Column filler for chromatography, column for chromatography, manufacturing method for column for chromatography, chromatography analyzer, and analysis method

Abstract

The purpose of the present disclosure is to provide: a column filler which is for chromatography and suitable for the separation and the like of a mixture containing various analytes; a column for chromatography; a manufacturing method for a column for chromatography; a chromatography analyzer; and an analysis method.　This column filler for chromatography is characterized in that a silanol group of silica contained in the filler is modified with a group represented by chemical formula (I).

Description

Chromatography column packing material, chromatography column, method for manufacturing a chromatography column, chromatography analysis device, and analysis method

[0001] The present disclosure relates to a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analysis device, and an analysis method.

[0002] Chromatography is commonly used as a method for separating mixtures. In chromatography, a carrier used to transport the sample is a gas, liquid, supercritical fluid, or the like, depending on the characteristics of the components to be separated. Liquid chromatography is mainly used for the separation and analysis of plant components. Hydrophobic silica gel modified with octadecylsilyl groups (hereinafter sometimes referred to as "ODS groups") is widely used as a column packing material for liquid chromatography (Patent Document 1).

[0003] Japanese Patent Application Laid-Open No. 2003-172733

[0004] On the other hand, analytical columns using hydrophobic silica gel as the column packing material are good at retaining hydrophobic analytes but are poor at retaining hydrophilic or amphiphilic analytes, and therefore are not suitable for separating mixtures containing various analytes, such as mixtures containing hydrophilic or amphiphilic analytes.

[0005] Therefore, an object of the present disclosure is to provide a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analysis apparatus, and an analysis method that are suitable for separating mixtures containing various analytes.

[0006] To achieve the above object, the chromatography column packing material of the present disclosure is characterized in that the silanol groups of the silica contained in the packing material are modified with a group represented by the following chemical formula (I): In the above chemical formula (I), R １is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with a substituent; m is an integer of 1 or more; n is 0 or a positive integer; X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group, and one or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent; Y １ and Y ２ At least one of Y is a hydrophilic group, １ and Y ２ may be the same or different, and * indicates the bonding position.

[0007] A chromatography column packing material comprising silica modified with a reaction reagent represented by the following chemical formula (II): In the chemical formula (II), R １ is a methylene group, one or more hydrogen atoms of which may or may not be further substituted with a substituent; R ２ is a methyl group, a linear or branched alkyl group, and one or more hydrogen atoms of the alkyl group may or may not be further substituted with a substituent; m is an integer of 1 or more; n is 0 or a positive integer; X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group, and one or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent; Y １１ and Y １２ At least one of the groups is a functional group capable of forming a silanol group.

[0008] The chromatography column of the present disclosure is packed with the packing material of the present disclosure.

[0009] The method for manufacturing a chromatography column according to the present disclosure includes a modification step and a packing step, wherein the modification step is a step of modifying silanol groups of silica gel particles contained in a packing material, and the packing step is a step of packing the silica after the modification into a chromatography column.

[0010] The method for manufacturing a chromatography column according to the present disclosure includes a modification step and a packing step, wherein the modification step is a step of modifying silanol groups of monolithic silica contained in a packing material, and the packing step is a step of packing the silica before the modification into a chromatography column.

[0011] The chromatography analysis device of the present disclosure includes a chromatograph, an eluent, and an analytical column, wherein the analytical column is the chromatography column of the present disclosure.

[0012] The analytical method of the present disclosure uses the analytical device of the present disclosure.

[0013] According to the present disclosure, it is possible to provide a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analysis apparatus, and an analysis method that are suitable for separating a mixture containing various analytes.

[0014] Figure 1 is a plot of columns 1 to 15, with methylene group selectivity α on the vertical axis and the reaction reagent delivery time (reaction time) on the horizontal axis. Figure 2 is a chromatogram of components contained in black tea (Darjeeling) separated using columns 1 and 15. Figure 3 is a chromatogram of components contained in five types of tea separated using column 1. Figure 4 is a chromatogram comparing the difference in separation ability due to differences in mobile phases when components contained in black tea (Darjeeling) were separated using column 1. Figure 5 is a chromatogram of components contained in asparagus (rutin) separated using column 1.

[0015] Next, the present disclosure will be described in more detail using examples, but the present disclosure is not limited to the following description.

[0016] In this disclosure, unless otherwise defined, technical terms, scientific terms, and other terms have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs. When the meaning of a term is defined, the meaning of the term is in accordance with that definition.

[0017] In this disclosure, "mass" may be read as "weight" unless otherwise specified. For example, "mass ratio" may be read as "weight ratio" unless otherwise specified, and "mass %" may be read as "weight %" unless otherwise specified.

[0018] Furthermore, in the present disclosure, when isomers such as tautomers or stereoisomers (e.g., geometric isomers, conformational isomers, and optical isomers) exist in substituents or compounds (e.g., a group represented by the above chemical formula (I) or a compound represented by the below-described chemical formula (II)), any of these isomers can be used in the present disclosure unless otherwise specified. Furthermore, when a compound can form a salt, the salt can also be used in the present disclosure unless otherwise specified. The salt may be an acid addition salt or a base addition salt. Furthermore, the acid that forms the acid addition salt may be an inorganic acid or an organic acid, and the base that forms the base addition salt may be an inorganic base or an organic base. The inorganic acid is not particularly limited, but examples thereof include sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, carbonic acid, hydrobromic acid, hydroiodic acid, hypofluorite acid, hypochlorous acid, hypobromous acid, hypoiodous acid, fluorite acid, chlorous acid, bromous acid, iodous acid, fluorine acid, chlorine acid, bromine acid, iodic acid, perfluorine acid, perchlorine acid, perbromine acid, and periodic acid. The organic acid is also not particularly limited, but examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. The inorganic base is not particularly limited, but examples thereof include ammonium hydroxide, alkali metal hydroxides, alkaline earth metal hydroxides, carbonates, and bicarbonates, and more specifically, examples thereof include sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, and calcium carbonate. The organic base is also not particularly limited, but examples thereof include ethanolamine, triethylamine, and tris(hydroxymethyl)aminomethane. The method for producing these salts is also not particularly limited, and they can be produced, for example, by a method in which the above-mentioned acid or base is appropriately added to the compound by a known method.

[0019] Furthermore, in the present disclosure, unless otherwise specified, chain-like substituents (e.g., hydrocarbon groups such as alkyl groups and unsaturated aliphatic hydrocarbon groups) may be linear or branched, and the number of carbon atoms therein is not particularly limited, but may be, for example, 1 to 40, 1 to 32, 1 to 24, 1 to 18, 1 to 12, 1 to 6, or 1 to 2 (2 or more in the case of unsaturated hydrocarbon groups). Furthermore, in the present disclosure, the number of ring members (the number of atoms constituting the ring) of cyclic groups (e.g., aryl groups, heteroaryl groups, etc.) is not particularly limited, but may be, for example, 5 to 32, 5 to 24, 6 to 18, 6 to 12, or 6 to 10. Furthermore, when isomers exist in substituents, etc., any isomer may be used unless otherwise specified. For example, a simple reference to a "naphthyl group" may refer to either a 1-naphthyl group or a 2-naphthyl group.

[0020] <Chromatography Column Packing Material> First, the chromatography column packing material of the present disclosure will be described.

[0021] In one embodiment of the chromatography column packing material of the present disclosure, in the chemical formula (I), R １ is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with a substituent, but is preferably unsubstituted. When substituted with a substituent, the substituent is preferably substituted with, for example, a hydrophobic group.

[0022] In the chemical formula (I), m is an integer of 1 or more, and may be, for example, 10 or less, 5 or less, 3 or less, or 2 or less, and is preferably 1.

[0023] In the chemical formula (I), n is 0 or a positive integer, and may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, 9 or more, 11 or more, 13 or more, 15 or more, or 17 or more, or may be 29 or less, 27 or less, 25 or less, 23 or less, 21 or less, or 19 or less. The n may be, for example, 0 to 29, 0 to 3, or 2 to 3.

[0024] In the chemical formula (I), X represents a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group. One or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent. If substituted, the substituent may be, for example, a non-aromatic hydrocarbon group (which may be linear or branched, saturated or unsaturated, and may or may not contain a cyclic structure), an aromatic group (for example, an aromatic group (aryl group) containing no heteroatoms, a heteroaromatic group (heteroaryl group) containing heteroatoms, and may be a single ring or a fused ring), a halogen, an amino group, a nitro group, a sulfo group, or a cyano group, and at least one hydrogen atom of each substituent may or may not be further substituted with another optional substituent.

[0025] The linear or branched alkyl group in X is, for example, a linear or branched alkyl group having 1 or more, 4 or more, 5 or more, 8 or more, or 10 or more carbon atoms, and is a linear or branched alkyl group having 30 or less, 24 or less, 18 or less, or 12 or less carbon atoms.

[0026] In the chemical formula (I), Y １ and Y ２ At least one of the groups is a hydrophilic group, for example, Y １ and Y ２ may both be hydrophilic groups, and Y １ and Y ２ Only one of the groups may be a hydrophilic group, or the other may be a hydrophobic group. Examples of the hydrophilic group include a hydroxy group, a carboxy group, and an amino group, and these groups are ionized to form -O － , -COO － , -NH ３ ＋Examples of the hydrophobic group include a methyl group, a linear or branched alkyl group, an alkenyl group, an aryl group, an alkylaryl group, and a polycyclic aryl group. At least one hydrogen atom of each substituent may be further substituted with another optional substituent, or may not be substituted. Y １ and Y ２ may be the same or different.

[0027] In the chemical formula (I), * indicates a bonding position, which is, for example, a bonding position of a bond formed after dehydration condensation between a compound having a group represented by the chemical formula (I) and a silanol group of the silica.

[0028] The ratio of the silanol groups (a) contained in the silica after the modification to the silanol groups (b) contained in the silica before the modification may be, for example, 100 mol % or more, and the ratio can be expressed, for example, by the formula "a (mol) / b (mol) × 100".

[0029] The filler of the present disclosure is represented by the formula (I) above, １ and Y ２ For example, Y １ and Y ２ If at least one of Y is a hydroxyl group, the ratio is 100 mol % or more. １ and Y ２ When only one of the groups is a hydroxyl group, the ratio is 100 mol %, and Y １ and Y ２ When all of the silanol groups contain hydroxyl groups, the proportion exceeds 100 mol %. When the proportion is 100 mol % or more, the silanol groups of the packing material of the present disclosure are modified with hydroxyl groups, which are hydrophilic groups, and therefore, when analyzing a mixture containing at least one hydrophilic or amphiphilic analyte, for example, the affinity for the hydrophilic or amphiphilic analyte is improved, making the packing material particularly effective for separating these analytes, etc.

[0030] The lower limit of the ratio may be, for example, 100 mol% or more, 110 mol% or more, or 116 mol% or more, and the upper limit may be 135 mol% or less, 130 mol% or less, or 125 mol% or less. The ratio is, for example, 100 to 135 mol%, 110 to 130 mol%, or 116 to 125 mol%.

[0031] The ratio can be calculated, for example, as follows: １ and Y ２ is a hydroxyl group, and Y １ and Y ２ The calculation method differs depending on whether only one of the above is a hydroxyl group or not.

[0032] First, Y １ and Y ２ The case where Y is a hydroxyl group will be described. １ and Y ２ When Aα is a hydroxyl group, the ratio can be calculated using, for example, liquid chromatography. First, the carbon content D (wt%) of the silica after the modification is calculated using the following formula (1): D (wt%) = Aα + B (1)

[0033] In the formula (1), A and B may be values ​​described in, for example, Kazuhiro Kimata et al. (1989). JOURNAL OF CHROMATOGRAPHIC SCIENCE, Vol. 27, 721-728. In this case, A and B are "A = 69.90 wt %" and "B = -87.35 wt %, respectively."

[0034] In addition, α in the above formula (1) can be measured as follows: First, the elution time (t Ｒ ) are analyzed. Ｒ ), and the elution time of one volume of solvent on the column (t ０) and the retention coefficients (k( Ｒ )) is obtained. Ｒ ) = (t Ｒ -t ０ ) / t ０ (2)

[0035] The retention coefficients of amylbenzene and butylbenzene (k( Ｒ )), α is calculated using the following formula (3): α=k( アミルベンゼン ) / k( ブチルベンゼン ) (3)

[0036] The carbon content D is the amount of carbon in the silica 1 m ２ The number of moles of substituents bonded per unit area, i.e., the density of the substituents E (μmol / m ２ ) the relationship of the following formula (4) holds. For example, by plotting D and E described in the above document, x in the following formula (4) is determined to be 0.1848 and y to be -0.3744. ２ )=xD+y (4)

[0037] Here, the amount of unmodified silanol groups on the silica surface is, for example, 8 μmol / m according to the literature (Yoshihisa Sudo et al. (2011). CHROMATOGRAPHY, Vol. 32 No. 2). ２ Therefore, if we do not consider the silanol groups newly generated by the bonding of the substituents, the amount of silanol groups present on the silica surface after modification is "8-E (μmol / m ２ )"

[0038] Here, the silanol group newly generated by bonding of the substituents is Y in the chemical formula (I). １ and Y ２ When both of them are silanol groups, "2 × E (μmol / m ２ )) Therefore, Y １ and Y ２ When both of these are hydroxyl groups, the amount of silanol groups present is (8-E) + (2 x E) = 8 + E (μmol / m ２ )) Therefore, Y１ and Y ２ When both of the groups are silanol groups, the percentage F (mol %) of silanol groups remaining on the silica surface after the modification reaction can be calculated based on the following formula (5): F (mol %) = (8 + E) / 8 × 100 (5)

[0039] On the other hand, Y in the chemical formula (I) １ and Y ２ When only one of the groups is a hydroxyl group, the silanol group newly generated by bonding of the substituents is "E (μmol / m ２ )) Therefore, Y １ and Y ２ When only one of the groups is a hydroxyl group, the amount of silanol groups present is determined as follows: (8-E) + E = 8 (μmol / m ２ Therefore, in this case, the proportion F (mol %) of silanol groups remaining on the silica surface after the modification reaction is 100 (mol %), as "F (mol %) = 8 / 8 × 100".

[0040] Although the above method has been described as an example of calculating the ratio, the method is not limited thereto. For example, the ratio may be calculated by various quantitative analyses. For example, quantitative analysis of the silanol groups before and after modification of the silica may be performed using energy dispersive X-ray spectroscopy (EDX, EDS), wavelength dispersive X-ray spectroscopy (WDS), X-ray photoelectron spectroscopy (XPS, ESCA), scanning electron microscope with energy dispersive X-ray spectroscopy (SEM-EDS), mass spectroscopy (MS), infrared spectroscopy, Raman spectroscopy, or the like, and the ratio may be calculated from the results of the quantitative analysis.

[0041] In one embodiment of the chromatography column packing material of the present disclosure, in the chemical formula (II), R １ is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with a substituent, but is preferably unsubstituted. When substituted with a substituent, the substituent is preferably substituted with, for example, a hydrophobic group.

[0042] In the chemical formula (II), m is an integer of 1 or more, and may be, for example, 10 or less, 5 or less, 3 or less, or 2 or less, and is preferably 1.

[0043] In the chemical formula (II), n is 0 or a positive integer, and may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, 9 or more, 11 or more, 13 or more, 15 or more, or 17 or more, or may be 29 or less, 27 or less, 25 or less, 23 or less, 21 or less, or 19 or less. The n may be, for example, 0 to 29, 0 to 3, or 2 to 3.

[0044] In the chemical formula (II), X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group. One or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent. If substituted, the substituent may be, for example, a non-aromatic hydrocarbon group (which may be linear or branched, saturated or unsaturated, and may or may not contain a cyclic structure), an aromatic group (for example, an aromatic group (aryl group) containing no heteroatoms, a heteroaromatic group (heteroaryl group) containing heteroatoms, and may be a single ring or a fused ring), a halogen, an amino group, a nitro group, a sulfo group, or a cyano group, and at least one hydrogen atom of each substituent may or may not be further substituted with any other substituent.

[0045] The linear or branched alkyl group in X is, for example, a linear or branched alkyl group having 1 or more, 4 or more, 5 or more, 8 or more, or 10 or more carbon atoms, and is a linear or branched alkyl group having 30 or less, 24 or less, 18 or less, or 12 or less carbon atoms.

[0046] In the chemical formula (II), Y １１ and Y １２At least one of the groups may be a functional group capable of forming a silanol group. For example, at least one of the groups may be a straight-chain or branched alkoxy group, a halogen atom, or a hydrophilic group, and one or more hydrogen atoms of the alkoxy group may or may not be further substituted with a substituent. If substituted, the group may be, for example, a non-aromatic hydrocarbon group (which may be straight-chain or branched, saturated or unsaturated, and may or may not contain a cyclic structure), an aromatic group (for example, an aromatic group (aryl group) containing no heteroatoms, a heteroaromatic group (heteroaryl group) containing heteroatoms, and may be a monocyclic or fused ring), a halogen, an amino group, a nitro group, a sulfo group, or a cyano group, and at least one hydrogen atom of each substituent may or may not be further substituted with another optional substituent. The straight-chain or branched alkoxy group may, for example, have 1 or more, 3 or more, or 5 or more carbon atoms, or 10 or less, 8 or less, or 6 or less. Examples of the linear or branched alkoxy group include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, and a tert-butoxy group. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Examples of the hydrophilic group include a hydroxy group, a carboxy group, an amino group, a dimethylamino group, and a diethylamino group, and ionized groups thereof, such as -O － , -COO － , -NH ３ ＋ Examples of the hydrophobic group include a methyl group, a linear or branched alkyl group, an alkenyl group, an aryl group, an alkylaryl group, and a polycyclic aryl group. At least one hydrogen atom of each substituent may be further substituted with another optional substituent, or may not be substituted. Y １１ and Y １２ may be the same or different.

[0047] Examples of the reaction reagent represented by the chemical formula (II) are shown in Compounds 1-1 to 11-25 below. Here, Compound 1 is a group of compounds in which m = 1, n = 1, and X = a methyl group in the chemical formula (II). Compound 2 is a group of compounds in which m = 1, n = 3, and X = a methyl group in the chemical formula (II). Compound 3 is a group of compounds in which m = 1, n = 7, and X = a methyl group in the chemical formula (II). Compound 4 is a group of compounds in which m = 1, n = 11, and X = a methyl group in the chemical formula (II). Compound 5 is a group of compounds in which m = 1, n = 17, and X = a methyl group in the chemical formula (II). Compound 6 is a group of compounds in which m = 1, n = 21, and X = a methyl group in the chemical formula (II). Compound 7 is a group of compounds represented by the chemical formula (II) where m = 1, n = 29, and X = a methyl group. Compound 8 is a group of compounds represented by the chemical formula (II) where m = 1, n = 1, and X = a branched alkyl group. Compound 9 is a group of compounds represented by the chemical formula (II) where m = 1, n = 3, and X = a branched alkylamine. Compound 10 is a group of compounds represented by the chemical formula (II) where m = 1, n = 11, and X = an aryl group. Compound 11 is a group of compounds represented by the chemical formula (II) where m = 1, n = 11, and X = a cyano group.

[0048]

[0049] For other explanations, the explanation of the chemical formula (I) can be cited.

[0050] <Chromatography Column> Next, the chromatography column of the present disclosure and a method for producing the chromatography column will be described.

[0051] As described above, the column of the present disclosure is a chromatography column packed with the packing material of the present disclosure. The column is, for example, a packed column prepared by modifying silanol groups of silica gel particles or the like and then packing the silica into a column, or a capillary column prepared by preparing (packing) silica such as monolithic silica in a column and then modifying silanol groups of the silica.

[0052] When the column of the present disclosure is a packed column, the column size is not particularly limited. The column length may be, for example, 50 mm or more, 75 mm or more, 100 mm or more, 125 mm or more, or 150 mm or more, and may be 1000 mm or less, 750 mm or less, 500 mm or less, 300 mm or less, 250 mm or less, or 200 mm or less. The column inner diameter may be, for example, 1.0 mm or more, 1.5 mm or more, 2.0 mm or more, 3.0 mm or more, or 4.0 mm or more, and may be 20 mm or less, 10 mm or less, 7.5 mm or less, 6.0 mm or less, or 4.5 mm or less.

[0053] When the column of the present disclosure is a capillary column, the column size is not particularly limited. The column length may be, for example, 150 mm or more, 250 mm or more, or 700 mm or more, and may be 5000 mm or less, 3000 mm or less, or 2000 mm or less. The column inner diameter may be, for example, 0.01 mm or more, 0.05 mm or more, or 0.1 mm or more, and may be 1 mm or less, 0.5 mm or less, or 0.2 mm or less. The capillary column may have a long, thin tubular shape, and for example, a groove of a size corresponding to the capillary tube formed by microfabrication on a glass plate may be used as the column tube.

[0054] The silica contained in the filler is, for example, silica gel particles or monolithic silica. The silica gel particles are, for example, silica gel particles after silanol groups have been modified, and the monolithic silica is, for example, monolithic silica before silanol groups have been modified.

[0055] The column of the present disclosure is, for example, a chromatography column for analyzing plant-derived components. Examples of the plant include, but are not limited to, ginkgo, fennel, turmeric, Corydalis chinensis, Scutellaria baicalensis, Coptis chinensis, Zedoary, licorice, cinnamon bark, Magnolia officinalis, Cornus officinalis, Dioscorea chinensis, Rehmannia Root, Lithospermum Root, Peony Root, Ginger Root, Kankyo, Cnidium Rhizome, Swertia Root, Atractylodes Root, Perilla Root, Rhubarb, Rhubarb, Chinese Locust, Chinese Locust, Clove, Tangerine Peel, Angelica Root, Eucommia ulmoides, Ginseng, Pinellia Root, Poria Coccinea, Bushi, Peony Peel, Rhizome, Ginger, and other herbal medicines listed in the Japanese Pharmacopoeia; ISO / TR 23022-2018 Traditional Chinese Medicine Examples of plant-derived ingredients include herbal medicines listed in the "Kampo Medicine" (Kampo Medicines for Chinese Medicine); tea leaves such as green tea, matcha, and black tea; and foods such as coffee beans, sesame seeds, onions, yuzu, mandarin oranges, bell peppers, chrysanthemum, perilla, grapes, blueberries, spinach, and broccoli. The plant-derived ingredients include, for example, at least one of a hydrophilic compound and an amphipathic compound, and may also include, for example, a hydrophobic compound. The plant-derived ingredients are not particularly limited, but include compounds having structures containing phenols and their glycosides, coumarins and their glycosides, flavonoids and their glycosides, chalcones and their glycosides, anthocyanidins and their glycosides, anthraquinones and their glycosides, indoles and their glycosides, nitriles and their glycosides, steroids and their glycosides, and alkaloids and their glycosides.

[0056] Next, a method for producing a chromatography column according to the present disclosure will be described. As described above, the method for producing a chromatography column according to the present disclosure includes a modification step and a packing step. When the column according to the present disclosure is a packed column, the method for producing a chromatography column according to the present disclosure may, for example, perform the packing step after the modification step. When the column according to the present disclosure is a capillary column, the method for producing a chromatography column according to the present disclosure may, for example, perform the modification step after the packing step. The method for producing a chromatography column according to the present disclosure may further include a drying step. The modification step may, for example, include a heating step, a reaction reagent washing step, a substitution step, and a substitution reagent washing step, which will be described later.

[0057] The column of the present disclosure can be produced, for example, by the following method.

[0058] First, a column in which silica has been formed in advance (hereinafter, sometimes simply referred to as a "column before modification") is dried, or silica before being packed into a column is dried (drying step). The silica may be, for example, monolithic silica or silica gel particles. The drying step removes, for example, moisture present on the surface of the silica. The drying may be, for example, heat drying, reduced-pressure drying, or reduced-pressure heat drying. The reduced-pressure conditions during the drying may be, for example, 1 kPa or more, 5 kPa or more, or 10 kPa or more, and may be 100 kPa or less, 80 kPa or less, or 50 kPa or less. The heating temperature during the drying may be, for example, 80°C or more, 100°C or more, or 120°C or more, and may be 300°C or less, 200°C or less, or 150°C or less. The drying time may be, for example, 1 hour or more, 2 hours or more, or 5 hours or more, and may be 24 hours or less, 18 hours or less, or 12 hours or less.

[0059] Next, in the case of a capillary column, the dried unmodified column is connected to a liquid delivery device and heated while delivering a reaction reagent (heating step). The heating step modifies the silica with the reaction reagent. The heating temperature during the modification may be, for example, 60°C or higher, 80°C or higher, or 100°C or higher, and may be 200°C or lower, 180°C or lower, or 150°C or lower. The heating time during the modification may be, for example, 3 hours or higher, 6 hours or higher, or 10 hours or higher, and may be 48 hours or lower, 36 hours or lower, or 24 hours or lower.

[0060] The liquid delivery device is, for example, one that can deliver the reaction reagent into the column by pressurization, and examples thereof include a syringe pump, a pump for liquid chromatography, etc. The liquid delivery may be, for example, by pressurizing a gas.

[0061] Next, in the case of a packed column, the silica gel particles are dried under the above conditions, weighed into a flask or the like, and then modified with a reaction reagent. The amount of the reaction reagent may be, for example, any amount in the range of 10% by weight to 500% by weight relative to 100% by weight of silica gel. The heating temperature and heating time during modification with the reaction reagent are the same as those in the heating step for the capillary column. The modified silica gel is filtered and washed by a known method, and then the silica gel is packed into a column tube to obtain the packed column.

[0062] As described above, the reaction reagent may be a reaction reagent represented by the following chemical formula (II).

[0063]

[0064] In the chemical formula (II), m is an integer of 1 or more, and may be, for example, 10 or less, 5 or less, 3 or less, or 2 or less, and is preferably 1.

[0065] In the chemical formula (II), n is 0 or a positive integer, and may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 7 or more, 9 or more, 11 or more, 13 or more, 15 or more, or 17 or more, or may be 29 or less, 27 or less, 25 or less, 23 or less, 21 or less, or 19 or less. The n may be, for example, 0 to 29, 0 to 3, or 2 to 3.

[0066] In the chemical formula (II), X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group. One or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent. If substituted, the substituent may be, for example, a non-aromatic hydrocarbon group (which may be linear or branched, saturated or unsaturated, and may or may not contain a cyclic structure), an aromatic group (for example, an aromatic group (aryl group) containing no heteroatoms, a heteroaromatic group (heteroaryl group) containing heteroatoms, and may be a single ring or a fused ring), a halogen, an amino group, a nitro group, a sulfo group, or a cyano group, and at least one hydrogen atom of each substituent may or may not be further substituted with any other substituent.

[0067] The linear or branched alkyl group in X is, for example, a linear or branched alkyl group having 1 or more, 4 or more, 5 or more, 8 or more, or 10 or more carbon atoms, and is a linear or branched alkyl group having 30 or less, 24 or less, 18 or less, or 12 or less carbon atoms.

[0068] In the chemical formula (II), Y １１ and Y １２ At least one of the Y is a functional group capable of forming a silanol group. １１ and the Y １２For example, at least one of the groups may be a functional group capable of forming a silanol group through the substitution step described below. For example, at least one of the groups may be a straight-chain or branched alkoxy group, a halogen atom, or a hydrophilic group, and one or more hydrogen atoms of the alkoxy group may or may not be further substituted with a substituent. If substituted, the group may be, for example, a non-aromatic hydrocarbon group (which may be straight-chain or branched, saturated or unsaturated, and may or may not contain a cyclic structure), an aromatic group (for example, an aromatic group (aryl group) containing no heteroatoms, a heteroaromatic group (heteroaryl group) containing heteroatoms, and may be a monocyclic or fused ring), a halogen, an amino group, a nitro group, a sulfo group, or a cyano group, and at least one hydrogen atom of each substituent may or may not be further substituted with another optional substituent. The straight-chain or branched alkoxy group may, for example, have 1 or more, 3 or more, or 5 or more carbon atoms, or 10 or less, 8 or less, or 6 or less. Examples of the linear or branched alkoxy group include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, and a tert-butoxy group. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. Examples of the hydrophilic group include a hydroxy group, a carboxy group, an amino group, a dimethylamino group, and a diethylamino group, and ionized groups thereof, such as -O － , -COO － , -NH ３ ＋ Examples of the hydrophobic group include a methyl group, a linear or branched alkyl group, an alkenyl group, an aryl group, an alkylaryl group, and a polycyclic aryl group. At least one hydrogen atom of each substituent may be further substituted with another optional substituent, or may not be substituted. Y １１ and Y １２ may be the same or different.

[0069] In the above chemical formula (II), R １is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with a substituent, but is preferably unsubstituted. When substituted with a substituent, the substituent is preferably substituted with, for example, a hydrophobic group.

[0070] In the above chemical formula (II), R ２ is a methyl group or a straight-chain or branched alkyl group, and one or more hydrogen atoms of the alkyl group may or may not be further substituted with a substituent. When substituted, the alkyl group may be, for example, a non-aromatic hydrocarbon group (which may be straight-chain or branched, saturated or unsaturated, and may or may not contain a cyclic structure), an aromatic group (for example, an aromatic group not containing a heteroatom (aryl group) or a heteroaromatic group containing a heteroatom (heteroaryl group), and may be a single ring or a fused ring), a halogen, an amino group, a nitro group, a sulfo group, or a cyano group, and at least one hydrogen atom of each substituent may or may not be further substituted with any other substituent. The linear or branched alkyl group is, for example, a linear or branched alkyl group having 1 to 12 carbon atoms, and examples thereof include a methyl group, an ethyl group, a propyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, a decyl group, an undecyl group, and a dodecyl group (a lauryl group).

[0071] When the silica is modified by the chemical formula (II), for example, R ２ The modification is carried out by a condensation reaction between --O-- and the silanol groups of the silica.

[0072] Examples of the reaction reagent include the above-mentioned Compounds 1-1 to 11-25.

[0073] The reaction reagent may or may not contain other components. Examples of the other components include a solvent and a catalyst. Examples of the solvent include alcohols such as water, methanol, ethanol, isopropyl alcohol (IPA), n-butyl alcohol, sec-butyl alcohol, isobutyl alcohol, and t-butyl alcohol (TBA); ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, and cyclopentanone; esters such as methyl acetate, ethyl acetate, and butyl acetate; ethers such as diisopropyl ether, propylene glycol monomethyl ether, 2-methoxyethanol, ethyl cellosolve, and butyl cellosolve; glycols such as ethylene glycol and propylene glycol; aliphatic hydrocarbons such as hexane, heptane, and octane; aromatic hydrocarbons such as benzene, toluene, and xylene; and nitriles such as acetonitrile. Examples of the catalyst include an amine catalyst. Examples of the amine catalyst include diethylamine, triethylamine, diisopropylethylamine, phenethylamine, pyridine, lutidine, 4-dimethylaminopyridine, phenethylamine, piperidine, morpholine, and 1,4-diazabicyclo[2.2.2]octane.

[0074] After the heating step, for example, a step of washing the unreacted reaction reagent may be included (reaction reagent washing step). The reaction reagent washing step is, for example, a step of feeding a solvent into the column after the heating step. The solvent is, for example, the same as the solvent contained as the other component. The time for feeding the solvent is not particularly limited as long as it is a time that allows the unreacted reaction reagent to be washed away, and is, for example, 1 hour or more, 6 hours or more, or 12 hours or more, and 72 hours or less, 48 ​​hours or less, or 24 hours or less.

[0075] Y in the chemical formula (II) １１ and Y １２ However, for example, when the silica contains a group other than a hydrophilic group, the process may further include a step of substituting the group other than a hydrophilic group with a hydrophilic group (substitution step). The substitution step is, for example, a step of further hydrolyzing the silica after the modification, and the Y in the chemical formula (II) bonded to the modified silica is further hydrolyzed. １１and Y １２ may be a step of substituting at least one of the above with a hydroxyl group (i.e., a step of forming a silanol group).

[0076] The substitution step is, for example, a step of pumping a substitution reagent into the column after the heating step. The substitution reagent may contain, for example, a solvent, an acid, etc. The solvent is, for example, the same as the solvent contained as the other component. The acid may be, for example, an organic acid or an inorganic acid. The organic acid is not particularly limited, but examples thereof include acetic acid, formic acid, difluoroacetic acid, and trifluoroacetic acid. Examples of the inorganic acid include sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, hypofluorite acid, hypochlorous acid, hypobromous acid, hypoiodite acid, fluorite acid, chlorous acid, bromous acid, iodite acid, fluorine acid, chlorine acid, bromine acid, iodic acid, perfluorine acid, perchloric acid, perbromine acid, and periodic acid. The pH of the substitution reagent is preferably acidic, for example, pH 4 or less, pH 3 or less, pH 2 or less, or pH 1 or less.

[0077] The temperature in the substitution step is, for example, 20° C. or higher, 40° C. or higher, or 60° C. or higher, and 120° C. or lower, 100° C. or lower, or 80° C. or lower. The liquid transfer time in the liquid transfer step is not particularly limited as long as it is a time period that allows the groups other than hydrophilic groups to be substituted with hydrophilic groups, and is, for example, 1 hour or longer, 3 hours or longer, or 6 hours or longer, and 48 hours or shorter, 24 hours or shorter, or 12 hours or shorter.

[0078] The substitution step may be followed by a washing step (a replacement reagent washing step). The replacement reagent washing step is, for example, a step of pumping a solvent into the column after the substitution step. The solvent may be, for example, the same as the solvent contained as the other component. The time for the solution pumping is not particularly limited as long as it is a time that allows the unreacted reaction reagent to be washed away, and is, for example, 1 hour or more, 3 hours or more, or 6 hours or more, and 48 hours or less, 24 hours or less, or 12 hours or less.

[0079] <Chromatography Analysis Apparatus> Next, the chromatography analysis apparatus (also referred to as a "chromatograph") of the present disclosure will be described.

[0080] The chromatography analyzer is, for example, a liquid chromatography analyzer. The liquid chromatography analyzer includes, for example, a liquid delivery unit, a gradient elution unit, a sample injection unit (injector), a detection unit, a data processing unit, etc. The analytical column is preferably connected, for example, to any location between the sample injection unit and the detection unit. The gradient elution unit is preferably connected, for example, to any location between the liquid delivery unit and the sample injection unit.

[0081] The number of analytical columns is not particularly limited, and may be determined in consideration of, for example, analytical conditions, etc., and one or two or more analytical columns may be used.

[0082] The eluent is, for example, a three-liquid or more eluent combining water and two or more water-soluble organic solvents. The water-soluble organic solvents may be, for example, two or more, three or more, four or more, or five or more, or eight or less, seven or less, or six or less. The number of liquid systems in the eluent varies depending on the number of solvents combined, but a three-liquid or more eluent is preferred. Examples of the water-soluble organic solvent include alcohol-based solvents and nitrile-based solvents. The water-soluble organic solvent may include, for example, at least one of the alcohol-based solvents and the nitrile-based solvents. Examples of the alcohol-based solvent include methanol, ethanol, isopropyl alcohol (IPA), n-propyl alcohol, n-butyl alcohol, sec-butyl alcohol, isobutyl alcohol, t-butyl alcohol (TBA), 2-methoxyethanol, etc., with methanol being preferred. Examples of the nitrile-based solvent include acetonitrile, etc.

[0083] The gradient elution section performs gradient elution by changing the solvent composition using the eluent as a mobile phase, for example. The solvent composition of the eluent may be changed at a flow rate of 20 nL to 100 mL / min, 100 nL to 1 mL / min, or 300 nL to 0.01 mL / min, for example. Furthermore, when the eluent is a three-liquid or more liquid system, the gradient elution may be performed by changing the solvent composition of at least one of the eluents, for example, from 0 to 100%, 10 to 90%, or 20 to 80%, from the start of elution to the end of elution.

[0084] The chromatography analysis device of the present disclosure may further include, for example, another analysis device such as a mass spectrometer. The other analysis device may be integrated with the analysis device of the present disclosure, or may be separate devices that are connected to each other for use.

[0085] <Analysis Method> Next, the analysis method of the present disclosure will be described.

[0086] As described above, the analytical method of the present disclosure is an analytical method using the analytical device of the present disclosure.

[0087] The analytical method of the present disclosure is, for example, a method for analyzing an analyte containing at least one of a hydrophilic compound and an amphipathic compound. The analytical method of the present disclosure is, for example, a method for analyzing a plant-derived component. The plant and the plant-derived component are, for example, the same as those described for the column of the present disclosure.

[0088] Next, examples of the present disclosure will be described, but the present disclosure is not limited to the following examples.

[0089] <Quantitative Determination of Hydrophilic Groups> [Example 1] In Example 1, a chromatography column 1 (hereinafter sometimes referred to as "column 1") containing the chromatography column packing material of the present disclosure was produced.

[0090] First, a monolithic silica capillary (inner diameter 0.1 mm, length 750 mm, manufactured by Shinwa Chemical Industry Co., Ltd.) was dried under reduced pressure at 140°C for 6 hours. The dried monolithic silica capillary was then connected to a pump including a heating furnace, and a reaction was carried out at 100°C while a reaction reagent was pumped into the monolithic silica capillary. The reaction reagent was a mixture of 23 mL of toluene, 2 mL of a toluene solution containing 10% by volume of phenethylamine acting as a catalyst, and 2 mL of octadecyltriethoxysilane. The reaction reagent was pumped for 40 hours. Toluene was then pumped at room temperature for 24 hours to wash away unreacted reaction reagent. Next, a substitution reagent was pumped into the reacted monolithic silica capillary at 60°C, and the unreacted leaving groups of the reacted octadecyltriethoxysilane were hydrolyzed to obtain silanol groups. The substitution reagent was a mixture of 80 mL of acetonitrile, 20 mL of ultrapure water, and 0.1 mL of formic acid, and had a pH of approximately 2. The time for pumping the substitution reagent was 20 hours. Finally, to wash and remove the substitution reagent, methanol was pumped as an organic solvent at room temperature for 24 hours to produce column 1. The end of column 1 was cut to a length of 700 mm.

[0091] The α (methylene group selectivity) of column 1 was measured by high performance liquid chromatography (HPLC). The HPLC conditions were set as follows: Uracil, butylbenzene, and amylbenzene were used as samples, with the respective concentrations set to 0.05 mg / mL, 0.5 μL / mL, and 0.5 μL / mL. The uracil peak was measured by t ０ The peaks of butylbenzene and amylbenzene are respectively t Ｒ The α of column 1 and the proportion (%) of silanol groups remaining on the silica surface after the modification reaction were calculated using the above-mentioned formulas (1) to (5). The results are shown in Table 1 below.

[0092] (HPLC measurement conditions) Apparatus: High-performance liquid chromatograph (JASCO Corporation) Mobile phase: Water / methanol = 20 / 80 (v / v) mixed solvent Column flow rate: 0.5 μL / min Elution conditions: Isocratic Column temperature: 30°C Detection wavelength: 220 nm

[0093] [Examples 2 to 14 and Comparative Example 1] In these examples, chromatography columns 2 to 14 (hereinafter sometimes referred to as "columns 2 to 14") containing the chromatography column packing material of the present disclosure and a comparative chromatography column (hereinafter sometimes referred to as "column 15") were produced as Examples 2 to 14 and Comparative Example 1. In Examples 2 to 14, columns 2 to 14 were produced in the same manner as in Example 1, except that the delivery time of the reaction reagent was changed to the time shown in Table 1 below.

[0094] In Comparative Example 1, a column (column 15) of Comparative Example 1 was produced in the same manner as in Example 1, except that a mixture of 9 mL of toluene and 1 mL of octadecyldimethyl(dimethylamino)silane was used as the reaction reagent instead of the reaction reagent used in Example 1.

[0095]

[0096] As shown in Table 1, in columns 1 to 14, the silanol groups of the silica contained in the packing material were modified with a substituent containing a hydrophilic group (silanol group), and the proportion of silanol groups remaining on the silica surface after the modification reaction was 100 mol % or more. On the other hand, in column 15, the silanol groups of the silica contained in the packing material were modified with a substituent not containing a hydrophilic group (silanol group), and the proportion of silanol groups remaining on the silica surface after the modification reaction was less than 100 mol %.

[0097] 1 is a plot of methylene group selectivity α on the vertical axis and the reaction reagent delivery time (reaction time) on the horizontal axis for columns 1 to 15. As shown in FIG. 1, the value of α changed from 1.30 to 1.40 when the delivery time of the reaction reagent containing octadecyltriethoxysilane was between 15 and 40 hours, and it was found that columns with α values ​​in the range of 1.40 to 1.48 could be stably produced when the delivery time exceeded 40 hours.

[0098] Furthermore, the hydrophilic groups (silanol groups) in the packing material before modification (untreated silica), Column 1 (Example 1), and Column 15 (Comparative Example 1) were quantified before and after modification. Quantification was performed using a scanning electron microscope equipped with an energy dispersive X-ray analyzer (manufactured by JEOL Ltd., product name: JSM-7100). The results are shown in Table 2 below.

[0099]

[0100] Untreated silica has an atomic ratio of oxygen atoms (O) to silicon atoms (Si) (hereinafter referred to as "O / Si ratio") of about 2, i.e., SiO ２ It was found that the packing material in column 1 had a higher O / Si ratio than untreated silica. In other words, it was found that the amount of hydroxyl groups in column 1 had increased. On the other hand, the packing material in column 15 had a lower O / Si ratio than untreated silica. In other words, it was found that the amount of hydroxyl groups in column 15 had decreased.

[0101] <Separation of Components Contained in Black Tea> Components contained in black tea (Darjeeling) were separated using Column 1 and Column 15 according to the following procedures and conditions.

[0102] (Preparation of analytical sample) First, 10 mg of commercially available tea leaves (Darjeeling tea) was placed in a sample tube, and a 50 / 50 (v / v) water / methanol mixed solvent was added to a concentration of 10 mg / mL. The mixture was then stirred for 10 seconds using a vortex mixer. After stirring, the mixture was allowed to stand for 10 minutes. 50 μL of the supernatant after standing and 950 μL of a 0.2% by volume aqueous solution of phosphoric acid were added to a 2 mL sample bottle, which was then shaken to obtain a 0.5 mg / mL analytical sample.

[0103] (HPLC measurement conditions) Apparatus: High-performance liquid chromatograph (Thermo Fisher Scientific Co., Ltd.) Mobile phase A: 0.2% by volume phosphoric acid added aqueous solution Mobile phase B: 0.2% by volume phosphoric acid added methanol Pump flow rate: 0.5 μL / min Elution conditions: 5% B - 50% B (90 min), 50% B - 95% B (1 min), 95% B (9 min) Column temperature: 50°C Detection wavelength: 203 nm Sample concentration: 0.5 mg / mL Sample injection volume: 1 μL

[0104] Figure 2 is a chromatogram showing the separation of components contained in black tea (Darjeeling) using columns 1 and 15. In Figure 2, the vertical axis represents the detection value (mAU) and the horizontal axis represents the elution time (minutes).

[0105] As shown in Figure 2, when Column 1 was used, peaks representing components contained in black tea were detected over a wide range. That is, Column 1, which contains the chromatography column packing material of the present disclosure, was able to adsorb and separate a wide range of black tea components. On the other hand, Column 15 only weakly adsorbed the components contained in black tea, causing them to elute quickly. Thus, Column 1, which contains many silanol groups remaining on the silica surface of the chromatography column packing material, was found to be suitable for retaining and separating plant components.

[0106] <Separation of Components Contained in Various Types of Tea> Components contained in tea were separated using column 1 according to the following procedures and conditions.

[0107] (Preparation of analytical samples) Analytical samples were prepared in the same manner as in the above-mentioned "Separation of components contained in black tea", except that commercially available tea bags (Earl Grey), green tea, roasted green tea, and sweet tea were used instead of commercially available tea bags (Darjeeling), and analytical samples of 0.5 mg / mL were obtained for five types of tea (Darjeeling, Earl Grey, green tea, roasted green tea, and sweet tea).

[0108] (HPLC Measurement Conditions) The measurement conditions were the same as those for "Separation of Components Contained in Black Tea" described above.

[0109] Figure 3 shows chromatograms obtained when components contained in five types of tea (Darjeeling, Earl Grey, green tea, roasted green tea, and sweet tea) were separated using column 1. In each chromatogram in Figure 3, the vertical axis represents the detection value (mAU) and the horizontal axis represents the elution time (minutes).

[0110] As shown in Figure 3, a large number of peaks of various sizes were detected in each analytical sample. It was also shown that Darjeeling, Earl Grey, green tea, and roasted green tea, all made from the same tea leaves, have different patterns of contained components. Furthermore, Amachā, made from tea leaves different from the other four types of tea, showed a different component pattern from the other four types of tea. Thus, it was found that a column containing the chromatography column packing material of the present disclosure can effectively adsorb and separate plant components and precisely separate them, making it useful for precise analysis of plant components and pattern analysis of plant components.

[0111] <Difference in separation ability due to mixed mobile phase> Components contained in black tea (Darjeeling) were separated using column 1 according to the following procedure and conditions, and changes in separation ability when a mixed mobile phase was used were confirmed. (Preparation of analytical sample) An analytical sample was prepared using the same procedure as in the above-mentioned "Separation of components contained in black tea" to obtain a 0.5 mg / mL analytical sample. (HPLC measurement conditions) Apparatus: High-performance liquid chromatograph (Thermo Fisher Scientific Inc.) Mobile phase A: 0.2% by volume phosphoric acid added aqueous solution Mobile phase B: Condition 1: 0.2% by volume phosphoric acid added methanol Condition 2: 0.2% by volume phosphoric acid added - methanol / acetonitrile = 60 / 40 (v / v) mixed solvent Pump flow rate: 0.5 μL / min Elution conditions: 5% B - 50% B (90 min), 50% B - 95% B (1 min), 95% B (9 min) Column temperature: 50°C Detection wavelength: 203 nm Sample concentration: 0.5 mg / mL Sample injection volume: 1 μL

[0112] Figure 4 shows chromatograms comparing the difference in separation ability due to different mobile phases when components contained in black tea (Darjeeling) were separated using Column 1. In each chromatogram in Figure 4, the vertical axis represents the detection value (mAU) and the horizontal axis represents the elution time (minutes).

[0113] As shown in Figure 4, gradient elution (condition 1) using a binary solvent of water and methanol, which is widely used in general HPLC, detected 62 peaks. On the other hand, gradient elution (condition 2) using a ternary solvent of water-methanol-acetonitrile detected 89 peaks. In other words, a column containing the chromatography column packing material of the present disclosure can effectively separate plant components using gradient elution with a binary solvent of water and methanol, but can separate more components using gradient elution with a ternary solvent of water-methanol-acetonitrile. This demonstrates that columns containing the chromatography column packing material of the present disclosure are useful for analyzing the components of plants, which contain a wide variety of components.

[0114] <Separation of Components Contained in Asparagus> Components contained in asparagus were separated using Column 1 according to the following procedures and conditions.

[0115] (Preparation of analytical sample) An analytical sample was prepared in the same manner as in the above-mentioned "Separation of components contained in black tea", except that commercially available asparagus dried under reduced pressure at 40°C was used instead of commercially available tea bag black tea (Darjeeling) leaves, and a 0.5 mg / mL analytical sample was obtained from the dried asparagus extract.

[0116] The rutin solution used as a standard was prepared as follows: 0.1 mg of rutin (Fujifilm Wako Pure Chemical Industries, Ltd.) was placed in a sample tube, and a 50 / 50 (v / v) water / methanol solvent was added to the tube to give a concentration of 0.1 mg / mL. The mixture was then stirred for 10 seconds using a vortex mixer. After stirring, the mixture was allowed to stand for 10 minutes. 10 μL of the supernatant and 990 μL of a 0.2% by volume aqueous solution of phosphoric acid were added to a 2 mL sample bottle, which was then shaken to obtain a 0.001 mg / mL analytical sample.

[0117] (HPLC measurement conditions) Apparatus: High-performance liquid chromatograph (Thermo Fisher Scientific Inc.) Mobile phase A: 0.2% by volume phosphoric acid added aqueous solution Mobile phase B: 0.2% by volume phosphoric acid added - methanol / acetonitrile = 60 / 40 (v / v) mixed solvent Elution conditions: 5% B - 50% B (90 min), 50% B - 95% B (1 min), 95% B (9 min) Column temperature: 50°C Detection wavelength: 203 nm Sample concentration: 0.5 mg / mL Sample injection volume: 1 μL

[0118] Figure 5 shows chromatograms obtained when a component (rutin) contained in asparagus was separated using Column 1. In each chromatogram in Figure 5, the vertical axis represents the detection value (mAU) and the horizontal axis represents the elution time (minutes).

[0119] As shown in Figure 5, the analysis of the rutin preparation allowed identification of rutin, which is abundant in asparagus. Thus, it was found that a column containing the chromatography column packing material of the present disclosure is useful not only for analyzing tea but also for analyzing other plant components, including asparagus. It was also found that the use of preparations allows for the identification and quantitative analysis of plant components.

[0120] As described above, a column containing the chromatography column packing material of the present disclosure enables component analysis with higher sensitivity than conventional columns.

[0121] Furthermore, because plant-derived components vary from plant to plant and exist in a wide variety, analysis using a general liquid chromatography analyzer is difficult. Columns using particles use extremely small particles for these analyses. Columns using such extremely small particles generate an extraordinary ultra-high pressure of approximately 1000 atmospheres during delivery, requiring a specialized, dedicated system that can withstand ultra-high pressure conditions. However, columns containing the chromatography column packing material of the present disclosure enable separation and analysis of a wide variety of components even using general liquid chromatography at approximately 100 atmospheres. Therefore, for example, depending on the application, a general liquid chromatography analyzer using a column containing the chromatography column packing material of the present disclosure and an ultra-high pressure liquid chromatography analyzer can be used complementarily.

[0122] Furthermore, a column containing the chromatography column packing material of the present disclosure is suitable for connection to a mass spectrometer that requires sample vaporization during component analysis, for example, because a small amount of solvent is discharged from the column along with the separated components. Therefore, a liquid chromatography mass spectrometer equipped with a column containing the chromatography column packing material of the present disclosure has higher sensitivity than a liquid chromatography mass spectrometer that uses a conventional column.

[0123] <Supplementary Notes> Some or all of the above embodiments and examples may be described as, but are not limited to, the following supplementary notes: (Supplementary Note 1) A chromatography column packing material, characterized in that silanol groups of silica contained in the packing material are modified with groups represented by the following chemical formula (I): (Chemical Formula I) In the above chemical formula (I), R １is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with a substituent; m is an integer of 1 or more; n is 0 or a positive integer; X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group, and one or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent; Y １ and Y ２ At least one of Y is a hydrophilic group, １ and Y ２ may be the same or different, and * indicates a bonding position. (Appendix 2) The filler according to Appendix 1, wherein the ratio of the silanol groups (a) contained in the silica after the modification to the silanol groups (b) contained in the silica before the modification is 100 mol % or more. (Appendix 3) In the chemical formula (I), Y １ and Y ２ The packing material according to Appendix 1 or 2, wherein either one of the following groups is a hydrophobic group. (Appendix 4) The packing material according to any one of Appendixes 1 to 3, wherein in X in the chemical formula (I), the linear or branched alkyl group is a linear or branched alkyl group having 1 to 30 carbon atoms. (Appendix 5) A chromatography column packing material, comprising silica modified with a reaction reagent represented by the following chemical formula (II): In the chemical formula (II), R １ is a methylene group, one or more hydrogen atoms of which may or may not be further substituted with a substituent; R ２is a methyl group, a linear or branched alkyl group, and one or more hydrogen atoms of the alkyl group may or may not be further substituted with a substituent; m is an integer of 1 or more; n is 0 or a positive integer; X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group, and one or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent; Y １１ and Y １２and at least one of the groups is a functional group capable of forming a silanol group. (Appendix 6) A chromatography column packed with the packing material according to any one of Appendices 1 to 5. (Appendix 7) A chromatography column according to Appendices 6 for analyzing plant-derived components. (Appendix 8) A method for producing a chromatography column according to Appendices 6 or 7, comprising a modifying step and a packing step, wherein the modifying step is a step of modifying silanol groups of silica gel particles contained in the packing material, and the packing step is a step of packing the silica after the modification into a chromatography column. (Appendix 9) A method for producing a chromatography column according to Appendices 6 or 7, comprising a modifying step, a packing step, and a packing step, wherein the modifying step is a step of modifying silanol groups of monolithic silica contained in the packing material, and the packing step is a step of packing the silica before the modification into a chromatography column. (Appendix 10) A chromatography analysis device comprising a chromatograph, an eluent, and an analytical column, wherein the analytical column is the chromatography column according to Appendices 6 or 7. (Appendix 11) The analytical device according to Appendix 10, wherein the chromatograph includes a gradient elution section, and the eluent is a three-liquid or more eluent system combining water and two or more water-soluble organic solvents, and the gradient elution section performs gradient elution by using the eluent as a mobile phase and changing the solvent composition of the eluent at a flow rate of 20 nL / min to 100 mL / min. (Appendix 12) The analytical device according to Appendix 11, wherein the water-soluble organic solvent includes at least one of an alcohol-based solvent and a nitrile-based solvent. (Appendix 13) An analytical method using the analytical device according to any one of Appendixes 10 to 12. (Appendix 14) A method for analyzing an analyte containing at least one of a hydrophilic compound and an amphipathic compound, using the analytical method according to Appendix 13. (Appendix 15) A method for analyzing a plant-derived component, using the analytical method according to Appendix 14.

[0124] This application claims priority based on Japanese Patent Application No. 2023-029001, filed February 27, 2023, the disclosure of which is incorporated herein in its entirety.

[0125] As described above, the present disclosure provides a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analyzer, and an analysis method suitable for separating mixtures containing various analytes. The packing material is particularly suitable for separating plant components containing many hydrophilic and amphiphilic analytes. The present disclosure is not particularly limited in its applications and can be used in a wide range of applications, such as the analysis, testing, and diagnosis of foods, pharmaceuticals, chemicals, etc.

Claims

1. The filler is characterized in that the silanol groups of silica contained in the filler are modified with a group represented by the following chemical formula (I): Chromatography column packing material. [Chemical formula I] In the above chemical formula (I), R １ is a methylene group, one or more hydrogen atoms of which may or may not be further substituted with a substituent; m is an integer of 1 or more, n is 0 or a positive integer, X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group, and one or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent; Y １ and Y ２ At least one of Y is a hydrophilic group. １ and Y ２ may be the same or different, * indicates the bond position.

2. 2. The filler according to claim 1, wherein the ratio of silanol groups (a) contained in the silica after the modification to the silanol groups (b) contained in the silica before the modification is 100 mol % or more.

3. In the above chemical formula (I), Y １ and Y ２ 2. The filler according to claim 1, wherein either one of the groups is a hydrophobic group.

4. In the above chemical formula (I), In X, the linear or branched alkyl group is a linear or branched alkyl group having 1 to 30 carbon atoms. The filler of claim 1.

5. The silica modified with a reaction reagent represented by the following chemical formula (II) is included: Chromatography column packing material. 【Chemistry II】 In the above chemical formula (II), R １ is a methylene group, one or more hydrogen atoms of which may or may not be further substituted with a substituent; R ２ is a methyl group, a linear or branched alkyl group, one or more hydrogen atoms of which may or may not be further substituted with a substituent; m is an integer of 1 or more, n is 0 or a positive integer, X is a methyl group, a linear or branched alkyl group, an amino group, an amido group, a cyano group, an aryl group, an alkylaryl group, a carboxy group, or a carbamoyl group, and one or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxy group, or the carbamoyl group may or may not be further substituted with a substituent; Y １１ and Y １２ At least one of them is a functional group capable of forming a silanol group.

6. A chromatography column packed with the packing material according to any one of claims 1 to 5.

7. The chromatography column according to claim 6, which is used for the analysis of plant-derived components.

8. The method includes a modification step and a filling step, The modification step is a step of modifying silanol groups of silica gel particles contained in a filler, The packing step is a step of packing the modified silica into a chromatography column. A method for producing the chromatography column according to claim 6.

9. A modification step, a filling step, and The modification step is a step of modifying a silanol group of the monolithic silica contained in the filler, The packing step is a step of packing the unmodified silica into a chromatography column. A method for producing the chromatography column according to claim 6.

10. A chromatograph, an eluent, and an analytical column; The analytical column is a chromatography column according to claim 6. Chromatography analyzer.

11. The chromatograph includes a gradient elution section; the eluent is a three-liquid or more eluent comprising water and two or more water-soluble organic solvents; The gradient elution section performs gradient elution by changing the solvent composition of the eluent at a flow rate of 20 nL / min to 100 mL / min using the eluent as a mobile phase. The analytical device according to claim 10.

12. The water-soluble organic solvent includes at least one of an alcohol-based solvent and a nitrile-based solvent. The analytical device according to claim 11.

13. An analysis method using the analysis device according to claim 10.

14. A method for analyzing an analyte containing at least one of a hydrophilic compound and an amphipathic compound, by the method according to claim 13.

15. A method for analyzing a plant-derived component by the method according to claim 14.

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