JPH0454496B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a modified porous inorganic material and a packing material for chromatography comprising the same.

[Prior art]

Porous inorganic materials have extremely large surface areas and adsorption activities, and have recently been widely used as column packing materials for liquid chromatography, gas chromatography, etc., in addition to catalysts for various catalytic reactions. Such porous inorganic materials include silica gel, alumina, silica alumina, zeolite,
These include diatomaceous earth and the like, but silica gel and alumina, especially silica gel, are widely used as packing materials for liquid chromatography.

Porous inorganic materials can be used as adsorption system fillers without modification due to their large surface area and strong adsorption activity. However, since the adsorption activity of inorganic porous fillers such as silica gel and alumina varies greatly depending on their water content, care must be taken when handling them. On the other hand, recently, the surface of silica gel has been coated with octadecyltrichlorosilane (ODS),
So-called reversed-phase packing materials, which are treated with octyltrichlorosilane and modified with _C18 or _C8 alkyl groups, have become widely used. ODS
- Reversed-phase packing materials such as silica have extremely stable activity against eluents such as water-methanol and water-acetonitrile, so they are easy to handle and can be used with various buffer solutions. .

Apart from this, fillers in which the surface of silica gel is modified with aminopropyl groups, cyanopropyl groups, diol groups, etc. are also commercially available. Furthermore, columns with optically active functional groups bonded to the silica surface and columns with fillers bonded with crown ether or the like are also commercially available. however,
Even when these columns are used, there are still many substances that are difficult to separate, and there is a desire to develop a packing material with better properties. Furthermore, these commercially available columns have essentially only one type of group on the surface of the packing material as a functional group effective for separation. It is expected that the ability to identify objects will increase. From this point of view, there are currently no columns on the market that have introduced functional groups that are substantially effective in separating two or more types.

〔the purpose〕

As a result of extensive research into selective separation methods using porous inorganic materials, the present inventors have found that two or more functional groups that are substantially involved in separation are regularly introduced onto the surface of porous inorganic materials. For example, the present invention was developed by focusing on the improvement of selective separation performance for specific substances.

〔composition〕

According to the present invention, some of the amino groups introduced in advance into the pores of a porous inorganic substance are represented by the general formula -NHCO-A-COOH (wherein A represents a divalent hydrocarbon group). Provided is a modified porous inorganic material having a structure substituted with amide groups, and characterized in that the amide groups are present at regular intervals via adjacent amino groups.

The modified porous inorganic material of the present invention comprises a step of reacting an acid halide having an ester group with an amino group present in the pores of the porous inorganic material, and removing the ester group contained in the reacted compound. It is produced by a step of separating the carboxyl groups to form carboxyl groups within the pores. In this case, the acid halide having an ester group to be reacted with the amino group has a size that provides steric hindrance when reacting with the amino group in relation to the pore diameter of the porous inorganic substance, and Reactively bonds with only part of the group.

Porous inorganic materials include silica gel, alumina, silica-alumina, zeolite, diatomaceous earth, etc., and silica gel, alumina, especially silica gel, and alumina, especially silica gel are suitable as packing materials for liquid chromatography. The pore characteristics of silica gel greatly depend on its preparation conditions, and in particular its surface area and pore size influence its separation characteristics in chromatography. In the present invention, as an example, a material having a pore diameter of 40 to 100 Å is used for modification. If a material with a pore diameter of 40 Å or less is used, steric hindrance during the reaction will be even greater. OH groups exist on the surfaces of silica gel and alumina, and various modifications are performed via these OH groups. When producing the product of the present invention, the starting material is preferably one in which an aminopropyl group is introduced by reacting the OH group with aminopropyltriethoxysilane. The amount introduced was determined by elemental analysis (C, H, N), and the amount of the compound introduced into the porous inorganic material hereinafter was determined by elemental analysis not otherwise specified. This silica gel modified with an aminopropyl group and a column filled with this silica gel are commercially available for use in liquid chromatography and are well known. The product of the present invention has an ester group in its molecule, and provides steric hindrance when chemically bonding with a porous inorganic material, thereby reducing the amino group present as the first functional group in the pores of the porous inorganic material. The acid halide is bonded as a compound that binds substantially only a portion of it, rather than all of it, and the ester group is then removed under appropriate conditions to create a porous structure with the carboxyl group as the second functional group. These materials are novel because they are obtained by disposing them at certain intervals on the surface of an inorganic material. Further, if necessary, another compound which may contain a detachable group is further bonded to the first amino group remaining unreacted in the pores of the porous inorganic material, and another third functional Groups can also be introduced and, if necessary, further different functional groups can be introduced by repeating this step in sequence.

In preferred embodiments for manufacturing the products of the invention, the porous inorganic materials include silica gel and alumina, with silica gel being particularly preferred. 1st
The reactive functional group is preferably an aminoalkyl group bonded via an OH group present on the surface of the porous inorganic material, particularly an amino group of an aminopropyl group. Methods for introducing amino groups into silica gel are known, and are easily carried out, for example, by reacting silica gel and aminopropyltriethoxysilane in toluene under reflux.

In a preferred embodiment for producing the products of the invention, a compound having a removable group and large enough to provide steric hindrance during chemical bonding is used as the first reactive functional group in the porous inorganic material. It is an acid chloride having an ester group that can be bonded to an amino group, through which it can be chemically bonded to the amino group present in the porous inorganic material. In this case, hydrochloric acid is eliminated by the acylation reaction between the amino group and the acid chloride, and the ester compound is introduced via the amide bond. This reaction is carried out in a water-free inert organic solvent at 0 to 200°C.
Preferably, the reaction is carried out at the reflux temperature of the solvent. The inert organic solvents include, for example, aliphatic hydrocarbons such as pentane, hexane, heptane, cycloaliphatic hydrocarbons such as cyclopentane, cyclohexane, chlorinated hydrocarbons such as dichloromethane, chloroform, tetrachloride, etc. Carbon, trichloroethane, chlorobenzene, and other aromatic hydrocarbons such as benzene, toluene, xylene, etc. are included, with benzene and toluene being preferred, and toluene being particularly preferred. Furthermore, an acid binder may be used in the acylation reaction in this case.

In a preferred embodiment for producing the product of the present invention, the acid chloride which has an ester group and a group that binds to the amino group of the porous inorganic substance and provides steric hindrance during the bonding is, for example, a It is obtained by subjecting an acid anhydride of a basic acid to an esterification reaction with an alcohol to produce a carboxylic acid containing an ester group, and then chlorinating the hydroxyl group of the carboxyl group. In this case, the esterification reaction is preferably carried out at 0 to 200°C in an inert organic solvent containing no water.
Preferably, the reaction is carried out at the reflux temperature of the solvent. The inert organic solvents include, for example, aliphatic hydrocarbons such as pentane, hexane, heptane, cyclic aliphatic hydrocarbons such as cyclopentane, cyclohexane, chlorinated hydrocarbons such as dichloromethane, chloroform, tetrachloride, etc. Carbon, trichloroethane, chlorobenzene, and other aromatic hydrocarbons such as benzene, toluene, xylene, etc. are included, with benzene and toluene being preferred, and toluene being particularly preferred.

Chlorination of carboxylic acids containing ester groups is carried out, for example, by chlorination with thionyl chloride, thereby giving acid chlorides. Chlorination with thionyl chloride is preferably carried out in a water-free inert organic solvent at 0 DEG to 200 DEG C., preferably at the reflux temperature of the solvent. This inert organic solvent contains
For example, fatty acid hydrocarbons, such as pentane, hexane, heptane, cycloaliphatic hydrocarbons, such as cyclopentane, cyclohexane, chlorinated hydrocarbons, such as dichloromethane, chloroform, carbon tetrachloride, trichloroethane, chlorobenzene, etc., aromatic group hydrocarbons, such as benzene,
It includes toluene, xylene, etc., with benzene and toluene being preferred, and toluene being particularly preferred. Examples of dibasic acid anhydrides include succinic anhydride, maleic anhydride, phthalic anhydride, and the like.

The acid halide having an ester group capable of bonding to the amino group existing in the pores of the porous inorganic substance used in the present invention must have a molecular structure large enough to provide steric hindrance when bonding with the amino group. Therefore, it is bonded to only some of the amino groups at substantially certain intervals, and the unbonded portions of the amino groups remain at substantially certain intervals in the porous inorganic material. That is, the amide group formed by the reaction between the acid halide and the amino group is surrounded by unreacted amino groups, so the amide group forms a porous inorganic material at regular intervals via the adjacent amino groups. introduced into matter. Such an acid halide with a large molecular structure is used when producing an acid halide having an ester group using a dibasic acid anhydride and an alcohol as starting materials. , preferably by using an alcohol having 10 to 36 carbon atoms. The acid anhydride and alcohol can be added in any quantitative ratio, but are preferably added in stoichiometric amounts. Thionyl chloride is preferably used in excess. The product is easily obtained by removing the solvent, such as by evaporation, and does not require further purification. An example of a reaction process in which an acid anhydride and an aliphatic alcohol are used as starting materials and chlorination is carried out using thionyl chloride is shown below.

In the formula, A represents a divalent hydrocarbon group, such as an alkylene group, an alkenylene group, or an arylene group. R is a hydrocarbon group having 8 or more carbon atoms, such as an alkyl group, an alkenyl group, an alkynyl group,
Represents a cycloalkyl group or an aryl group.

Further, the reaction between the acid halide and the amino group in the porous inorganic material can be expressed by the following formula.

In the above formula, X represents a porous inorganic substance, and A and R have the same meanings as above.

As can be seen from the above reaction formula, acid halides have large molecules and provide steric hindrance to the reaction, reacting only with a portion of the amino groups that make the porous inorganic material, and reacting with the positions adjacent to the amide bonds. has an amino group, and the amino groups are not adjacent to each other.
As a result, amide groups are regularly introduced into the porous inorganic material at regular intervals.

The acid halide is used in an amount of 0.01 to 10 mol, preferably 0.1 to 5 mol, per mol of amino group in the porous inorganic material.
It can be used in molar amounts, particularly preferably from 0.5 to 3 molar.

After the acid halide is bonded to the amino group as the first reactive functional group as described above, the ester group as the removable group present in the reacted acid halide is bonded by an appropriate method. Detach it with
A carboxyl group is formed as a second functional group. In this way, amino groups and carboxyl groups can be introduced at regular intervals into the pores of the porous inorganic material. In a preferred embodiment of the invention, this ester group can be removed by acid or alkali hydrolysis in a suitable solvent. Suitable solvents include water, polar organic solvents,
For example, methanol, ethanol, propanol,
Examples include isopropanol and butanol, acetone, acetonitrile, N,N-dimethylformamide, and mixtures thereof. Preferably, mixtures of water and these polar organic solvents, especially alcohols, are used. As acids or alkalis, customary inorganic or organic acids or bases may be used. In the present invention, it is preferable to hydrolyze with an acid, and hydrochloric acid is particularly preferable. Accordingly, in a particularly preferred embodiment of the invention, hydrochloric acid solutions of 0.1 to 6N, preferably 0.2 to 4N, particularly preferably 0.5 to 3N, in a water-ethanol mixed solvent are used under reflux.
Hydrolyze for 10-60 minutes. This hydrolysis results in
The alcohol group forming the ester group of the bonded compound is detached, creating a carboxyl group at the end on the surface of the porous inorganic material, and thus, in addition to the amino group, different reactive functional groups separate the carboxyl groups at certain intervals. It can be installed by placing

Next, if necessary, a third functional group can be introduced by bonding a compound different from the one described above to the reactive functional group remaining in the pores of the porous inorganic material. In this case, if the compound contains a removable group, this can be removed to introduce a third functional group. If appropriate, further different functional groups can be introduced into the porous inorganic material by repeating this step in sequence.

〔effect〕

The modified porous inorganic material of the present invention contains at least both an amino group and a carboxyl group-containing amide group in its pores. in this case,
Unreacted amino groups are present adjacent to the carboxyl group-containing amide groups, thereby introducing the amide groups into the porous inorganic material at regular intervals.

Since the modified porous inorganic material of the present invention has the above-described structure, it can be advantageously used as an adsorbent or filler that independently exerts the functions of both its amino group and carboxyl group. For example, when the modified porous inorganic material of the present invention is used as a packing material for liquid chromatography, it can be caused to interact regularly with the substance to be analyzed rather than randomly. For example, when the substance to be analyzed is an amino acid having an amino group and a carboxyl group in the molecule, the amino group and carboxyl group in the modified porous inorganic material of the present invention are independent of the amino group and carboxyl group of the amino acid, respectively. Because of its strong action, the amino acid can be adsorbed and separated at a high adsorption rate.

The modified porous inorganic material of the present invention can be used as a column for chromatography, especially a column for high performance liquid chromatography, by filling it into a column by an appropriate known method.

〔Example〕

Next, the present invention will be explained in more detail with reference to Examples.

Reference example 1 Silica gel (SI-40 for column chromatography manufactured by Merck, particle size: 0.063 to 0.200 mm, pore size 40
(hereinafter abbreviated as SI-40) was placed in a 200 ml flat bottom flask equipped with a reflux condenser.
100 ml of toluene was added, followed by 3.0 g of aminopropyltriethoxysilane, and the mixture was reacted for 1 hour under reflux of the toluene solvent while stirring with a magnetic stirrer. The silica gel was then separated from the solvent by filtration, washed three times with 50 ml of benzene and three times with 50 ml of dichloromethane, and dried overnight at 100°C in a vacuum dryer to obtain aminopropyl silica (hereinafter abbreviated as AP silica). Obtained.
This product was subjected to elemental analysis to determine the introduced aminopropyl group.

Elemental analysis value:
C: 6.58%; H: 2.47%; N: 1.65%.

From the nitrogen content, it was found that 1.18 mmol of aminopropyl groups were bound per gram of silica gel.

Different silica gels (Merck Co., Ltd. SI-60 for column chromatography, 0.063 to 0.200 mm, pore size 60
An aminopropyl group was bonded to silica gel in the same manner as above except that SI-60 (hereinafter abbreviated as SI-60) was used.

Elemental analysis value:
C: 5.50%; H: 1.81%; N: 1.53%.

From the nitrogen content, it was found that 1.09 mmol of aminopropyl groups were bound per gram of silica gel.

Different silica gels (Merck Co., Ltd. SI-100 for column chromatography, particle size: 0.063 to 0.200 mm,
An aminopropyl group was bonded to silica gel in the same manner as above, except that the pore size was 100 Å (hereinafter abbreviated as SI-100).

Elemental analysis value:
C: 4.07%; H: 1.27%; N: 1.12%.

From the nitrogen content, it was found that 0.80 mmol of aminopropyl groups were bound per gram of silica gel.

Reference Example 2 Preparation of ester group-containing acylating agent 5.0 g (0.05 mol) of succinic anhydride and 7.91 g (0.05 mol) of n-decanol were placed in an eggplant-shaped flask equipped with a reflux condenser, and 200 ml of toluene was added thereto. The reaction was carried out under reflux for 3 hours. Take a portion of this and use acetonitrile:water = 1:1 as an eluent.
Analysis of succinic anhydride by high performance liquid chromatography using an ODS-silica gel column revealed that almost no unreacted succinic anhydride was detected, indicating that the reaction was quantitative. In addition, the NMR spectrum revealed a carboxyl group peak at around 11.3 ppm based on TMS. After the reaction product was cooled in the flask, about 4 equivalents (about 14.5 ml) of thionyl chloride was added to the flask and heated under reflux for 2 hours. The solvent was removed on a rotary evaporator, redissolved twice with 100 ml of benzene, and the benzene was removed on a rotary evaporator each time, followed by drying at 80° C. in a vacuum dryer overnight. The product is
The NMR spectrum revealed that the peak around 11.3 ppm had disappeared, indicating that the carboxyl group was chlorinated. Therefore, this compound is a compound called n-decylic acid (chlorocarbonyl)-propionate (C ₁₀ T ₂₁ OCOC ₂ H ₄ COCl), and will be abbreviated as E ₁ below.

The above method was repeated except that 10.72 g (0.05 mol) of n-tetradecanol was used instead of n-decanol, and n-tetradecylic acid (chlorocarbonyl)-propionate (C ₁₄ H ₂₉ OCOC ₂ H ₄ COCl ) was obtained. This compound is hereinafter abbreviated as _E2 .

Octadecanol instead of n-decanol
_The above method _was repeated except that 13.52 g (0.05 mol) was used to obtain octadecyl acid ( _{chlorocarbonyl} )-propionate ( _{C18H37OCOC2H4COCl} ). This compound is hereinafter abbreviated as _E3 .

Example 1 [Reaction step] Ester group-containing acid chloride from Reference Example 2
A toluene solution containing E ₁ at 0.25 mmol/ml was prepared. Dilute 25 ml of this solution with 25 ml of toluene and add 5.0 g of AP silica Si-40 from Example 1 to this solution.
In addition, the acylation reaction of AP silica was carried out for 1 hour under reflux while stirring with a magnetic stirrer.
After the reaction was completed, the silica gel was cooled to room temperature, separated by filtration, washed three times with 50 ml of benzene and three times with 50 ml of dichloromethane, and placed in a vacuum dryer at 100°C.
It was dried overnight. Elemental analysis revealed that 0.47 mol of E ₁ was bound per mol of amino group.

The above method was repeated except that E ₂ was used instead of E ₁ . As a result of elemental analysis of the obtained silica gel, 0.42 mol of E ₂ per mol of amino group.
were combined.

The above procedure was repeated except that E ₃ was used instead of E ₁ . As a result of elemental analysis of the obtained silica gel, 0.34 mol of E ₃ per 1 mol of amino group was found.
were combined.

Next, instead of AP silica SI-40, use AP silica SI−
An acylation reaction was carried out with E ₁ , E ₂ and E ₃ using E 60 in the same manner as above. As a result of elemental analysis, amino group 1
0.57, 0.49 and 0.49 moles of E ₁ per mole, respectively;
E ₂ and E ₃ were combined.

Next, instead of AP silica SI-40, use AP silica SI−
Acylation reactions were carried out with E ₁ , E ₂ and E ₃ in the same manner as above using E 100. As a result of elemental analysis, amino group 1
0.68, 0.60 and 0.60 moles of E ₁ per mole, respectively;
E ₂ and E ₃ were combined.

These results revealed that silica gel with a smaller pore diameter is more likely to experience steric hindrance, and acid chloride containing an alcohol with a larger number of carbon atoms via an ester group is more likely to experience steric hindrance.

Example 2 [Desorption step] AP silica SI treated with _E2 in Example 1
3.0 g of -40 was added to 30 ml of a 2N hydrochloric acid solution of water:ethanol = 1:1, and hydrolysis was carried out under reflux while stirring with a magnetic stirrer. After hydrolysis, it was cooled to room temperature, the silica gel was separated by filtration, and 3 times with 50 ml of a solution of water:ethanol = 1:1.
and 50 ml of methanol three times, and dried overnight at 100°C in a vacuum dryer. As a result of comparing the elemental analyzes before and after hydrolysis, per molecule of bound _E2 ,
Since 14.2 carbon atoms were eliminated and the value of nitrogen atoms remained almost unchanged, hydrolysis did not affect other functional groups, and all _C14 alcohols were selectively eliminated, and the silica gel surface A carboxyl group could be generated at the upper end. The presence of this carboxyl group was also confirmed by the absorption peak near 1700 cm ^-1 in the IR spectrum.

The same procedure as above was repeated using 3.0 g of AP silica SI-60 treated with E2 instead of AP silica SI- ₄₀ . As a result of elemental analysis, 13.7 carbon atoms were released per molecule of bonded E ₂ , and the value of nitrogen atoms remained almost unchanged, so hydrolysis did not affect other functional groups and C ₁₄ All of the alcohol was selectively eliminated, and carboxyl groups were generated at the ends of the silica gel surface. The presence of carboxyl groups was also confirmed from the IR spectrum.

The same procedure as above was repeated using 3.0 g of AP silica SI-100 treated in the same manner instead of AP silica SI-40 treated with _E2 . As a result of elemental analysis, there are 14.4 carbon atoms per bonded E ₂ molecule.
Since each individual is eliminated and the value of the nitrogen atom is almost unchanged, hydrolysis does not affect other functional groups, and only _C14 alcohols are selectively eliminated, resulting in the free radicals on the silica gel surface. We were able to generate a carboxyl group. The presence of carboxyl groups was also confirmed from the IR spectrum.

Example 3 Two types of silica gel for high performance liquid chromatography (HPLC) (manufactured by Merck & Co., Ltd.) were used instead of the silica gel for column chromatography in Reference Example 1.
Amino groups and carboxyl groups were introduced in the same manner as in Reference Examples 1 and 2 and Examples 1 and 2 using LiChrosorb SI-60 (particle size 10 μm). These two types
The silica gel for HPLC differs only in particle size from the corresponding silica gel for column chromatography, but has the same properties, and the modification of the present invention could be carried out in the same way.

This modified silica gel was processed into 4i.d.
Fill a 250mm stainless steel column with slurry,
It was used as a column for HPLC. FIG. 1 shows, as an example, a chromatogram obtained when protein-removed serum was analyzed using a column modified with SI-60 silica gel. The eluent is acetonitrile:water=
A 80:20 solution adjusted to pH 6.0 with a phosphate buffer was used. In the chromatogram, 1 is creatinine and 2 is creatine.

Example 4 3.0 g of produced silica gel SI-40 from Example 2
Add 50 ml of toluene to this mixture, and add 2.5 mL of p-nitrobenzoyl chloride to 1 g of silica gel.
It was added in an amount of millimole and reacted for 1 hour under reflux while stirring with a magnetic stirrer. After the reaction was completed, it was cooled to room temperature, the silica gel was separated by filtration, and the silica gel was extracted with benzene three times and with dichloromethane three times.
It was washed twice and dried in a vacuum dryer at 100°C overnight. Through this reaction, it was possible to further introduce 0.26 mmol of p-nitrobenzoyl groups per 1 g of silica gel.

2.0 g of the produced silica gel was added to 20 ml of 10% stannous chloride solution and 5 ml of concentrated hydrochloric acid, and the mixture was allowed to react at room temperature for 2 hours while stirring with a magnetic stirrer. This allowed 80% of the nitro groups to be reduced to amino groups.

The above method was repeated except that SI-60 was used instead of SI-40 as the silica gel produced from Example 2, and it was possible to introduce 0.23 mmol of p-nitrobenzoyl groups per 1 g of silica gel. Furthermore, approximately 80% of nitro groups could be reduced to amino groups.

The above method was repeated except that SI-100 was used instead of SI-40 as the silica gel produced from Example 2, and it was possible to introduce 0.21 mmol of p-nitrobenzoyl groups per 1 g of silica gel. Furthermore, approximately 80% of nitro groups could be reduced to amino groups.

The methods of Reference Examples 1 and 2 and Examples 1 and 2 were repeated except that alumina for column chromatography (manufactured by Merck, pore size: 90 Å, neutral, particle size: 0.063 to 0.200 mm) was used instead of silica gel. The alumina was modified. As a result of elemental analysis,
Aminopropyl groups were introduced in an amount of 0.43 mmol per gram of alumina, and for example, _E2 was bonded in an amount of 0.47 mol per mol of amino groups.

[Brief explanation of the drawing]

FIG. 1 shows a chromatogram obtained when serum was analyzed by high performance liquid chromatography using a silica gel column modified according to the present invention.

Claims (1)

[Claims] 1. A part of the amino group introduced in advance into the pores of a porous inorganic substance is expressed by the general formula -NHCO-A-COOH (wherein A represents a divalent hydrocarbon group). 1. A modified porous inorganic material having a structure substituted with amide groups as shown above, and wherein the amide groups are present at regular intervals via adjacent amino groups. 2. A part of the amino group introduced in advance into the pores of the porous inorganic substance is converted into a carboxyl group-containing amide represented by the general formula -NHCO-A-COOH (wherein A represents a divalent hydrocarbon group). A packing material for chromatography comprising a modified porous inorganic substance having a structure substituted with a group and present at regular intervals.