JPH1054828A - Filler for liquid chromatography and separating method using the filler - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a filler for liquid chromatography, which can specifically hold boron-acid derivative and can be used as the filler for reversed-phase liquid chromatography, and to provide a method for advantageously separating the boron-acrid derivative by using the filler or a separating method by the reversed-phase liquid chromatography method. SOLUTION: The filler for liquid chromatography is composed of porous carrier, which is chemically modified by aromatic functional group, in which hydrogen group is coupled with at least one set of neighboring carbon atoms on an aromous ring. Furthermore, by using the filler for the liquid chromatography such as this, boron-acid derivative is separated by the liquid chromatography method, or the specified compound is separated by the reversed- phase liquid chromatography method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel packing material for liquid chromatography and a separation method using the packing material, and more particularly to a separation characteristic of a packing material for reversed-phase liquid chromatography and to specifically retain only boronic acid derivatives. A liquid chromatography packing material that has separation characteristics and also enables the separation of compounds that cannot be separated with conventional alkyl group-bonded packing materials in reverse phase liquid chromatography, and the use of such packing materials. The separation method used.

[0002]

2. Description of the Related Art Conventionally, a liquid chromatography technique can apply a wide range of separation conditions, does not require exposing a sample to high temperatures,
Since it is capable of separating compounds and can exhibit high separation ability, it has been widely used for purification and separation of various compounds ranging from biologically related substances to organic compounds in general. Since the separation characteristics of substances by this liquid chromatography technique are greatly affected by the characteristics of the packing material packed in the column, the separation of various compounds can be performed by selecting the packing material according to the separation target. You can do it.

[0003] By the way, generally, as a filler used in such a liquid chromatography technique, a reversed-phase silica gel filler represented by an alkyl group-bonded type silica gel made of silica gel chemically modified with a functional group having an alkyl group is used. Liquid chromatography (hereinafter referred to as “reverse phase chromatography”) packing materials are widely used, but the packing material for reversed phase chromatography retains a target compound mainly by hydrophobic interaction. In addition, they are separated according to the difference in their interactions, and are applied to separate compounds over a very wide range from biological substances to hydrocarbon compounds.

[0004] However, such a packing for reversed phase chromatography is a highly versatile packing that can hold various compounds, so that the target compound present in the sample and the other compounds are separated from each other. In some cases, it may be retained at the same level, and it is often difficult to retain only the target compound. Therefore, a conventional packing material for reversed phase chromatography can be used to quickly remove a desired trace amount of useful components from a sample containing a large number of contaminants, such as a biological sample. When trying to separate, there is a problem that it is difficult to obtain an appropriate separation.

[0005] In the case of using an alkyl group-bonded packing material which has been widely used in the conventional reversed-phase chromatography, such packing material exhibits separation characteristics mainly by hydrophobic interaction. Therefore, although compounds having different hydrophobicity can be effectively separated, it is difficult to separate compounds having similar hydrophobicity.

Therefore, in order to solve the above-mentioned problems of the packing material for reversed phase chromatography, development of packing materials capable of specifically retaining and separating only the target compound has been attempted. In order for only the target compound to be specifically retained and separated by the agent, it is necessary that the filler and the target compound specifically interact with each other. And as such interacting specifically,
Combinations of biological substances such as enzymes and substrates, antigens and antibodies, or hormones and receptors can be exemplified.

More specifically, for example, when the interaction between a substrate and an enzyme is used and the target compound to be separated is an enzyme, first, the substrate of the enzyme to be separated is bound to a carrier as a stationary phase. A filler is prepared. Next, a sample solution containing the enzyme to be separated is separated using the packing material according to the procedure of a usual liquid chromatography technique. Then, since only the target enzyme specifically interacts with the substrate serving as the stationary phase, only the target enzyme is retained in the filler, and the other compounds are not retained in the filler. .
If the mobile phase solvent is kept flowing in that state, other compounds except the enzyme retained in the packing material will be removed from the column. After the removal of the other compounds from the column is completed, the target enzyme can be separated by using an appropriate means to suppress the interaction between the enzyme and the substrate.

However, such a filler capable of specifically retaining only the target compound is most suitable for separating the target compound, but can be used for separating compounds other than the target compound. However, there is a problem that the range of application as a filler is significantly narrowed, and the utility value is almost lost.

As described above, the conventional packing material for reversed phase chromatography can be applied to the separation of a wide range of compounds, but it is difficult to separate a specific compound with high efficiency. On the other hand, in a filler that can specifically retain only a specific compound,
While it is possible to separate a specific compound with high efficiency, the range of compounds that can be separated is significantly narrowed. In other words, these two types of fillers have properties that are mutually contradictory, and thus each have advantages and disadvantages. Therefore, a filler that has the advantages of these two types of fillers can separate a specific compound more efficiently than a conventional filler for reversed-phase chromatography, and can also remove only a specific compound. It can be applied to the separation of more compounds than a filler that can be specifically retained, and is expected to be an excellent filler that can overcome each other's problems.

On the other hand, with the rise of fine chemical technology in recent years, the number of compounds that need to be separated and purified has increased, and boronic acid derivatives to which a boronic acid group has been bonded are also targeted for such separation and purification. It is coming. That is, this boronic acid derivative is used as an interface for recognizing a carbohydrate having a cis diol structure, since the boronic acid group has a specific discriminating ability for the cis diol structure,
It is also used for medical purposes such as killing specific cells by incorporating a boronic acid derivative into cancer cells and irradiating the cells with low-energy neutrons. The boronic acid derivative needs to be highly purified in order to sufficiently exhibit its properties when used in such advanced technology.

Incidentally, such a boronic acid derivative is
In the case of purifying using a liquid chromatography technique, a conventional packing material for reversed-phase chromatography will be used as a packing material. Since it is expected that a boronic acid derivative that cannot be sufficiently separated will appear in the packing for phase chromatography, only the boronic acid derivative can be enhanced by utilizing the specific interaction with the boronic acid group. It is believed that a filler that separates efficiently is needed.

However, like boronic acid derivatives,
For compounds that do not exist in the living body, the interaction between the enzyme and the substrate or other biologically-related substances cannot be used. However, at present it is not realized.

Accordingly, the boronic acid derivative can be specifically retained, and the boronic acid derivative can be separated more efficiently than the conventional packing material for reversed-phase chromatography. In addition, it can be applied to the separation of compounds other than boronic acid derivatives, and it is also possible to separate compounds that cannot be separated with alkyl group-bonded packing materials that are widely used in reverse phase chromatography. There is a need for the development of new fillers.

[0014]

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a packing material for reverse phase chromatography, which can specifically retain a boronic acid derivative. To provide a packing material for liquid chromatography which can also be used as a separation method, and a separation method capable of advantageously separating a boronic acid derivative using such an excellent packing material, and further, a reverse phase chromatography method. Accordingly, it is an object of the present invention to provide a separation method which can advantageously separate various substances other than the boronic acid derivative.

[0015]

In order to solve such a problem, the present inventors have repeatedly searched for and evaluated various functional groups, and as a result, a hydroxyl group was formed on at least one pair of adjacent carbon atoms on an aromatic ring. By using a packing material composed of a porous carrier chemically modified with an aromatic functional group bound thereto, it not only has separation characteristics as a packing material for reversed-phase chromatography, but also specializes boronic acid derivatives. They have also found that they also have a separation property that keeps them stable. Also,
In addition, the filler having such a structure has a strong interaction not only with hydrophobic interaction but also with a compound having an electron-withdrawing group or a hydrogen-bonding compound. Having found that it is possible to separate compounds that cannot be separated with an alkyl group-bonded packing material that is widely used in chromatography, a novel packing material for reversed phase chromatography and a separation method using the same have been established. It was.

That is, the present invention relates to a porous carrier chemically modified with an aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on an aromatic ring. A filler for liquid chromatography, characterized by being constituted by the above, is the gist of the invention.

In such a packing for liquid chromatography according to the present invention, by having such a predetermined structure, the interaction between the packing and the boronic acid derivative can be increased. Therefore, the boronic acid derivative can be more strongly retained.

Further, since such a packing material can retain a compound by hydrophobic interaction, it can be advantageously used as a packing material for reversed-phase chromatography, and is therefore advantageously applied to separation of a wide range of compounds. What you get.

Further, in the packing material for liquid chromatography according to the present invention, an aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on an aromatic ring is introduced into a predetermined carrier. As a result, the interaction with the retained compound is not merely a hydrophobic interaction, so that the compound having similar hydrophobicity that could not be separated well with the conventional packing material for reversed phase chromatography was used. Can also be advantageously applied.

According to one preferred embodiment of the packing material for liquid chromatography according to the present invention, the porous carrier is silica gel or a porous polymer.

In the present invention, when a boronic acid derivative is separated by a liquid chromatography technique, at least one pair of adjacent carbon atoms on an aromatic ring has a hydroxyl group as a filler that specifically retains the boronic acid derivative. The present invention also provides a separation method characterized by using a filler composed of a porous carrier chemically modified with an aromatic functional group to which is bonded. And
As described above, when the boronic acid derivative is separated by the liquid chromatography technique, a filler capable of specifically retaining the boronic acid derivative is used, so that the boronic acid derivative can be advantageously separated.

Further, the present invention provides a method for separating a filler by an aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on an aromatic ring upon separation by a reversed-phase liquid chromatography technique. The separation method composed of the modified porous carrier is also the gist thereof.

According to one preferred embodiment of the separation method according to the present invention, the porous carrier is silica gel or a porous polymer.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS As the filler according to the present invention, as described above, an aromatic functional group having a structure in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on an aromatic ring (hereinafter referred to as an aromatic functional group). , Simply referred to as aromatic functional groups)
As long as the porous carrier is chemically modified, any form may be used. However, in general, an Ar-A-porous carrier material (where Ar: aromatic functional group, A : Spacer group), a chemical bond type filler having a structure represented by the formula:

The above-mentioned aromatic functional group is chemically bonded to the porous carrier material which gives the target filler by binding the aromatic functional group via a predetermined spacer group (A). Any known porous carrier material can be used as long as it can be bound and held, and is usually silica gel, porous glass, glass beads,
Inorganic carriers such as alumina, titania, and zirconia, and porous polymers such as polystyrene gel, dextran gel, polyacrylamide gel, agarose gel, polyvinyl acetate gel, and polymethyl methacrylate gel will be used. Water-soluble polymers are preferably used. This is because silica gel has high mechanical strength, in addition to being easily chemically modified due to high reactivity of silanol groups, and is easily available, and This is because the porous polymer is stable to acids and alkalis.

As the aromatic functional group for chemically modifying the porous carrier material, any aromatic functional group can be used as long as a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on the aromatic ring. What you get. The aromatic ring constituting such an aromatic functional group may be monocyclic or polycyclic, and may be substituted on the aromatic ring as long as the effects of the present invention can be obtained. It may have a group. And examples of such aromatic rings include benzene, pentalene, indene, naphthalene, azulene, heptalene, biphenylene, acenaphthylene, fluorene, phenalene, phenanthrene, anthracene, fluoracene, acephenanthrylene, aceanthrylene, triphenylene, pyrene , Naphthacene, pleiaden, picene, perylene, pentaphene,
Pentacene, tetraphenylene, benzofluoranthine, hexaphen, hexacene, rubicene, coronene,
Trinaphthylene, heptafen, heptacene, pyranthrene, decacyclene, ovalen and the like can be mentioned.

Further, the spacer group represented by A, which is bonded between the aromatic functional group and the porous carrier material, has a large holding power to the compound by the filler, especially to the boronic acid. The moiety is expressed by a hydroxyl group bonded to at least one pair of adjacent carbon atoms on the aromatic ring constituting the aromatic functional group, and is hardly affected by the difference in the spacer group. Various known atomic groups can be used. And
Examples of such a spacer group include a linear hydrocarbon group such as a methylene group, an ethylene group, and a propylene group.
Even if an atomic group into which a functional group including a sulfur atom or a nitrogen atom or the like is used in addition to an ester bond and a carbonyl group is used,
No problem. Further, here, a structure having a spacer group is exemplified, but the structure is not limited to this.The aromatic functional group is directly bonded to the porous carrier material without using a spacer group. Even
No problem.

By the way, the amount of the stationary phase (aromatic functional group) in the filler according to the present invention affects the retention time of the solute. In general, when the amount increases, the solute and the stationary phase become incompatible with each other. Are increased, resulting in increased solute retention. Therefore, the use of such a filler having a large number of stationary phases bonded thereto exerts a strong retention of a target compound, for example, a boronic acid derivative, and as a result, the separation of those compounds is effectively performed. You will get. That is, in order to obtain a filler having a larger holding power for the target compound,
It is desirable that as many aromatic functional groups as possible be bound to the porous support. On the other hand, the lower limit of the amount of the stationary phase in the filler according to the present invention depends on the conditions,
Although not uniquely determined, it is generally sufficient that the amount is sufficient to effectively exert the holding power for the target compound.

Further, as the number of hydroxyl groups bonded to the aromatic functional group increases, the hydrophobic interaction between the stationary phase and the compound becomes weaker. The number of hydroxyl group bonds is particularly limited since the interaction with the compound having an attractive group is strengthened, and an effect other than the hydrophobic interaction is developed, so that the holding power of the filler does not decrease. Not something.

Here, the packing material for liquid chromatography according to the present invention as described above can be produced by bonding an aromatic functional group to a porous carrier using various known reactions. For example, when silica gel is used as a porous carrier, generally, 1) a method in which a silylating agent having an aromatic functional group is reacted with the silica gel carrier, and 2) an organometallic compound having an aromatic functional group is used. A method of reacting with a silica gel carrier,
3) After the reactive functional group is introduced into the silica gel carrier, it is manufactured by utilizing a reaction such as a method of binding an aromatic functional group to the functional group.

More specifically, in the above method (1) using a silylating agent, first, a compound having an aromatic functional group of interest and a compound having a double bond are reacted, and then the double bond is formed. The silylating agent is obtained by adding hydrosilane (H-Si) to the carbon-carbon double bond part of the alkene having a platinum catalyst. In this reaction, the addition of the anti-Markovnikov type proceeds mainly, and the reaction proceeds so that the silicon atom is bonded to the terminal of the alkene molecule. This method has a high conversion rate to the silylating agent,
Since there are few side reactions, there is an advantage that a silylating agent having a target structure can be easily obtained.However, a compound having a double bond may be polymerized, and particularly a polymerizable polymer such as used for polymer synthesis. In a compound having a high similarity to a monomer-like double bond, the polymerization reaction may proceed more than the addition reaction. The method for synthesizing a silylating agent does not use an addition reaction of hydrosilane.
A method of reacting an organochlorinated silicon compound having one or more reactive functional groups (mainly chlorine) with an organometallic reagent such as a Grignard reagent can also be used.

In the silylating agent obtained above, at least one of the four substituents bonded to the silicon atom is a silica gel such as chlorine, bromine, methoxy, ethoxy or alkylamino. It must be a functional group that reacts with silanol groups present on the surface of the support. There are also silylating agents having two or three reactive functional groups, all of which can be applied to chemical modification of a silica gel carrier with an aromatic functional group.

Then, the silylating agent having an aromatic functional group obtained above is reacted with a silanol group present on the surface of the silica gel carrier, whereby the silylating agent is chemically modified with the desired aromatic functional group. The resulting porous carrier (silica gel) can be easily obtained.

The method using an organometallic compound of the above 2) is a method reported by Locke et al. [DC Locke, J. et al.
J. Schermud and B. Banner, "Anal.Chem.", 44, 90 (1
972)]. In this method, first, a reagent such as thionyl chloride capable of replacing a hydroxyl group with chlorine is allowed to act on the silanol group of the silica gel carrier to replace the silanol group with chlorine. Then, the chlorine-substituted silica gel carrier is reacted with an organometallic compound that has been converted to a Grignard reagent having an intended aromatic functional group, whereby the aromatic functional group is bonded to the silica gel carrier and introduced. Thus, the hydroxyl group in the silanol group can be replaced with an aromatic functional group.

Further, the method of 3) for introducing a functional group having reactivity into a carrier and then bonding an aromatic functional group to the functional group according to the method of the above 3) is to convert the organometallic compound shown in the above 2) with the carrier. This method is similar to the reaction method, but in this method, first, a compound having a reactive functional group such as an amino group is reacted and bonded with a silanol group of a silica gel carrier, and then, a functional group capable of reacting with the amino group For example, an aromatic functional group having a carboxyl group and the silica gel carrier into which the reactive functional group has been introduced are combined with N, N′-
By condensing with a condensing agent such as dicyclohexylcarbodiimide, the intended filler can be obtained. In this example, an amide bond is finally formed, but the reaction of a combination of other functional groups forming an ester bond, an ether bond, or the like can be similarly performed.

As described above, the filler obtained by chemically modifying the silica gel carrier with an aromatic functional group can be produced by various synthetic methods. In the case where the filler is commercially available, it is preferable to produce the filler by the method 3) because the synthesis process can be simplified.

When the porous polymer is used as the porous carrier, the intended filler is produced by the same method as in the method 3) in the method of chemically modifying the silica gel carrier with an aromatic functional group. You can do it. More specifically, first, a compound having a reactive functional group such as an amino group is reacted and bonded with a porous polymer in advance,
Introduce reactive functional groups into the porous polymer. Then, a compound having a functional group capable of reacting with the amino group, for example, a carboxyl group and having an aromatic functional group is prepared, and the porous polymer having the reactive functional group introduced therein is N, N ′. By condensing with a condensing agent such as -dicyclohexylcarbodiimide or N, N'-diisopropylcarbodiimide, the intended filler can be obtained.

The present invention is also characterized in that the boronic acid derivative is separated using the packing material for liquid chromatography according to the present invention as described above. Derivatives can now be separated, but operations or conditions similar to those in the related art will be adopted for such a boronic acid derivative separation operation by liquid chromatography.

That is, in such a separation operation, the boronic acid derivative is supplied as a solution dissolved in a predetermined solvent to the column packed with the packing material according to the present invention. By the way, as the predetermined solvent used here, methanol, ethyl acetate, tetrahydrofuran, chloroform, water and the like are usually used.

The boronic acid derivative supplied in the form of a solution into the separation column is held by a filler,
Subsequently, the boronic acid derivative is sequentially eluted and separated by the mobile phase solvent passed through the separation column. As the mobile phase solvent used here, a solvent used in ordinary reversed-phase liquid chromatography, that is, a polar solvent is used. Specific examples of such a polar solvent include water, methanol, acetonitrile, and formamide. Or a mixed solvent thereof.

Further, the present invention is characterized in that the above-mentioned filler is used in the separation by the reversed-phase chromatography technique, whereby various compounds can be effected by the reversed-phase chromatography technique. Although the separation can be performed objectively, the same operation or conditions as those in the related art are employed in such a separation operation by the reverse phase chromatography technique.

[0042]

EXAMPLES In order to clarify the present invention more specifically, some examples of the present invention will be shown below.
It goes without saying that the present invention is not subject to any restrictions by the description of such embodiments. In addition, in addition to the following examples, the present invention, in addition to the above-described specific description, various changes, corrections, and modifications based on the knowledge of those skilled in the art without departing from the spirit of the present invention. It should be understood that improvements can be made.

Example 1 {Synthesis of Various Packing Materials for Liquid Chromatography}
─ As described above, the packing material for liquid chromatography according to the present invention can be synthesized by using various conventionally known techniques.In the present embodiment, a commercially available amino group-bonded silica gel packing material is used. Since it can be used as a porous carrier and can simplify the synthesis process of the filler, an amino-bonded porous carrier and an aromatic functional group having a functional group capable of reacting with such an amino group Are combined to form a desired filler. In addition, various bonding forms such as an amide bond, a urea bond, and a urethane bond can be selected as the bonding form between the amino group and the aromatic functional group. Since the method of forming an amide bond is simple, the form of the amide bond is applied.

However, in such a method for forming an amide bond, when a carboxylic acid derivative having a phenolic hydroxyl group is bonded to an amino group, the amide bond-forming carboxylic acid derivative represented by the following formula (a) is formed. In addition to the reaction (amidation reaction), it is expected that a phenolic hydroxyl group reacts with the acid anhydride to form an ester bond, and the side reaction shown in the following chemical formula (b) proceeds. Therefore, conditions for suppressing a side reaction caused when an amide bond is formed will be examined. In the following chemical formula 1, Ar represents an aromatic ring.

(A) HO—Ar—COOH + H ₂ N— (carrier) →
HO-Ar-CONH- (carrier) (b) HO-Ar-COOH + HO-Ar-COO
H → HO-Ar-COO-Ar-COOH

More specifically, first, phenol is used as the compound having a phenolic hydroxyl group, benzoic acid is used as the compound having a carboxyl group, and hexylamine having an amino group is used instead of the amino group-bonded porous carrier. Were prepared and reacted. More specifically, phenol: 0.47 g, benzoic acid: 0.61
g and hexylamine: 0.51 g were dissolved in N, N'-dimethylformamide: 20 mL, and N, N'-diisopropylcarbodiimide: 0.6 was used as a condensing agent.
3 g was added, and the condensation reaction (amidation reaction) was allowed to proceed under uniform solution conditions (reaction solution A). In addition, the reaction further proceeded with the addition of 1-hydroxybenzotriazole: 0.68 g during the condensation reaction (reaction solution B). It is generally known that 1-hydroxybenzotriazole promotes an amidation reaction.

Next, the reaction products obtained in each of the above cases were analyzed by high performance liquid chromatography to measure the peak area of the esterified product and the peak area of the amidated product. The analysis by high performance liquid chromatography was performed using a column of Cosmosil 5
C18-MS (inner diameter: 4.6 mmφ, length: 150 m
m) using a mixed solvent of methanol: 0.1% trifluoroacetic acid aqueous solution = 60: 40 (volume ratio) as a mobile phase solvent.

Then, using the peak area value of the amidated compound and the peak area value of the esterified compound measured above, the formation ratio of the amidated compound and the formation ratio of the esterified compound are calculated according to the following formulas. The results are shown in Table 1 below. Production rate of amidated product = peak area value of amidated product /
(Peak area value of amidated product + peak area value of esterified product) × 100 (%) Production ratio of esterified product = 100−Production ratio of amidated product (%)

[0049]

[Table 1]

As is evident from the results shown in Table 1, when the condensation reaction was allowed to proceed without adding 1-hydroxybenzotriazole, the esterified product which was a by-product was obtained. 7% was produced. On the other hand, when 1-hydroxybenzotriazole is added and the condensation reaction is allowed to proceed, almost no esterified product, which is a by-product, is produced, and the amide, which is the main reaction product, is produced almost quantitatively. 99.8% of the compound was produced. As described above, when an amide compound is synthesized by reacting an amino group and a carboxylic acid having a phenolic hydroxyl group, the addition of 1-hydroxybenzotriazole promotes the formation of an amidated product, As a result, it was confirmed that an undesirable side reaction (esterification reaction) can be effectively suppressed.

Therefore, in this example, when a carboxylic acid derivative is reacted with an amino group-bonded porous carrier in order to synthesize a packing material for liquid chromatography, 1-hydroxybenzotriazole is used. The reaction conditions to be added were adopted.

Next, based on the above experimental results, the corresponding carboxylic acid derivative was reacted with Cosmosil 5NH ₂ (manufactured by Nacalai Tesque, Inc.), which is a commercially available carrier (filler) having an aminopropyl group introduced into silica gel, based on the above experimental results. By doing so, each filler having a stationary phase structure shown in Table 2 below (2,3-OH type filler, 3,4-OH type filler, 3,4,5-OH type filler, 3 , 4-OH-1 type filler, CH ₃ type filler, 3,5-OH type filler). More specifically, a method of synthesizing a filler will be described in detail below, taking as an example a case where a 3,4-OH type filler is synthesized. In Table 2 below, 2,3-OH
Type filler, 3,4-OH type filler, 3,4,5-OH type filler, 3,4-OH-1 type filler, CH ₃ type filler,
The structure represented by “Si.” On the carrier side of the 3,5-OH type filler is, to be precise, “Si≡”, “Si (OC ₂ H
₅ ) = ”,“ Si (OH) = ”,“ Si (OC ₂ H ₅ )
₂ - "," _{Si (OH) (OC 2 H} 5) - "," Si
(OH) _2- ”.

[0053]

[Table 2]

First, 3,4-dihydroxybenzoic acid:
0.86 g, 1-hydroxybenzotriazole: 0.1.
76 g of N, N'-dimethylformamide: 20 mL
Was dissolved. In this solution, Cosmosil 5NH ₂ :
After adding 2.0 g and dispersing, 0.71 g of N, N'-diisopropylcarbodiimide was added and stirred at room temperature. Then, the gel obtained from such a reaction solution after a lapse of one night is suction-filtered and washed with N, N′-dimethylformamide, methanol and chloroform, and then dried at 50 ° C. to form a gel adjacent to the benzene ring. A 3,4-OH type filler chemically modified with a functional group in which two hydroxyl groups are bonded to the matching carbon atoms at the 3- and 4-positions was obtained. Note that Cosmosil 5NH ₂ is a filler obtained by chemically modifying porous spherical silica gel having a particle diameter of 5 μm and a pore diameter of 12 nm with aminopropyltriethoxysilane, and further chemically modifying it with hexamethyldisilazane.

Further, in the same manner as in the synthesis of the 3,4-OH type filler, the 2,3-OH type filler, 3,3
4,5-OH type filler, 3,4-OH-1 type filler, C
For H ₃ type fillers and 3,5-OH type fillers,
It synthesize | combined using the hydroxyl-substituted carboxylic acid derivative of a respectively corresponding structure instead of 3,4- dihydroxybenzoic acid.

Incidentally, 3,4-
The OH-P type filler was prepared in the same manner as in the synthesis of the 3,4-OH type filler except that a porous polymer carrier having an amino group introduced therein was used instead of Cosmosil 5NH ₂ : 2.0 g. This is the filler obtained. Incidentally, the porous polymer carrier into which the amino group was used used was glycidyl methacrylate and ethylene glycol dimethacrylate as a cross-linking agent, toluene as a diluent, and α, α′-azobisisomethol as a polymerization initiator. It is obtained by copolymerizing in the presence of an aqueous solution in which butyronitrile and polyvinyl alcohol as a suspension stabilizer are dissolved to obtain a porous spherical polymer, which is further heated and refluxed in ammonia. It is.

Further, a C ₁₈ type filler (octadecyl group-bonded type filler; Cosmosil 5C18-MS: manufactured by Nacalai Tesque KK) and a Diol type filler (diol type filler; Co
smosil 5Diol: manufactured by Nacalai Tesque, Inc.) is a filler that has been conventionally used.
Are also shown.

The fillers shown in Table 2
In the number, the number described before "-OH" is
The position where the carbonyl group of the aromatic ring is bonded was taken as the 1st position
The bonding position of the hydroxyl group at that time is shown. In addition, "-O
H ”and subsequent numbers indicate the methylene spacer
Indicates the number of groups. Furthermore, it is indicated after "-OH".
P indicates that it is a filler using a polymer carrier
I have. Furthermore, "CH _Three"Is a hydroxyl-substituted benzene ring
CH instead of_ThreeIndicates that the group is the filler used.
doing.

Table 3 below shows the carbon content of the various fillers synthesized above (excluding the 3,4-OH-P type filler) and the amount of the aromatic functional group bonded to the filler. Is shown. For comparison, the carbon content of the Cosmosil 5NH ₂ type filler and the amount of amino groups bonded to the surface of the filler are also shown.

[0060]

[Table 3]

As shown in Table 3, the 2,3-OH type filler, 3,4-OH type filler,
4,5-OH type filler and CH ₃ type filler of Comparative Example, 3,
A filler synthesized from a Cosmosil 5NH ₂ type filler such as a 5-OH type filler has an aromatic functional group bonded to the amino group introduced into the Cosmosil 5NH ₂ type filler at a rate of 15 to 70%. The amount of binding is
Differences were noted due to the attached aromatic functionality.
For example, a hydroxyl group is bonded to the 2-position of a benzene ring,
In the case of the 3-OH type filler and the 3,4,5-OH type filler in which three hydroxyl groups are bonded on the benzene ring, a decrease in the amount of aromatic functional groups bonded was observed. This is considered to be because the amount of binding to the carrier was reduced due to steric hindrance based on the steric size of the aromatic functional group.

Example 2 << Retention Characteristics for Benzeneboronic Acid >> In this example, each packing material for liquid chromatography synthesized in the above-mentioned Example 1 was 4.6 mm in inner diameter.
φ, length: packed in a 150 mm chromatographic tube, and using it, acetonitrile: 0.02 M phosphate buffer (p
Using a mixed solvent of H4) = 50: 50 (volume ratio) as a mobile phase, benzene and a benzeneboronic acid derivative were actually separated by liquid chromatography.

More specifically, the liquid chromatography apparatus includes a liquid sending pump (LC-6A: manufactured by Shimadzu Corporation), a column packed with each of the above packing materials, and a sample injector (model 7125: Leodyne). UV detector (SPD-6A UV detector: manufactured by Shimadzu Corporation)
And the flow rate of the mobile phase solvent was all set to 1.0 mL / min. The measurement was performed with the detection sensitivity of the ultraviolet absorption detector set to 0.16 AUFS and the detection wavelength set to 254 nm. The temperature of the column was adjusted by a constant temperature water bath to 30 ° C. Furthermore, a data processor (CR-5A: manufactured by Shimadzu Corporation) was used for measuring the elution time.

Table 4 below shows the retention characteristics of each filler against benzene and benzeneboronic acid, using the k 'value as an index.

In general, when the retention characteristic of a column (filler) is exhibited, the time from the injection of a sample into the column to the top of the peak eluted from the column (retention time) is used. The time can be compared only when the flow rate of the mobile phase solvent to be sent is the same and the same column is used, so that the relative evaluation can be performed. In the case of comparison, when the size of the column is different, or when the liquid sending speed of the mobile phase solvent is different, it is impossible to make a relative comparison even if the retention times are compared. Therefore, in comparison of the results of chromatographic separation under different conditions, usually, the value represented by k ′ in the following equation, that is, the ratio of the retention time of the retained solute in the column to the elution time of the unretained solute, Thus, the retention characteristics (retention power) of each solute are evaluated. _{k '= (t r -t 0} ) / t 0

[0066] However, t _r: retention time of the solute to be held,
t ₀ : elution time of unretained solute. Then, by using such a k ′ value, it is possible to ignore the influence of a difference in column length, thickness, flow rate, and the like, and thus it becomes possible to perform pure evaluation of the characteristics of the filler. is there.

Then, one boronic acid group is bonded to the benzene ring.
Each filler for benzeneboronic acid with combined structure
The specificity of retention is determined by the retention characteristics of benzene
(K ' _AOf benzeneboronic acid by fillers for
Characteristic (k '_B) Ratio: separation coefficient (α = k ′)_B/
k '_A). Specifically, benzene is
No boronic acid group, mainly by hydrophobic interaction
Large separation factor due to retained compounds
Value, the more specific to benzeneboronic acid
It shows that it is a filler having the power to perform.

[0068]

[Table 4]

As apparent from the results in Table 4, the 2,3-OH type filler of the present invention, which was synthesized using an amino group-bonded type silica gel filler as a porous carrier, 3,4
-OH type filler, 3,4,5-OH type filler, and 3,
4-OH-1 type filler has a separation coefficient of 20 or more,
It was shown to have a specifically strong retention of benzeneboronic acid. Further, the 3,4-OH-P type filler of the present invention synthesized using a porous polymer carrier also shows a large separation coefficient of 10.88, and is specific for benzeneboronic acid. It was shown to have strong holding power. On the other hand, the carrier is chemically modified with an aromatic functional group bonded to carbon atoms that are not adjacent to each other, although a CH ₃ type filler having no aromatic ring or hydroxyl group or having two hydroxyl groups on the aromatic ring. The resulting 3,5-OH-type filler had a separation factor of 1 or less, indicating that it had only a weak retention of benzeneboronic acid. Further, a C ₁₈ type filler which is a conventionally used filler which can be applied to separation of a wide range of compounds, and a hydroxyl group is bonded to carbon atoms adjacent to each other, but a hydroxyl group is bonded to an aromatic ring. Even in the case of the Diol-type packing material in which the carrier is chemically modified with no organic functional group, it showed a separation coefficient of 2 or less and only a weak holding power. In general, it is known that a boronic acid group has a specific interaction with a compound having a diol-type functional group. It is considered that a specific holding power cannot be obtained with a filler having a hydroxyl group bonded to adjacent carbon atoms other than the aromatic ring.

From a comparison between the separation coefficient of the 2,3-OH type filler and the separation coefficient of the 3,4-OH type filler, two hydroxyl groups bonded to adjacent carbon atoms in the aromatic ring were compared. The difference in the separation coefficient due to the difference in the bonding position is about three times, and the separation coefficient between the 3,4-OH type filler and 3,4,5-O
From the comparison with the separation coefficient of the H-type filler, the difference in the separation coefficient due to the difference in the number of hydroxyl groups bonded to the carbon atom in the aromatic ring is about 1.7 times, and further, the 3,4-OH type From the comparison between the separation coefficient of the filler and the separation coefficient of the 3,4-OH-1 type filler, the difference in the separation coefficient due to the difference in the length of the spacer connecting the aromatic ring functional group having a hydroxyl group and the silica gel filler is shown. Was shown to be about 1.1 times. In addition, 3, 4
From the comparison between the separation coefficient of the -OH type filler and the separation coefficient of the 3,4-OH-P type filler, the difference in the separation coefficient due to the difference in the porous carrier chemically modified with the aromatic functional group is about 2. .6
It was shown to be double.

From these facts, the bonding position of the hydroxyl group bonded to at least one pair of adjacent carbon atoms on the aromatic ring, the number of hydroxyl groups bonded, and the bonding between the aromatic ring functional group having a hydroxyl group and the porous carrier are performed. Depending on the length of the spacer or the carrier chemically modified with a functional group, the specificity for benzeneboronic acid is considered to cause a difference of less than three times. However, the separation coefficient (10.88 to 80.42) of the filler of the present invention example in which a plurality of hydroxyl groups are chemically modified with functional groups bonded to adjacent carbon atoms in the aromatic ring,
In comparison with the separation coefficient of the filler of the comparative example (0.12 to 1.64), the separation coefficient of the filler of the present invention gives a value of 6 to 670 times the separation coefficient of the filler of the comparative example. However, the specific retention properties for benzeneboronic acid are:
It is presumed to be mainly expressed by a structure in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on the aromatic ring, and the contribution of other factors is considered to be relatively small.

Accordingly, from the above results, even if the filler other than the filler used in the present embodiment is used, the aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on the aromatic ring. It is thought that if the carrier is chemically modified with a group, a high specificity for benzeneboronic acid can be obtained.

Example 3 {Effect of pH on Retention of Benzeneboronic Acid}
充填 The specific strong retention of benzeneboronic acid by a filler chemically modified with an aromatic functional group having a hydroxyl group bonded to at least one pair of adjacent carbon atoms on the aromatic ring is as follows:
It is thought to be based on the interaction between the boronate anion generated by dissociation of the boronic acid group and the hydroxyl group bonded to at least one pair of adjacent carbon atoms on the aromatic ring of the filler. For this reason, the relationship between the pH value of the mobile phase, which is expected to affect the dissociation of the hydroxyl group on the boronic acid group and the aromatic ring, and the separation coefficient for the benzeneboronic acid derivative by the filler were investigated.

More specifically, each p as shown in Table 5 below
5 of phosphate buffer solution and acetonitrile giving H value
Using a mixed solvent of 0:50 (volume ratio) as a mobile phase, 3,4-
Using OH type filler, benzene and benzeneboronic acid were separated, and their retention characteristics (k ′ value) were measured. The results are shown in Table 5 below. Further, the separation coefficient of benzeneboronic acid with respect to benzene was calculated from the retention characteristics, and the results are shown in Table 5 below. The number in parentheses after the pH value in Table 5 indicates the actual pH value of the mobile phase measured after mixing the phosphate buffer solution and acetonitrile.

[0075]

[Table 5]

As is apparent from the results shown in Table 5, the separation coefficient of benzeneboronic acid with respect to benzene is
It was shown that the pH changed significantly with the change in pH. More specifically, the retention characteristic (k 'value) of benzene does not change due to the difference in the pH of the mobile phase, but the retention characteristic (k' value) of benzeneboronic acid shows a large value at pH 9 to 10, and the separation is accordingly accompanied. The coefficient also showed the maximum value at pH 9 to 10. The reason for this is that the boronic acid group is known to be easily dissociated as it becomes more alkaline, and in the acidic mobile phase, the dissociation of the boronic acid is reduced and the interaction with the hydroxyl group on the aromatic ring is reduced. It is considered that it is difficult to form. Further, a decrease in the separation coefficient was observed at pH 11 or higher, which is considered to be due to the fact that the hydroxyl group bonded to the carbon atom on the aromatic ring was dissociated, and it became difficult to form a bond with the boronate anion. Can be

Further, the separation coefficient at pH 10 is pH
When the separation coefficient reaches 150 times or more of the separation coefficient in 3, and when the boronic acid derivative is separated, by changing the pH value of the mobile phase used, benzeneboronic acid is specifically retained by the filler, and It is understood that elution of the existing benzeneboronic acid can be easily controlled.

Example 4 {Retaining Properties for Various Boronic Acid Derivatives} Using a 3,4-OH type filler, acetonitrile: 0.1.
Various types of boron shown in Table 6 below, using 02M phosphate buffer solution (pH3) = 50: 50 (volume ratio) or acetonitrile: 0.02M phosphate buffer solution (pH4) = 50: 50 (volume ratio) as a mobile phase. The acid derivative was separated, and its retention characteristics were measured. The results are shown in Table 6 below.

[0079]

[Table 6]

From the results shown in Table 6, the retention characteristics of the boronic acid derivative were similar to those in Example 3 except that the pH of the mobile phase was
Therefore, it is considered that the filler according to the present invention specifically retains boronic acid derivatives other than benzeneboronic acid.

As for the effect of the substituent of the boronic acid derivative on the retention characteristics, the boronic acid derivative substituted with an electron-withdrawing group such as a nitro group exhibited a retention characteristic larger than that of benzeneboronic acid. In contrast, derivatives substituted with electron donating groups such as methyl groups showed smaller retention properties than benzeneboronic acid. This is because even under the same mobile phase conditions, a boronic acid derivative substituted with an electron-withdrawing functional group has a more stable boronate anion and is more likely to interact with a filler. The reason for this is that while the holding power of the filler increases, the boronic acid derivative substituted with the electron-donating functional group hardly interacts with the filler, so that the holding power of the filler decreases. Conceivable. Therefore, it is considered that the difference in the retention characteristics for each boronic acid derivative shown in Table 6 is mainly caused by the difference in the chemical properties of the retained compounds. According to the filler according to the present invention, the boronic acid derivatives are different from each other. It is suggested that can be effectively separated.

Example 5 << Effect of Acid or Base on Retention of Benzeneboronic Acid >> Transferring a solvent prepared using an acid or base shown in Table 7 below without using a phosphate buffer. 3,4-OH as phase
The retention characteristics when separating benzene and benzeneboronic acid were measured using a P-type filler, and the separation coefficient of the benzeneboronic acid derivative with respect to benzene was determined from the retention characteristics. Are also shown.

[0083]

[Table 7]

As is evident from the results shown in Table 7, even if the mobile phase is composed of a solvent prepared using an organic acid or an organic base such as trifluoroacetic acid, acetic acid, N-methylmorpholine or triethylamine, Example 3
As in the case of using the phosphate buffer, the retention characteristics (k ′ value) of benzene are almost the same regardless of the change in pH value, but the retention characteristics of benzeneboronic acid (k ′ value) )
Are separated using a mobile phase that provides a pH value at which an interaction between the boronate anion and a hydroxyl group bonded to at least one pair of adjacent carbon atoms on the aromatic ring is likely to be formed,
The longer the benzeneboronic acid is retained, the higher the pH
It was confirmed that the change in the retention characteristics of the boronic acid derivative due to the change in the value was caused even when the pH value was adjusted without using a phosphate buffer. Furthermore, benzeneboronic acid has a relatively low retention property when separated using an organic solvent prepared without water as a mobile phase, and when an organic solvent to which trifluoroacetic acid is added is used as a mobile phase. On the other hand, when the organic solvent to which N-methylmorpholine is added is used as the mobile phase, the retention characteristics are relatively large. Therefore, the filler according to the present invention chemically modified with an aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on the aromatic ring can be used even when a non-aqueous mobile phase is used. It is understood that the derivatives can be effectively separated.

As described above, Embodiments 2, 3, 4, and 5
From the results, the filler according to the present invention chemically modified with an aromatic functional group having a hydroxyl group bonded to at least one pair of adjacent carbon atoms on the aromatic ring, by adjusting the pH value of the mobile phase, It is possible to control the retention properties for the boronic acid derivative, and thus adjust the pH value of the mobile phase so that the filler retains the boronic acid derivative more specifically or vice versa. It has been shown that the filler has the property of facilitating the elution of the boronic acid derivative used.

Example 6 {Evaluation of Retention Characteristics of Mono-Substituted Benzene} Here, a chemically modified aromatic functional group having a hydroxyl group bonded to at least one pair of adjacent carbon atoms on an aromatic ring was used. Separation characteristics when the packing material according to the present invention is used as a packing material for reversed phase chromatography will be examined.

More specifically, various mono-substituted benzenes were separated using a 3,4-OH type filler or a C ₁₈ type filler conventionally used in reverse phase chromatography,
The retention characteristics were determined. Then, find the logarithmic value,
The results are shown in Table 8 below. Log of various monosubstituted benzenes
The value of P is also shown in Table 8 below. Where L
The value of og P is a distribution coefficient between octanol and water of a compound, and a larger value indicates a compound having higher hydrophobicity. In addition, since the magnitude of hydrophobicity of both fillers is remarkably different, when separating various monosubstituted benzenes with a 3,4-OH type filler, methanol: water = 30: When a mixed solvent of 70 (volume ratio) was used and various monosubstituted benzenes were separated with a C ₁₈ type filler, a mixed solvent of methanol: water = 60: 40 (volume ratio) was used as a mobile phase.

[0088]

[Table 8]

From the results shown in Table 8, the logarithm (Log) of the retention characteristics of benzene (substituent: -H), toluene (substituent: -CH ₃ ) and ethylbenzene (substituent: -CH ₂ CH ₃ ) was obtained. From the comparison of k ′ values), it can be seen that a compound having more methylene groups (—CH ₂ —), which are hydrophobic groups, in both the 3,4-OH type filler and the C ₁₈ type filler can be more strongly retained. It was confirmed that the packing material according to the present invention can exhibit the separation characteristics as a packing material for reversed-phase chromatography because of the characteristic properties of the packing material for phase chromatography.

Further, the correlation between the logarithm of the retention characteristic of monosubstituted benzene and the Log P value shows that in the case of a filler having an alkyl group bonded to silica gel, such as a _C18 type filler,
The correlation coefficient was 0.99, indicating that the separation characteristics of the _C18- type filler were mainly governed by hydrophobic interactions, whereas the 3,4-OH-type filler according to the present invention was In Comparative Example 1, the correlation coefficient was a relatively low value of 0.69, suggesting that the retention characteristics of the 3,4-OH type filler were not solely due to hydrophobicity. In addition, the retention characteristics of a compound to which an electron-withdrawing functional group such as a nitro group, an ester, or a cyano group is bonded are larger than the retention characteristics predicted from the hydrophobicity of the compound. It is believed that the fillers according to the invention have separation properties due to electronic interactions in addition to hydrophobic interactions.

Example 7 {Retaining Property for Compound Having Electron-Withdrawing Group}
３ ， Methanol: water = 20: 80 (volume ratio) as mobile phase, 3,4-OH type filler, 3,4,5-OH type filler, CH ₃ type filler, and C ₁₈ type filler Are separated into benzene and nitrobenzene, and the benzene and nitrobenzene retention characteristics (k ′ _A ), the retention characteristics for nitrobenzene bonded with a nitro group, which is an electron-withdrawing group (k ′ _C ), and the nitrobenzene for benzene , And the separation coefficient (k ′ _C / k ′ _A ) of
It was shown to. Here, since benzene is a compound having no functional group that gives charge transfer, the separation coefficient (k ′ _C /
It is considered that the larger the value of k ' _A ), the stronger the interaction with the compound having an electron-withdrawing group.

[0092]

[Table 9]

As shown in Table 9, 3,4-OH
The separation coefficient of the mold filler is 3.30 times the separation coefficient of the _C18 filler.
4 times as high as 1.8 times the separation factor of CH ₃ type packing material without aromatic ring or hydroxyl group, and hydroxyl group is bonded to at least one pair of adjacent carbon atoms on aromatic ring. It is suggested that the action of the aromatic functional group can exert the property of strongly interacting with the compound having the electron-withdrawing group. Also, 3,4-OH type filler and 3,4
When compared with the 5-OH type filler, a filler chemically modified with an aromatic functional group having an increased number of hydroxyl groups on an aromatic ring, such as a 3,4,5-OH type filler, has a hydrophobic property. On the other hand, it was shown that the separation coefficient was increased and the separation ability was improved because the interaction with the compound having an electron-withdrawing group was increased while the property was easily lowered. In Table 9, in all the fillers, separation was performed using the same mobile phase. However, the concentration of methanol contained in the mobile phase was reduced to maintain the 3,4-OH type filler. By increasing, based on increased hydrophobic interaction, the substantial resolution of benzene and nitrobenzene is
It is believed to be greater than that of the _C18 type filler.

Example 8 << Hydrogen Bonding Property of Filler >> Caffeine having a carbonyl group or a nitrogen-containing heterocyclic ring structure in the molecule is a hydrogen bonding compound. By examining the retention properties, it is possible to evaluate the interaction of the filler with the hydrogen bonding compound. Therefore, in the present embodiment, the retention characteristics for caffeine will be examined.

More specifically, methanol: water = 20: 8
0 (volume ratio) as the mobile phase,
H-type fillers, 3, 4, 5-OH-type filler, with each of CH ₃ type filler and C ₁₈ type filler, to separate the phenol and caffeine, phenol retention characteristics (k '
_D ) and caffeine retention characteristics (k ' _E ) were measured, and the separation factor of caffeine from phenol (k' _E /
k ′ _D ). The results are shown in Table 10 below. In addition, phenol is less susceptible to the effect of hydrogen bonding than caffeine, and is a compound that is mainly retained by hydrophobic interaction. Is considered to be a filler that interacts strongly with

[0096]

[Table 10]

As is apparent from the results shown in Table 10, the separation coefficient of the 3,4-OH type filler is about 64 times that of the CH ₃ type filler, and is smaller than that of the C ₁₈ type filler. About 2
This was a very large value of twice. Therefore,
The filler according to the present invention, for a hydrogen bonding compound,
It was suggested that it exerted a stronger interaction. As described above, the filler according to the present invention is considered to be a filler that has an interaction with a compound having a hydrogen bond in addition to an interaction with a compound having a hydrophobic interaction or an electron-withdrawing group. Further, when the separation coefficients of the 3,4-OH type filler and the 3,4,5-OH type filler were compared, it was found that the 3,4,5
The -OH type filler has an increased number of hydroxyl groups on the aromatic ring, so that the hydrophobicity is reduced, but the interaction with the hydrogen bonding compound is increased. The resolution was shown to be improved. Further, among the fillers shown in Table 10, the difference between the retention characteristics of caffeine and phenol is the largest.
It is a 3,4-OH type filler, and it has been shown that caffeine and phenol can be separated more advantageously than a C ₁₈ type filler.

Example 9 {Separation of isomers} Using each of 3,4-OH type filler, CH ₃ type filler and C ₁₈ type filler, mixing shown in the following Table 11 was carried out. Using a solvent as a mobile phase, 3
Separation of methyl acetophenone in which two isomers are present was carried out, and the retention characteristics were measured. Then, the separation coefficient of each isomer was determined, and the results are shown in Table 11 below. Table 1 below
In 1, the mixing ratio of the mobile phase is shown by volume ratio. In Table 11, the m-isomer / o-isomer is the separation coefficient of the m-isomer with respect to the o-isomer, and the p-isomer / o-isomer is the separation coefficient of the p-isomer with respect to the o-isomer. The p-isomer / m-isomer represents the separation coefficient of the p-isomer from the m-isomer.

[0099]

[Table 11]

As is clear from the results in Table 11,
Separation coefficient of meta isomer from ortho isomer of methyl acetophenone in CH ₃ type filler or C ₁₈ type filler (m
-/ O-) are 1.11 and 1.00, respectively;
While it can hardly be separated, 3,4-OH
The separation coefficient (m− / o−) of the meta isomer with respect to the ortho isomer of the mold filler was 1.22, indicating that the ortho isomer and the meta isomer can be separated. Separation coefficient of para isomer to ortho isomer (p- / o
-) Also showed the highest value when it was separated using a 3,4-OH type filler. Further, regarding the separation coefficient (p− / m−) of the para isomer with respect to the meta isomer,
3,4-OH type filler is equivalent to C ₁₈ type filler (0.9
The reciprocal of 6 gave a separation factor of 1.04). Therefore, if a liquid chromatography technique is carried out using the packing material according to the present invention, isomers which are difficult to separate with a C ₁₈ type packing material, which is an alkyl group-bonded packing material conventionally applied to the separation of a wide range of compounds. Can be advantageously separated.

In each of the above embodiments, a column having an inner diameter of 4.6 mmφ and a length of 150 mm is used. However, the present invention is not limited to such a column. The inner diameter and length can be appropriately selected within a range in which the target compound is separated. Also, regarding the method of sending the mobile phase solvent,
The method is not limited to a method using a liquid sending pump such as high performance liquid chromatography (HPLC), but is a flash chromatography method employing a pressurized liquid sending method using compressed air, an open column chromatography using gravity-driven gravity flow. It is needless to say that the present invention can be applied to a thin-layer chromatography technique utilizing a solvent transfer by a capillary technique or a capillary phenomenon.

The packing material for liquid chromatography according to the present invention can be used not only for liquid chromatography, but also as an adsorbent for boronic acid derivatives.

[0103]

As is apparent from the above description, according to the packing material for liquid chromatography according to the present invention, the boronic acid derivative is advantageously retained and separated by performing liquid chromatography separation using the packing material. You can do it. Further, such a filler can also be used as a general-purpose filler for reversed-phase chromatography, and can advantageously retain and separate various compounds. Furthermore, in the filler according to the present invention, since a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on the aromatic ring, not only the hydrophobic interaction but also the electronic property is maintained. It is expressed by interactions and hydrogen bonding interactions.

Further, according to the method for separating a boronic acid derivative according to the present invention, since a packing material for liquid chromatography which can advantageously retain the boronic acid derivative is used as the packing material, the boronic acid derivative can be efficiently used. It is possible to separate them.

Further, in the separation method by the reversed phase chromatography method according to the present invention, since the packing having excellent characteristics as described above is used, when the conventional packing for reversed phase chromatography is used. Can be efficiently separated due to differences in electronic interactions and hydrogen bonding interactions, even if they have similar hydrophobic properties that could not be separated.

Claims (5)

[Claims] 1. A liquid chromatograph comprising a porous carrier chemically modified with an aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on an aromatic ring. Filler for graphy. 2. The packing material for liquid chromatography according to claim 1, wherein the porous carrier is silica gel or a porous polymer. 3. When separating a boronic acid derivative by a liquid chromatography technique, a hydroxyl group is bound to at least one pair of adjacent carbon atoms on an aromatic ring as a filler that specifically retains the boronic acid derivative. Using a filler composed of a porous carrier chemically modified with an aromatic functional group. 4. The method of claim 1, wherein the separation is carried out by a reversed phase liquid chromatography method. The packing material is formed of a porous material chemically modified with an aromatic functional group in which a hydroxyl group is bonded to at least one pair of adjacent carbon atoms on an aromatic ring. A separation method characterized by comprising a hydrophilic carrier. 5. The method according to claim 3, wherein the porous carrier is silica gel or a porous polymer.