structure containing an alkyl group such as an octadecyl group and a heteroalkyl group such as an aminopropyl group, an aromatic hydrocarbon group, and the like. Examples thereof include a structure containing an aromatic heterocyclic group, and particularly when separating an organic electronic material, an aromatic hydrocarbon group having a conjugated π electron or a structure containing an aromatic heterocyclic group is preferable. For example, carbazolyl group, azacarbazolyl group, phenanthrenyl group, phenanthrolinyl group, benzofuranyl group, dibenzofuranyl group, azadibenzofuranyl group, thiophenyl group, benzothiophenyl group, azadibenzothiophenyl group, dibenzothiophenyl group, Examples thereof include, but are not limited to, a pyridyl group, a pyrimidinyl group, a triazinyl group, an indolyl group, a benzoimidazolyl group, a benzotriazolyl group, an imidazolyl group, a triazolyl group and a benzothiazolyl group.

The chemical modifier according to the present invention is a 5- or 6-membered aromatic heterocycle, or a condensation of each.

It is preferable to have a modifying group containing a ring as a structure. Further, the chemical modifier preferably has a plurality of condensed aromatic heterocycles, and may further have a plurality of aromatic heterocycles.

Above all, it is preferable that the chemical modifying agent has a modifying group containing a structure represented by the following general formulas (a-1) to (a-16).

In the formula, X independently represents a carbon atom or a hetero atom, and at least one is preferably a hetero atom, and as the hetero atom, an oxygen atom, a nitrogen atom, and a sulfur atom are particularly preferably used. M represents a metal atom, and Ir and Pt are particularly preferably used. n represents an integer of 1 to 3. Each X may have a substituent independently or may form a ring with each other. Further, a ring may be formed between the substituents of X.

As a modifying group containing the structures represented by the general formulas (a-1) to (a-16), a more preferable modifying group is a structure represented by the following general formulas (b-1) to (b-24). Examples include modifying groups containing.

In the formula, Y independently represents either a carbon atom or a nitrogen atom, and W independently represents a hetero atom, and as the hetero atom, any one of nitrogen atom, sulfur atom, and oxygen atom is particularly preferably used. Be done. M represents a metal atom, and Ir and Pt are particularly preferably used. n represents an integer of 1 to 3. In the general formula (b-24), at least one of Y represents a heteroatom. Each Y may have a substituent independently, or may form a ring with each other. Further, a ring may be formed between the substituents of Y. R represents a substituent, and the substituents may form a ring with each other.

Examples of the substituent represented by R include a hydrogen atom and an alkyl group (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a (t) butyl group, a pentyl group, a hexyl group, an octyl group and a dodecyl group. Tridecyl group, tetradecyl group, pentadecyl group, etc.), cycloalkyl group (eg, cyclopentyl group, cyclohexyl group, etc.), alkenyl group (eg, vinyl group, allyl group, etc.), alkynyl group (eg, propargyl group, etc.), aromatic Hydrocarbon ring group (also called aryl group, for example, phenyl group, p-chlorophenyl group, mesityl group, tolyl group, xsilyl group, naphthyl group, anthryl group, azulenyl group, acenaphthenyl group, fluorenyl group, phenanthryl group, indenyl group, Pyrenyl group, biphenylyl group, etc.), heterocyclic group (eg, epoxy ring, aziridine ring, thiirane ring, oxetane ring, azetidine ring, thietan ring, tetrahydrofuran ring, dioxolan ring, pyrrolidine ring, pyrazolidine ring, imidazolidine ring, oxazolidin ring , Tetrahydrothiophene ring, sulfolan ring, thiazolidine ring, ε-caprolactone ring, ε-caprolactam ring, piperidine ring, hexahydropyridazine ring, hexahydropyrimidine ring, piperazine ring, morpholin ring, tetrahydropyran ring, 1,3-dioxane ring. , 1,4-dioxane ring, trioxane ring, tetrahydrothiopyran ring, thiomorpholin ring, thiomorpholin-1,1-dioxide ring, pyranose ring, diazabicyclo [2,2,2] -octane ring, etc.), aromatic complex Ring group (pyridyl group, pyrimidinyl group, furyl group, pyrrolyl group, imidazolyl group, benzoimidazolyl group, pyrazolyl group, pyrazinyl group, triazolyl group (for example, 1,2,4-triazol-1-yl group, 1,2,3) -Triazole-1-yl group, etc.), oxazolyl group, benzoxazolyl group, thiazolyl group, isooxazolyl group, isothiazolyl group, frazayl group, thienyl group, quinolyl group, benzofuryl group, dibenzofuryl group, benzothienyl group, dibenzothienyl Group, indrill group, indroindrill group, carbazolyl group, carbolinyl group, diazacarbazolyl group (indicating one of the carbon atoms constituting the carbolin ring of the carbolinyl group replaced with a nitrogen atom), quinoxalinyl group, Pyridadinyl group, triazinyl group, quinazolinyl group, phthalazinyl group, etc.), halogen atom (for example, chlorine atom, bromine atom) , Iodine atom, fluorine atom, etc.), alkoxyl group (eg, methoxy group, ethoxy group, propyloxy group, pentyloxy group, hexyloxy group, octyloxy group, dodecyloxy group, etc.), cycloalkenyl group (eg, cyclopentyloxy) Group, cyclohexyloxy group, etc.), aryloxy group (eg, phenoxy group, naphthyloxy group, etc.), alkylthio group (eg, methylthio group, ethylthio group, propylthio group, pentylthio group, hexylthio group, octylthio group, dodecylthio group, etc.) , Cycloalkylthio group (eg, cyclopentylthio group, cyclohexylthio group, etc.), arylthio group (eg, phenylthio group, naphthylthio group, etc.), alkoxycarbonyl group (eg, methyloxycarbonyl group, ethyloxycarbonyl group, butyloxycarbonyl group, etc.) , Octyloxycarbonyl group, dodecyloxycarbonyl group, etc.), aryloxycarbonyl group (eg, phenyloxycarbonyl group, naphthyloxycarbonyl group, etc.), sulfamoyl group (eg, aminosulfonyl group, methylaminosulfonyl group, dimethylaminosulfonyl group, etc.) , Butylaminosulfonyl group, hexylaminosulfonyl group, cyclohexylaminosulfonyl group, octylaminosulfonyl group, dodecylaminosulfonyl group, phenylaminosulfonyl group, naphthylaminosulfonyl group, 2-pyridylaminosulfonyl group, etc.), ureido group (eg, methyl Ureid group, ethyl ureido group, pentyl ureido group, cyclohexyl ureido group, octyl ureido group, dodecyl ureido group, phenyl ureido group, naphthyl ureido group, 2-pyridyl amino ureido group, etc.), acyl group (for example, acetyl group, ethyl carbonyl group) , Apropylcarbonyl group, pentylcarbonyl group, cyclohexylcarbonyl group, octylcarbonyl group, 2-ethylhexylcarbonyl group, dodecylcarbonyl group, phenylcarbonyl group, naphthylcarbonyl group, pyridylcarbonyl group, etc.), acyloxy group (eg, acetyloxy group, Ethylcarbonyloxy group, butylcarbonyloxy group, octylcarbonyloxy group, dodecylcarbonyloxy group, phenylcarbonyloxy group, etc.), amide group (for example, methylcarbonylamino group, ethylcarbonylamino group, dimethylcarbonylamino group, propylcarbonylamino) Group, Pentyl Carbonyl Luamino group, cyclohexylcarbonylamino group, 2-ethylhexylcarbonylamino group, octylcarbonylamino group, dodecylcarbonylamino group, phenylcarbonylamino group, naphthylcarbonylamino group, etc.), carbamoyl group (for example, aminocarbonyl group, methylaminocarbonyl group) , Dimethylaminocarbonyl group, propylaminocarbonyl group, pentylaminocarbonyl group, cyclohexylaminocarbonyl group, octylaminocarbonyl group, 2-ethylhexylaminocarbonyl group, dodecylaminocarbonyl group, phenylaminocarbonyl group, naphthylaminocarbonyl group, 2- Pyridylaminocarbonyl group, etc.), sulfinyl group (eg, methylsulfinyl group, ethylsulfinyl group, butylsulfinyl group, cyclohexylsulfinyl group, 2-ethylhexylsulfinyl group, dodecylsulfinyl group, phenylsulfinyl group, naphthylsulfinyl group, 2-pyridylsulfinyl group Etc.), alkylsulfonyl group or arylsulfonyl group (eg, methylsulfonyl group, ethylsulfonyl group, butylsulfonyl group, cyclohexylsulfonyl group, 2-ethylhexylsulfonyl group, dodecylsulfonyl group, phenylsulfonyl group, naphthylsulfonyl group, 2-pyridyl (Sulfonyl group, etc.), amino group (eg, amino group, ethylamino group, dimethylamino group, butylamino group, cyclopentylamino group, 2-ethylhexylamino group, dodecylamino group), anilino group, diarylamino group (eg, diphenyl) Amino group, dinaphthylamino group, phenylnaphthylamino group, etc.), naphthylamino group, 2-pyridylamino group, etc.), nitro group, cyano group, hydroxy group, mercapto group, alkylsilyl group or arylsilyl group (for example, trimethylsilyl group) , Triethylsilyl group, (t) butyldimethylsilyl group, triisopropylsilyl group, (t) butyldiphenylsilyl group, triphenylsilyl group, trinaphthylsilyl group, 2-pyridylsilyl group, etc.), alkylphosphino group or aryl Phosphino group (dimethylphosphino group, diethylphosphino group, dicyclohexylphosphino group, methylphenylphosphino group, diphenylphosphino group, dinaphthylphosphino group, di (2-pyridyl) phosphosphino group), alkylphosphoryl group or Arylphosphoryl group (dimethylphosphoryl group, Diethylphosphoryl group, dicyclohexylphosphoryl group, methylphenylphosphoryl group, diphenylphosphoryl group, dinaphthylphosphoryl group, di (2-pyridyl) phosphoryl group), alkylthiophosphoryl group or arylthiophosphoryl group (dimethylthiophosphoryl group, diethylthiophosphoryl group) , Dicyclohexylthiophosphoryl group, methylphenylthiophosphoryl group, diphenylthiophosphoryl group, dinaphthylthiophosphoryl group, di (2-pyridyl) thiophosphoryl group). In addition, these substituents may be further substituted by the above-mentioned substituents, or they may be condensed with each other to further form a ring.

Further, an alkyl group, an aromatic hydrocarbon ring group, an aromatic heterocyclic group, a heterocyclic group, and a cycloalkyl group are preferable.

Hereinafter, specific examples of modifying groups including structures represented by the general formulas (a-1) to (a-16) and the general formulas (b-1) to (b-24) will be illustrated. In (c-1) to (c-115), − * represents a connection point with a linker portion described later.

As the modifying group according to the present invention, a structure containing a heteroaryl group is suitable. In the present invention, such a structure in which a ring containing a heteroaryl and a metal atom form a complex is also regarded as a condensed aromatic heterocycle. To give an example of such a structure, for example, in a structure such as tris (2-phenylpyridinato) iridium, since three phenylpyridine ligands form a complex with a metal, as a whole, Considered as one heteroaryl ring. Other such structures include tris (8-quinolinolato) aluminum, 8-hydroxyquinolinolatritium, tris (2,2'-bipyridyl) ruthenium dichloride, and tris [1-phenylisoquinoline-C ² , N] iridium. Although it is also found in complex compounds, it is not limited to the structures exemplified above.

These chemically modifying agents or modifying groups can be synthesized by known methods.

It is also preferable that the porous fine particles are surface-modified in advance. That is, it may be chemically modified in two steps.

Specifically, it is preferable that the modifying group is chemically bonded to the porous fine particles that have been surface-modified by the surface modifying group. Further, it is also possible to use a porous fine particle chemically modified with a surface modifying group and further subjected to an end capping treatment.

Examples of the end capping agent include disilazan compounds such as hexamethyldisilazane, hydrogensilane compounds such as diethylmethylsilane and triethylsilane, alkoxysilane compounds such as trimethylmethoxysilane, pentamethyldisiloxane and hexamethylcyclotrisiloxane, 1, A siloxane compound such as 1,3,3-tetramethyldisiloxane, a chlorosilane compound such as trimethylchlorosilane, and the like can be preferably used.

It is also preferable that the porous fine particles are surface-modified in advance with a chemical modifier having a chemical structure different from that of the chemical modifier. As a result, for example, by keeping the test material away from the residual silanol groups of silica gel, the effect of suppressing the interaction with the silanol groups that the test material does not anticipate and promoting the interaction with the modifying group can be obtained.

Further, it is also a preferable embodiment that the above-mentioned modifying group and the surface-modified porous fine particles have a structure in which they are bonded via a divalent linking group.

Assuming that the group that surface-modifies the porous fine particles is bonded to the modifying group according to the present invention via a divalent linking group or a single bond, the following general formula (d-1) is used. Alternatively, it preferably has a structure represented by the general formula (e-1).

In the general formula (d-1), L ₁ and L ₂ represent a single bond or a divalent linking group, and m and o represent integers of 0 to 5, respectively. Ar is an aromatic hydrocarbon and represents a divalent linking group.

The divalent linking group represented by L ₁ and L ₂ includes a substituted or unsubstituted alkylene group, an arylene group or a heteroarylene group, −O−, −S−, −S (= O) ₂₋ , −. Examples thereof include N (R)-, a carboxyl ester group, a ketone carbonyl group, a sulfonicyl group, a carbamoyl group, a sulfamoyl group, an amide group and the like.

In the general formula (e-1), L ₁ represents a single bond or a divalent linking group, and m and o represent integers of 0 to 5, respectively. Ar represents a divalent linking group that is an aromatic hydrocarbon.

In formula (e-1), L ₁ has the same meaning as L ₁ in the general formula (d-1).

Examples of the divalent linking group which is an aromatic hydrocarbon represented by Ar include ortho, para or metaphenylene, naphthylene, biphenylene, anthraceneylene, terphenylene and the like.

R ₁ represents an element that independently connects to the porous fine particles, and carbon, oxygen, or nitrogen is particularly preferably used.

Specific structures of pre-surface-modified porous fine particles having a divalent linking group preferably used in the present invention are exemplified below (f-1) to (f-15), but the present invention is limited thereto. It is not something that is done. In (f-1) to (f-15), * represents a connecting portion with the modifying group, and the wavy line portion on the left side of the structural formula is a schematic diagram showing a connecting portion in which Si is bonded to porous fine particles. is there.

The concentration of the chemical modifier in the present invention with respect to the supercritical fluid can be appropriately selected and is not particularly limited, but is preferably 0.01 to 20% (volume ratio), particularly 0.1 to 10% (volume ratio). Is preferable.

The reaction temperature in the chemical modification reaction can be appropriately selected depending on the type of the porous fine particles and the chemical modification agent, and is not particularly limited as long as it is equal to or higher than the critical temperature of the supercritical fluid, but if the temperature becomes too high, chemical bonding has already occurred. If a chemically modifying group is present, its desorption may increase, so the upper limit thereof is preferably 400 ° C. The upper limit of the more preferable reaction temperature is 300 ° C., and the most preferable upper limit of the reaction temperature is 250 ° C.

There is no problem if the pressure of the supercritical fluid in the chemical modification reaction is equal to or higher than the critical pressure, and it can be appropriately selected according to the type and concentration of the chemical modification agent. The critical pressure is usually ~ 100 kg / cm ² . The reaction time is preferably 30 minutes to 120 hours, more preferably 2 to 24 hours. The flow rate of the fluid in the reaction vessel is not particularly limited, but is preferably 5 mL or less per minute in the supercritical fluid state.

[3] Filler With respect to the packing material for chromatography obtained by the present invention, residual active groups that have not been completely reacted by the present invention may be used by end-capping with different modified structures. Further, the operation of replacing the fine particles with a dry gas may be performed at the time of end capping. The structure of the endcapping may be any one that can react with the residual active group on the particles, and for example, an alkylsilyl group, an acyl group, an alkoxyl group, an alkylamino group and the like having a small exclusion volume are particularly preferable. It is not limited.

The filler obtained by the present invention is preferably used as a filler for column chromatography, particularly as a filler for high performance liquid chromatography or a filler for supercritical fluid chromatography, but is not limited thereto. For example, it may be used as a carrier or an adsorbent for thin layer chromatography, or may be used in the form of a filtration aid or a filter.

[4] Column The chromatographic column of the present invention is characterized by containing the porous fine particles of the present invention, and is used for column chromatography.

The column of the present invention has the effect of reducing so-called reading, tailing, and peak width broadening.

Usually, with respect to reading and tailing, a symmetry coefficient can be mentioned as an index showing the performance of a column in chromatography. The symmetry coefficient (tailing coefficient) is an index showing the degree of symmetry of the peak, and is represented by the following formula in FIG.

(Symmetry coefficient: S)
S = W (0.05h) / 2f
W (0.05h): Peak width at a height of 1/20 of the peak height h from the peak baseline)
f: The peak width of W (0.05h) is divided into two by a perpendicular line extending from the peak apex to the horizontal axis. If the distance S = 1 on the peak rising side, the peak is symmetrical and shows an ideal shape as a peak. .. When S> 1, the peak has a shape bulging to the right, that is, a tailing shape. When S <1, the peak has a shape bulging to the left, that is, a leading shape. Therefore, the closer S is to 1, the better the performance as a column. The symmetry coefficient of the column of the present invention is preferably 0.70 <S <1.30, more preferably 0.80 <S <1.20, and 0.85 <S <1.15. It is more preferable to have.

[4.1] Column chromatography (hereinafter, also referred to as "chromatography")
The most typical aspect of column chromatography according to the present invention is to use the porous fine particles of the present invention as a filler (stationary phase), adsorb compound A there, and use it as a mobile phase (solvent or eluent). In B), it is gradually eluted.

At this time, when the interaction (adsorption) between the modifying group existing on the surface of the porous fine particles and the inside of the pores and the compound A is antagonized by the interaction with the mobile phase (B), A is the porous fine particles. The adsorption-desorption equilibrium is repeated with the mobile phase B, and when the interaction with the porous fine particles is small, it is fast, and when the interaction is large, it is slow and elutes.

At this time, the larger the number of round trips between adsorption and desorption equilibrium, the greater the number of theoretical plates (that is, purification efficiency). Therefore, the purification efficiency by the chromatographic method is proportional to the length of the packing material (stationary phase), and the mobile phase It is proportional to the passing speed and also to the surface area of the filler (stationary phase).

High performance liquid chromatography has made this possible, and it is a rare method that can realize a high number of theoretical plates backed by this theory, which is widely used for component analysis and quality assurance of organic compounds. It is due to the fact that.

The reason why this chromatographic method is superior to recrystallization is that the polarity of mobile phase B can be changed arbitrarily. For example, the mobile phase may be a mixed solvent of a good solvent and a poor solvent from the beginning, and the number of theoretical plates can be further increased by using a gradient method in which the ratio of good solvents is gradually increased during purification.

In addition, since the temperature can be changed arbitrarily, the range of application of the solute that can be purified is extremely wide, and the greatest feature is that it can be used as an almost general-purpose purification method.

On the other hand, there are also drawbacks of the chromatographic method. As mentioned above, the fundamental principle for increasing the number of theoretical plates is to utilize the adsorption-desorption equilibrium.

For example, when the chromatography method is performed using only the solvent B'(that is, a good solvent) that strongly interacts with the compound A in the mobile phase, A and the mobile phase B'are more than the interaction between A and the porous fine particles. If the interaction between the two is strong, the number of round trips of the adsorption-desorption equilibrium is drastically reduced, and the purification effect is lowered.

That is, in order to enhance the purification effect, it is necessary to mix a large excess of the poor solvent C in addition to the good solvent B'to increase the number of round trips of the adsorption-desorption equilibrium. However, in this case, the purified and fractionated solution of compound A contains a large excess of C, and the biggest problem is that this must be concentrated.

For example, in order to obtain 1 g of A, the mixing ratio of the good solvent B'and the poor solvent C needs to be about 1:99 to 10:90, and generally about 10 L to 100 L of the poor solvent C is used. It will be necessary. Therefore, although HPLC preparative is applied to research and development, it is not used for mass production.

A means for solving the problem of poor solvent concentration is supercritical fluid chromatography (SFC), which is HPLC using supercritical carbon dioxide.

Supercritical carbon dioxide is carbon dioxide converted into supercritical fluid at high temperature and high pressure, and other substances can be made into such supercritical fluid, but it is in a supercritical state at relatively low pressure and temperature. Carbon dioxide is used exclusively for chromatographing and extraction.

This supercritical carbon dioxide has characteristics different from those of ordinary fluids and liquids. That is, by changing the temperature and pressure, the polarity can be continuously changed according to the polarity of what is desired to be melted.

For example, this supercritical carbon dioxide is also used when selectively extracting docosahexaenoic acid contained in fish heads, and when cleaning special clothing using an adhesive, the sebum is melted and adhered. Supercritical carbon dioxide, which does not dissolve, is used as the agent.

Although supercritical carbon dioxide can have various polarities in this way, the polarity of supercritical carbon dioxide formed in a region of relatively low temperature and pressure is about cyclohexane or heptane. In supercritical fluid chromatography (SFC) currently on the market, supercritical carbon dioxide of this degree of polarity is produced in the instrument, mixed with a good solvent and entered into the column, similar to normal HPLC. The compound is purified by the mechanism of.

In a column chromatography system using supercritical carbon dioxide, it enters the detector after passing through the column, but normally the high temperature and high pressure state is maintained until that stage, and carbon dioxide also exists as a supercritical fluid. After that, carbon dioxide becomes a gas until it is separated at normal temperature and pressure, and at the time of separation, it escapes from the solution by itself, so that concentration of a poor solvent becomes unnecessary. At this time, carbon dioxide can be recovered by a carbon dioxide recovery device equipped with a gas-liquid separation mechanism or the like described in References (Journal of the Society of Biotechnology, Vol. 88, No. 10, pp. 525-528, 2010). It can also be used as a supercritical fluid again.

Therefore, in the drug discovery industry, which requires the synthesis of a large number of new high-purity synthetic compounds, this supercritical fluid chromatography (SFC) has recently been actively utilized, and due to its influence, it is used for analysis. The selling price has dropped for both preparative use, and it has become quite common.

[4.2] Supercritical or subcritical chromatography method In the supercritical chromatography method, not only a supercritical fluid but also a subcritical fluid can be used. As the column used in the supercritical or subcritical chromatography method, a packed column, an open column, or a capillary column can be used.

<Column for chromatography>
The chromatographic column of the present invention is a column having the filler of the present invention capable of separating the target substance in the sample injected into the mobile phase.

As the filler, the porous fine particles of the present invention are used. It may be stored in the column in a state of being supported by an integrated filler housed in the column, or may be stored in the column as an integrated molded product made of the filler.

In the chromatography column of the present invention, as shown in FIG. 4, for example, a supercritical fluid 11 containing an organic solvent (including carbon dioxide), a pump 12, a modifier 13, and an injector 14 for injecting an organic compound to be separated are injected. , A device including a separation column 15, a detector 17, a pressure regulating valve 18, and the like can be used.

The temperature of the column 15 is adjusted in the column oven 16. As the filler, the porous fine particles of the present invention are used.

In the present invention, the supercritical fluid is a substance in a supercritical state.

Here, the supercritical state will be described. A substance changes between three states of gas, liquid and solid due to changes in environmental conditions such as temperature and pressure (or volume), which is determined by the balance between intermolecular force and kinetic energy. The horizontal axis represents temperature and the vertical axis represents the transition of gas-liquid solid state, which is called a state diagram (phase diagram). In it, the three phases of gas, liquid, and solid coexist and are in equilibrium. The point in is called the triple point. If the temperature is higher than the triple point, the liquid and its vapor are in equilibrium. The pressure at this time is the saturated vapor pressure and is represented by the evaporation curve (vapor pressure line). If the pressure is lower than the pressure represented by this curve, all the liquid is vaporized, and if a pressure higher than this is applied, all the vapor is liquefied. Even if the pressure is kept constant and the temperature is changed, when this curve is exceeded, the liquid becomes vapor and the vapor becomes liquid. This evaporation curve has an end point on the high temperature and high pressure side, and this is called a critical point. The critical point is an important point that characterizes a substance, and the interface between gas and liquid disappears in a state where liquid and vapor cannot be distinguished.

In a state higher than the critical point, it is possible to move between a liquid and a gas without causing a gas-liquid coexistence state.

A fluid that is above the critical temperature and above the critical pressure is called a supercritical fluid, and a temperature / pressure region that gives the supercritical fluid is called a supercritical region. Further, a state in which either the critical temperature or higher or the critical pressure or higher is satisfied is called a subcritical (expanded liquid) state, and a fluid in the subcritical state is called a subcritical fluid. Supercritical fluids and subcritical fluids are high-density fluids with high kinetic energy, exhibit liquid behavior in terms of dissolving solutes, and exhibit gaseous characteristics in terms of density variability. There are various solvent characteristics of supercritical fluids, but it is important that they have low viscosity, high diffusivity, and excellent permeability to solid materials.

In the supercritical state, for example, in the case of carbon dioxide, the critical temperature (hereinafter, also referred to as Tc) is 31 ° C., the critical pressure (hereinafter, also referred to as Pc) is 7.38 × 10 ⁶ Pa, and propane (Tc = 96.7). ° C., Pc = 43.4 × 10 ⁵ Pa), ethylene (Tc = 9.9 ° C., Pc = 52.2 × 10 ⁵ Pa), etc. Above this region, the fluid has a large diffusion coefficient and low viscosity, and is a substance. Efficient separation is possible because of the rapid transfer and arrival of concentration equilibrium and the high density of liquid. Moreover, recovery can be speeded up by using a substance that becomes a gas at normal pressure and room temperature, such as carbon dioxide. In addition, there are no various obstacles due to the residual trace amount of solvent that is unavoidable in the purification method using a liquid solvent.

As the solvent used as the supercritical fluid or the subcritical fluid, carbon dioxide, dinitrogen monoxide, ammonia, water, methanol, ethanol, 2-propanol, ethane, propane, butane, hexane, pentane and the like are preferably used. Of these, carbon dioxide can be preferably used.

One type of solvent used as a supercritical fluid or a subcritical fluid can be used alone, or a substance called a modifier (entrainer) for adjusting the polarity can be added.

Examples of the modifier include hydrocarbon solvents such as hexane, cyclohexane, benzene and toluene, halogenated hydrocarbon solvents such as methyl chloride, dichloromethane, dichloroethane and chlorobenzene, and alcohol solvents such as methanol, ethanol, propanol and butanol. , Ethyl solvent such as diethyl ether and tetrahydrofuran, acetal solvent such as acetaldehyde diethyl acetal, ketone solvent such as acetone and methyl ethyl ketone, ester solvent such as ethyl acetate and butyl acetate, carboxylic acid, acetic acid, trifluoroacetic acid and the like. Examples thereof include acid-based solvents, nitrogen-based solvents such as acetonitrile, pyridine, N, N-dimethylformamide, sulfur-based solvents such as carbon disulfide and dimethyl sulfoxide, and water, nitric acid, sulfuric acid and the like.

The operating temperature of the supercritical fluid or subcritical fluid is basically not particularly limited as long as it is above the temperature at which the organic compound used as the solute according to the present invention dissolves, but if the temperature is too low, it is in a supercritical fluid state or The operating temperature is preferably in the range of 20 to 600 ° C. because it becomes difficult to maintain the subcritical fluid state and the organic compound may be decomposed if the temperature is too high.

The working pressure of the supercritical fluid or subcritical fluid is not particularly limited as long as it is equal to or higher than the critical pressure of the substance to be used, but if the pressure is too low, the supercritical fluid of the organic compound used as the solute according to the present invention or The working pressure is 1 to 100 MPa because the solubility in subcritical fluid may be poor and if the pressure is too high, problems may occur in terms of durability of the manufacturing equipment and safety during operation. It is preferably within the range.

The device using the supercritical fluid or the subcritical fluid is limited as long as the organic compound used as the solute has a function of contacting the supercritical fluid or the subcritical fluid and dissolving in the supercritical fluid or the subcritical fluid. For example, a batch method in which a supercritical fluid or a subcritical fluid is used in a closed system, a distribution method in which a supercritical fluid or a subcritical fluid is circulated and used, or a composite method in which a batch method and a distribution method are combined. Etc. can be used.

In the supercritical or subcritical chromatography method according to the present invention, after injecting a sample into the mobile phase, the following before the tailing of the peak of the target substance having the slowest elution from the column is completely attenuated is as follows. It is preferable to inject the sample.

At this time, after injecting the sample into the mobile phase, the composition of the mobile phase may be changed or the composition may be constant. In particular, when a large amount of the compound to be separated is to be separated, the composition of the mobile phase can be changed.

The step of changing the composition of the mobile phase is to change the composition of the mobile phase containing a supercritical or subcritical fluid and a solvent. By changing the composition of the mobile phase by this step, the attenuation of peak tailing can be accelerated. Column adsorption supercritical or subcritical fluid chromatography shows tailing with marked peaks, especially when performing preparative operations to load relatively large amounts of compounds to be separated. If the next sample is injected before the tailing is attenuated, the tailing component will be mixed with the peak component of the next injected sample, and the purity of the separated compound will decrease, resulting in inconvenience. Therefore, the next sample must be injected after waiting for the tailing to completely decay. Therefore, it is possible to accelerate the timing of the next sample injection by accelerating the attenuation of tailing. However, in the present invention, by changing the composition of the mobile phase, the extruding of the peak component from the column is promoted, and tailing is performed. Can accelerate the decay of.

Changing the composition in the mobile phase produces the same effect as the step gradient method in liquid chromatography, and promotes the extrusion of peak components from the column, thereby accelerating the attenuation of tailing.

Since supercritical or subcritical chromatography uses a supercritical or subcritical fluid having high diffusivity and low viscosity, the flow velocity of the mobile phase is high and the column equilibration is quick. Therefore, even if the composition in the mobile phase changes temporarily, the column quickly restores to the environment before the change when the composition in the mobile phase is restored. Therefore, the next sample immediately after the tailing is attenuated. Can be injected. As a result, the amount of sample processed per hour can be increased, and efficiency and productivity are improved.

The step of changing the composition of the mobile phase may be any method as long as it can be performed by a supercritical or subcritical chromatography apparatus. For example, by increasing the solvent ratio in the mobile phase, the composition of the mobile phase can be changed, and by significantly changing the pressure and column temperature, the CO ₂ density in the mobile phase can be changed. , These are included in the composition change of the mobile phase.

The solvent is already contained in the mobile phase, but in addition to the solvent contained in the mobile phase, a solvent injection device is provided upstream of the column and downstream of the mobile phase generator to increase the solvent ratio in the mobile phase. Can be made to. The solvent injection device can be, for example, a solvent injection device including a loop pipe for holding the solvent to be injected, a flow path switching valve, and a solvent injection pump.

The loop pipe used in the solvent injection device is a pipe having a predetermined volume. Having a loop pipe is preferable because the quantitativeness of sample injection is improved and a larger amount of sample can be injected. In the present invention, the volume of the loop pipe varies depending on the conditions such as the type of column used in the supercritical or subcritical chromatography apparatus, the inner diameter of the column, the type of the target substance, the composition of the mobile phase, etc., but at once. Since it is necessary to inject a large amount of solvent, the loop pipe of the solvent injection device is larger than the loop pipe of the sample injection device, and one capable of holding a large amount of solvent is suitable.

The flow path switching valve used in the solvent injection device is not particularly limited as long as it is an openable / closable valve or cock provided in the flow path of the mobile phase. For example, a valve that uses a combination of a two-way valve and a butterfly valve, or a valve that switches the flow path of the mobile phase by using a three-way valve can be mentioned. As the solvent injection pump used in the solvent injection device, a high-pressure pump used for sample injection of a supercritical or subcritical chromatography device can be used.

When a solvent injection device is used, the solvent is injected by switching the flow path switching valve and sending the solvent to the mobile phase of the column by the solvent injection pump. It is preferable to inject the solvent instantaneously by the injection volume of the sample or more, preferably 2 times or more, more preferably 5 times or more. As the upper limit value, it is preferable to inject a solvent 30 times or less, preferably 20 times or less, more preferably 15 times or less the injection volume of the sample. With such a solvent injection amount, the attenuation of peak tailing is further accelerated.

The solvent injected from the solvent injection device is not particularly limited, and may be, for example, the same solvent as the solvent contained in the mobile phase or a different solvent. Further, the solvent to be injected may be one kind or two or more kinds.

In particular, a highly polar solvent is preferred in that it further accelerates tailing decay. Further, it is preferable to use a solvent having a higher polarity than the solvent contained in the mobile phase.

It is preferable that both the step of changing the composition of the mobile phase and the step of returning the composition of the mobile phase to before the change are performed instantaneously. The moment mentioned here may be a time sufficient to cause a change in the mobile phase.

The method of peak detection is not particularly limited, but the timing can be measured by a peak detected by a detector usually possessed by supercritical or subcritical chromatography, for example, an ultraviolet absorption spectrometer.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. In the examples, the indication of "parts" or "%" is used, but unless otherwise specified, it indicates "parts by mass" or "% by mass".

Example 1
<Preparation of porous fine particles 1-1>
² g of spherical silica gel (Lichospher-NH ₂ , registered trademark, manufactured by Merck) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, a specific surface area of 350 m ² / g, and an aminopropyl group modification degree of 1.44 mmol / g is placed in a glass container. , There 3- (carbazole-9-yl) benzoic acid (2.87 g, 10 mmol), EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (3.10 g, 20 mmol), tetrahydrofuran (30 mL) ) Was added. This mixed solution was immersed in an ultrasonic generator, and the reaction was carried out for 4 hours while applying ultrasonic waves. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 1-1 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 1-2>
Hereinafter, the production of the porous fine particles of the present invention will be specifically described. The reaction between the porous fine particles and the chemical modifier in the supercritical fluid was carried out using ACQUITYUPC2 (registered trademark) manufactured by Japan Waters Corp.

₂ g of spherical silica gel (Lichospher-NH ₂ , registered trademark, manufactured by Merck Co., Ltd.) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, and an aminopropyl group modification degree of 1.44 mmol / g is vacuum-dried at 110 ° C. It was moved into the glove box and the container was purged with dry nitrogen. After transferring this silica gel to the reaction vessel in the glove box and installing it in the apparatus, 3- (carbazole-9-yl) benzoic acid (2.87 g, 10 mmol) and EDC: 1-ethyl-3 were added to the reaction vessel. A mixed solution of − (3-dimethylaminopropyl) carbodiimide (3.10 g, 20 mmol) in 50 mL of tetrahydrofuran was used as a modifier so that it would be 3.0% (volume ratio) with respect to the supplied carbon dioxide. Then, the reaction was carried out by supplying the reaction tank to the reaction vessel under the conditions of a reaction temperature of 60 ° C., a fluid pressure of 250 kg / cm ² , and a flow rate of 0.15 mL / min. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 1-2 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 1-3>
The porous fine particles 1-3, which are the packing materials for chromatography of the comparative example, were prepared in exactly the same manner except that the purging after vacuum drying was performed in the air and all the operations in the glove box were performed in the air. Obtained.

Table I below shows the results of elemental analysis of silica gel obtained as porous fine particles 1-1 to 1-3 after drying under reduced pressure.

For elemental analysis, a carbon / hydrogen / nitrogen simultaneous quantifier CHN coder MT-6 (manufactured by Yanaco Technical Science Co., Ltd.) was used. In the table, the carbon content value represents a relative value, and the porous fine particles 1-1 were set to 1.00.

From Table I, it was found that the method of the present invention had the highest carbon content and the highest rate of introduction of modifying groups.

Example 2
The fillers obtained from the porous fine particles 1-1 to 1-3 of Example 1 were filled in a stainless steel column having an inner diameter of 4.6 mm and a length of 15 cm by a slurry filling method using isopropyl alcohol. Using this column, samples were analyzed by supercritical fluid chromatography. UPC2 manufactured by Japan Waters Corp. was used as the analyzer. A supercritical fluid carbon dioxide using 40% ethyl acetate as a mobile phase as a modifier was sent under the conditions of a flow velocity of 2.5 mL / min, a column temperature of 50 ° C., and a fluid pressure of 70 kg / cm ² , and organic electroluminescence (organic). A chromatogram was obtained by injecting 1 μL of a solution containing Ir (piq) ₃ (0.10 mg / mL) used as a dopant in the light emitting layer of the EL) material.

The retention coefficient, the separation coefficient α of Example 3, and the degree of separation R of Examples 5 and 6 were calculated by the following formulas.

Retention coefficient k: Calculated by applying the following formula, where the solvent shock is the hold-up time t ₀ and the retention time of the component is t.

k = (t−t ₀ ) / t ₀
Separation coefficient α: For component A and component B, when the retention coefficients were k _A and k _B , respectively, they were calculated by applying the following formula. However, the retention coefficient of the component that elutes later is k _B (k _A <k _B ).

α = k _B / k _A
Separation degree R: When components A and peak width retention time and W _A, t _A, respectively, W _B the retention time and peak width of the component B, respectively, and t _B, the separation of R is expressed as follows To. However, the retention time of the component that elutes later is t _B (t _A <t _B ).

_{R = (t B -t A)} / ((W A + W B) × 0.5)
In Table I, the retention coefficient values represent relative values, and the porous fine particles 1-1 were set to 1.00.

From the results of 1-1 to 1-3 in Table II, the filler of the present invention in which the filler using carbazole as a modifying group was reacted in a supercritical fluid using silica gel substituted with a dry gas was the most suitable. It was found to retain the compound well. That is, it can be seen that the filler has a large amount of modifying groups and is advantageous for retention.

Example 3
A filler using the porous fine particles 1-2 and 1-3 was filled into a stainless steel column having an inner diameter of 4.6 mm and a length of 15 cm by a slurry filling method using isopropyl alcohol. As a mobile phase, supercritical fluid carbon dioxide using 40%% ethyl acetate as a modifier was sent under the conditions of a flow velocity of 2.5 mL / min and a fluid pressure of 75 kg / cm ² , and the host of the light emitting layer of the organic EL material. MCP: 1,3-bis (N-carbazolyl) benzene (0.90 mg / mL) and Ir (piq) ₃ (0.10 mg / mL) used as a dopant for the light emitting layer. A chromatogram was obtained by injecting 1 μL of the liquid. The temperature was analyzed under three conditions from 35 to 75 ° C.

In the table, the separation coefficient α value represents a relative value, and column 3-2 is set to 1.00.

From Table III, it was found that the column using the porous fine particles 1-2 of the present invention as a filler was easily affected by the analysis temperature. It is known that the viscosity and diffusion coefficient of carbon dioxide, which has become a supercritical fluid, increases as the temperature rises. Along with this, it is considered that the transportability of the substance is likely to extend to the deep part of the pores due to the increase in the viscosity and diffusion coefficient of carbon dioxide. The filler using the porous fine particles 1-2 is chemically modified to the deep part of the pores, and the influence of the substance transport extending to the deep part of the pores is remarkable, but the filler of the porous fine particles 1-3 is deep in the pores. It is presumed that it is not easily affected by temperature because the chemical modification of the particles is insufficient.

Example 4
<Preparation of porous fine particles 4-1>
₂ g of spherical silica gel (Lichospher-NH ₂ , registered trademark, manufactured by Merck Co., Ltd.) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, and an aminopropyl group modification degree of 1.44 mmol / g is vacuum-dried at 110 ° C. It was moved into the glove box and the container was purged with dry nitrogen. After transferring this silica gel to the reaction vessel in the glove box and installing it in the apparatus, benzoic acid (1.22 g, 10 mmol) and EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (3-dimethylaminopropyl) carbodiimide ( A mixture of 3.10 g, 20 mmol) dissolved in 50 mL of tetrahydrofuran was used as a modifier so that the reaction temperature was 65 ° C. and the fluid pressure was 2.5% (volume ratio) with respect to the supplied carbon dioxide. The reaction was carried out by supplying the reaction vessel to the reaction vessel for 8 hours under the conditions of 250 kg / cm ² and a flow rate of 0.15 mL / min. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 4-1 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 4-2-4-20>
Porous fine particles 4-2 to 4-20 were prepared in the same manner as the porous fine particles 4-1 except that acetic acid was variously changed to the carboxylic acid corresponding to Table IV.

In Table IV, the modifying group structure of the porous fine particles 4-2 to 4-20 has the following structure. In the formula, * represents the connection with the corresponding modifying group structure, and the wavy line represents the connection with the remaining linker.

The fillers obtained from the porous fine particles 4-1 to 4-20 were filled in a stainless steel column having an inner diameter of 4.6 mm and a length of 15 cm by a slurry filling method using isopropyl alcohol. Using this column, as a mobile phase, supercritical fluid carbon dioxide using 40% ethyl acetate as a modifier was sent under the conditions of a flow rate of 3.0 mL / min, a column temperature of 50 ° C., and a fluid pressure of 70 kg / cm ^2. , MCP (0.90 mg / mL) and Ir (piq) ₃ (0.10 mg / mL) were injected in 1 μL to obtain a chromatogram. The results obtained are shown in Table IV.

However, the degree of separation R in the table is
Less than 0.5 is not separated ×,
0.5 or more and less than 1.5 △,
1.5 or more and less than 3.0 ○,
3.0 or higher is described as ⊚.

From Table IV, it is inferred that the column using the porous fine particles 4-1 to 4-20 of the present invention as a filler has good separation, and thus the filler is chemically modified to the deep part of the pores. To. It was also found that more favorable results can be obtained in the case of a modifying group containing a heteroatom.

Example 5
<Preparation of porous fine particles 5-1>
₂ g of spherical silica gel (Lichospher-NH ₂ , registered trademark, manufactured by Merck) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, and an aminopropyl group modification degree of 1.44 mmol / g was placed in a glass container, and 3- (2) was placed therein. -Phenyl) pyridylbenzoic acid (modifying group structure c-18, 2.75 g, 10 mmol) and EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (3.10 g, 20 mmol), tetrahydrofuran (30 mL) Was added. This mixed solution was immersed in an ultrasonic generator, and the reaction was carried out for 4 hours while applying ultrasonic waves. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 5-1 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 5-2>
₂ g of spherical silica gel (Lichospher-NH ₂ , registered trademark, manufactured by Merck Co., Ltd.) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, and an aminopropyl group modification degree of 1.44 mmol / g is vacuum-dried at 110 ° C. It was moved into the glove box and the container was purged with dry nitrogen. After transferring this silica gel to the reaction vessel in the glove box and installing it in the apparatus, 3- (2-phenyl) pyridylbenzoic acid (modifying group structure c-18, 2.75 g, 10 mmol) and EDC: A mixture of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (3.10 g, 20 mmol) dissolved in 50 mL of tetrahydrofuran was used as a modifier and 2.0% (volume) with respect to the carbon dioxide supplied. The reaction was carried out by supplying the mixture to the reaction vessel for 8 hours under the conditions of a reaction temperature of 60 ° C., a fluid pressure of 200 kg / cm ² , and a flow velocity of 0.20 mL / min. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 5-2, which is a chemically modified packing material for chromatography.

<Preparation of porous fine particles 5-3 to 5-15>
Porous fine particles 5-2 to 5-15 were prepared in the same manner as the porous fine particles 5-1 except that the modifying group structure c-18 was variously changed to the carboxylic acid corresponding to Table V.

The fillers obtained from the porous fine particles 5-1 to 5-15 were filled in a stainless steel column having an inner diameter of 4.6 mm and a length of 15 cm by a slurry filling method using isopropyl alcohol. Using this column, as a mobile phase, supercritical fluid carbon dioxide using 40% ethyl acetate as a modifier was sent under the conditions of a flow rate of 3.0 mL / min, a column temperature of 50 ° C., and a fluid pressure of 70 kg / cm ^2. , MCP (9.00 mg / mL) and Ir (piq) ₃ (1.00 mg / mL) were injected in 1 μL to obtain a chromatogram. The results obtained are shown in Table V.

However, the degree of separation R is
Less than 0.5 is not separated ×,
0.5 or more and less than 1.5 △,
1.5 or more and less than 3.0 ○,
3.0 or higher is described as ⊚.

From the results in Table V, it was found that when the filler of the present invention was used, good separation could be obtained even when a large amount of sample was injected.

Example 6
<Preparation of porous fine particles 6-1>
₂ g of spherical silica gel (Lichospher-NH ₂ , manufactured by Merck) having a pore size of about 100 Å, an average particle size of about 5 μm, and an aminopropyl group modification degree of 1.44 mmol / g was placed in a glass container, and 3- (9-phenylcarbazole-) was placed therein. 3-Il) Benzoic acid (3.63 g, 10 mmol) and EDC: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (3.10 g, 20 mmol), tetrahydrofuran (40 mL) were added. This mixed solution was immersed in an ultrasonic generator, and the reaction was carried out for 4 hours while applying ultrasonic waves. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 6-1 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 6-2>
₂ g of spherical silica gel (Lichospher-NH ₂ , registered trademark, manufactured by Merck Co., Ltd.) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, and an aminopropyl group modification degree of 1.44 mmol / g is vacuum-dried at 110 ° C. It was moved into the glove box and the container was purged with dry nitrogen. After transferring this silica gel to the reaction vessel in the glove box and installing it in the apparatus, 3- (9-phenylcarbazole-3-yl) benzoic acid (3.63 g, 10 mmol) and EDC: 1-ethyl are placed in the reaction vessel. A mixture of -3- (3-dimethylaminopropyl) carbodiimide (3.10 g, 20 mmol) dissolved in 50 mL of tetrahydrofuran was used as a modifier, and the ratio was 2.0% (volume ratio) with respect to the supplied carbon dioxide. The reaction was carried out by supplying the mixture to the reaction vessel for 8 hours under the conditions of a reaction temperature of 60 ° C., a fluid pressure of 250 kg / cm ² , and a flow rate of 0.20 mL / min. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 6-2 which were chemically modified packing materials for chromatography.

The fillers obtained from the porous fine particles 6-1 and 6-2 were filled in a stainless steel column having an inner diameter of 4.6 mm and a length of 15 cm by a slurry filling method using isopropyl alcohol. Using this column, as a mobile phase, supercritical fluid carbon dioxide using 40% ethyl acetate as a modifier was sent under the conditions of a flow rate of 3.0 mL / min, a column temperature of 50 ° C., and a fluid pressure of 70 kg / cm ^2. , Ir (piq) ₃ (0.10 mg / mL) was injected in 1 μL to obtain a chromatogram. In addition, these columns were applied to HPLC using hexane: isopropanol = 95: 5 (flow rate 1.0 mL / min) as the mobile phase, and the same analysis was performed. Table VI shows the relative values of the retention factors obtained from each analysis. In the table, the retention coefficient value was set to a relative value of 1.00 for column 6-1 / HPLC.

From Table VI, when comparing the retention coefficients when HPLC is applied and when SFC is applied to each column, the retention coefficient when SFC is applied is significantly improved in the column using the filler containing the porous particles of the present invention. It turned out. It is known that carbon dioxide, which has become a supercritical fluid, has a lower viscosity and a higher diffusion coefficient than a liquid, and it is considered that the transportability of a substance is likely to extend to the deep part of the pores. The filler of the column (6-2) is modified to the deep part of the pores, and the influence of the substance transport extending to the deep part of the pores is remarkable, but the filler of the column (6-1) is modified to the deep part of the pores. It is presumed that the performance does not reflect the feature that the mobile phase is a supercritical fluid because it is insufficient.

Example 7
<Preparation of porous fine particles 7-1>
2.0 g of spherical silica gel (Daiso Gel SP-100-5-P, manufactured by Daiso) having a pore diameter of about 100 Å, an average particle diameter of about 5 μm, and a specific surface area of 450 m ² / g was placed in a glass container, and 9- (3) was placed therein. -(7-Chlorodimethylsilylheptyl) phenyl) carbazole (4.34 g, 10 mmol), pyridine (1.19 g, 15 mmol), tetrahydrofuran (40 mL) were added. This mixed solution was immersed in an ultrasonic generator, and the reaction was carried out for 6 hours while applying ultrasonic waves. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 7-1 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 7-2>
2.0 g of spherical silica gel (Daiso Gel SP-100-5-P, manufactured by Daiso) having a pore diameter of about 100 Å and an average particle diameter of about 5 μm was vacuum dried at 110 ° C., and then the drying container was moved into the glove box. The container was purged with dried nitrogen. After transferring this silica gel to the reaction vessel in the glove box and installing it in the apparatus, 9- (3- (7-chlorodimethylsilylheptyl) phenyl) carbazole (4.34 g, 10 mmol) and pyridine (1) are placed in the reaction vessel. A mixture of .19 g, 15 mmol) dissolved in 50 mL of tetrahydrofuran was used as a modifier so that the reaction temperature was 60 ° C. and the fluid pressure was 250 kg so as to be 2.0% (volume ratio) with respect to the supplied carbon dioxide. The reaction was carried out by supplying the reaction vessel to the reaction vessel for 6 hours under the conditions of / cm ² and a flow rate of 0.20 mL / min. This silica gel was filtered using a glass filter (G4), and washed and filtered sequentially with 300 mL of tetrahydrofuran, 300 mL of methanol, and 300 mL of acetone. Further, the obtained silica gel was dried under reduced pressure to obtain porous fine particles 7-2 which were chemically modified packing materials for chromatography.

<Preparation of porous fine particles 7-3>
The filler 7-3 of Comparative Example was obtained in exactly the same manner except that the purging after vacuum drying was performed in the air and all the operations in the glove box were performed in the air.

Table VII shows the results of elemental analysis of the silica gel obtained from the porous fine particles 7-1 to 7-3 after being dried under reduced pressure.

From Table VII, the method of the present invention has the highest carbon content and is modified even when applied to a synthetic method that does not involve amide condensation, unlike the synthetic method of porous fine particles 1-1 to 1-3. It was found that the introduction rate of the group was high.

Example 8
The fillers obtained from the porous fine particles 7-1 to 7-3 were filled in a stainless steel column having an inner diameter of 4.6 mm and a length of 15 cm by a slurry filling method using isopropyl alcohol. Using this column, as a mobile phase, supercritical fluid carbon dioxide using 40% ethyl acetate as a modifier was sent under the conditions of a flow rate of 2.5 mL per minute, a column temperature of 50 ° C., and a fluid pressure of 70 kg / cm ^2. , Ir (piq) ₃ (0.10 mg / mL) was injected in 1 μL to obtain a chromatogram. Table VIII shows the relative values of the retention factors obtained from each analysis.

From the results of columns 7-1 to 7-3 in Table VIII, the filler of the present invention in which the filler using carbazole as a modifying group was reacted in a supercritical fluid using silica gel substituted with a dry gas was obtained. It was found to retain the compound best. That is, it can be seen that the filler has a large amount of modifying groups and is advantageous for retention.

Further, it was found that the method of the present invention is effective even when applied to a synthetic method not accompanied by amide condensation, unlike the synthetic method of porous fine particles 1-1 to 1-3.

Since the porous fine particles of the present invention provide excellent separation characteristics and have an increased retention capacity, they are suitably used as a packing material for chromatography and a column for chromatography.

11 Supercritical fluid 12 Pump 13 Modifier 14 Injector 15 Separation column 16 Column oven 17 Detector 18 Pressure regulating valve

Claims (7)

Porous fine particles whose surface is modified with a chemical modifier, and the inside of the pores existing in the porous fine particles is chemically modified. The porous fine particles according to claim 1, wherein the chemical modifying agent has a modifying group containing a 5-membered or 6-membered aromatic heterocycle or each condensed ring as a structure. The porous fine particles according to claim 1 or 2, wherein the porous fine particles are fine particles containing a metal oxide. The porous fine particles according to any one of claims 1 to 3, wherein the porous fine particles are fine particles containing silicon dioxide. A packing material for chromatography, which comprises the porous fine particles according to any one of claims 1 to 4. A column for chromatography, which comprises the porous fine particles according to any one of claims 1 to 4. A method for producing porous fine particles according to claim 1 to 3, wherein the porous fine particles are produced.
A step of replacing the system containing the porous fine particles with a dry gas after depressurizing,
A step of reacting the porous fine particles with the chemical modifier in a supercritical fluid to modify the particles,
A method for producing porous fine particles, which comprises at least.

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