Chromatography column packing material, chromatography column, method for manufacturing a chromatography column, chromatography analyzer, and analytical 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

[Technical Field]

[0001] This disclosure relates to a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analyzer, and an analytical method. [Background technology]

[0002] Chromatography is commonly used as a method for separating mixtures. In chromatography, the carrier used for sample transport is a gas, liquid, or supercritical fluid, depending on the characteristics of the components to be separated. However, 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). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2003-172733 [Overview of the project] [Problems that the invention aims to solve]

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

[0005] Therefore, the present disclosure aims to provide a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analyzer, and an analytical method suitable for separating mixtures containing various analytes.

Means for Solving the Problem

[0006] In order to achieve the above object, the column packing material for chromatography of the present disclosure is characterized in that the silanol groups of silica contained in the packing material are modified with groups represented by the following chemical formula (I). In the chemical formula (I), [Chem.] R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with substituents; 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 amide 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 substituents; At least one of Y1 and Y2 is a hydrophilic group, and Y1 and Y2 may be the same or different; * indicates the bonding position. * indicates the bonding position.

[0007] A column packing material for chromatography, comprising silica modified with a reaction reagent represented by the following chemical formula (II). Column packing material for chromatography [Chem.] In the chemical formula (II), R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be further substituted with substituents; R2 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 substituents; 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 amide group, a cyano group, an aryl group, an alkylaryl group, a carboxyl 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 carboxyl group, or the carbamoyl group may or may not be further substituted with substituents. Y 11 and Y 12 These are functional groups in which at least one of them can form a silanol group.

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

[0009] The method for manufacturing a chromatography column disclosed herein is: Including a modification step and a filling step, The modification step is a step of modifying the silanol groups of silica gel particles contained in the filler, The packing step is the step of packing the modified silica into a chromatography column.

[0010] The method for manufacturing a chromatography column disclosed herein is: Including a modification step, a filling step, The modification step is a step of modifying the silanol groups of monolithic silica contained in the filler, The packing step is the step of packing the silica before modification into a chromatography column.

[0011] The chromatography analyzer disclosed herein is Includes a chromatograph, eluent, and analytical column. The analytical column is the chromatography column of this disclosure.

[0012] The analytical method described herein uses the analytical apparatus described herein. [Effects of the Invention]

[0013] According to this disclosure, it is possible to provide a chromatography column packing material, a chromatography column, a method for producing a chromatography column, a chromatography analyzer, and an analytical method suitable for separating mixtures containing various analytes. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 shows 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] Figure 2 shows the chromatogram obtained when separating components contained in black tea (Darjeeling) using columns 1 and 15. [Figure 3] Figure 3 shows the chromatogram obtained when separating components contained in five types of tea using column 1. [Figure 4] Figure 4 is a chromatogram comparing the difference in separation ability based on the mobile phase when separating components contained in black tea (Darjeeling) using column 1. [Figure 5] Figure 5 is a chromatogram obtained by separating the component (rutin) contained in asparagus using column 1. [Modes for carrying out the invention]

[0015] Next, we will provide a more detailed explanation of this disclosure with examples. However, this disclosure is not limited in any way by the following explanation.

[0016] In this disclosure, technical terms, scientific and technological terms, and other such terms have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains, unless otherwise defined. If a definition is given for a term, that definition shall prevail.

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

[0018] Furthermore, in this disclosure, if substituents or compounds (for example, the group represented by chemical formula (I) or the compound represented by chemical formula (II) described later) have isomers such as tautomers or stereoisomers (e.g., geometric isomers, conformational isomers, and optical isomers), any of these isomers may be used in this disclosure unless otherwise specified. Also, if a compound can form a salt, the salt may be used in this disclosure unless otherwise specified. The salt may be an acid addition salt or a base addition salt. Furthermore, the acid forming the acid addition salt may be an inorganic acid or an organic acid, and the base forming the base addition salt may be an inorganic base or an organic base. The inorganic acid is not particularly limited, but examples include sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, carbonic acid, hydrobromic acid, hydroiodic acid, hypofluorite, hypochlorous acid, hypobromous acid, hypoiodic acid, hypofluorite, chlorous acid, bromous acid, iodic acid, fluorite, chloric acid, bromate, iodic acid, perfluorite, perchloric acid, perbromic acid, and periodic acid. The organic acid is also not particularly limited, but examples 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 include ammonium hydroxide, alkali metal hydroxide, alkaline earth metal hydroxide, carbonate, and bicarbonate. More specifically, examples 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 include ethanolamine, triethylamine, and tris(hydroxymethyl)aminomethane. The method for producing these salts is not particularly limited, and they can be produced, for example, by adding the aforementioned acids or bases to the compounds using known methods.

[0019] Furthermore, in this disclosure, linear substituents (e.g., hydrocarbon groups such as alkyl groups and unsaturated aliphatic hydrocarbon groups) may be linear or branched unless otherwise specified, and the number of carbon atoms 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 this disclosure, the number of ring members (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, if isomers exist for substituents, etc., any isomer may be used unless otherwise specified, for example, when simply referred to as "naphthyl group", it may be either a 1-naphthyl group or a 2-naphthyl group.

[0020] <Column packing material for chromatography> First, the chromatography column packing material of this disclosure will be described.

[0021] In one embodiment of the chromatography column packing material of the present disclosure, in chemical formula (I), R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with substituents, but it is preferable that they are not substituted. If substituted with substituents, it is preferable that the substituents are, for example, hydrophobic groups.

[0022] In the chemical formula (I) above, 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, and may be 29 or less, 27 or less, 25 or less, 23 or less, 21 or less, or 19 or less. For example, n may be between 0 and 29, between 0 and 3, or between 2 and 3.

[0024] In the above chemical formula (I), X is a methyl group, a linear or branched alkyl group, an amino group, an amide group, a cyano group, an aryl group, an alkylaryl group, a carboxyl group, or a carbamoyl group. One or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxyl group, or the carbamoyl group may or may not be further substituted with substituents. If substituted, these substituents may be, for example, non-aromatic hydrocarbon groups (which may be linear or branched, saturated or unsaturated, and which may or may not have a cyclic structure), aromatic groups (for example, aromatic groups that do not contain heteroatoms (aryl groups), heteroaromatic groups that contain heteroatoms (heteroaryl groups), which may be monocyclic or fused rings), halogens, amino groups, nitro groups, sulfo groups, or cyano groups, and at least one hydrogen atom of each substituent may or may not be further substituted with any other substituent.

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

[0026] In the above chemical formula (I), at least one of Y1 and Y2 is a hydrophilic group, for example, both Y1 and Y2 may be hydrophilic groups, only one of Y1 and Y2 may be a hydrophilic group, or the other may be a hydrophobic group. Examples of the hydrophilic group include a hydroxyl group, a carboxyl group, an amino group, etc., and when these are ionized, -O - , -COO - , -NH3 + The hydrophobic group may be, for example, a methyl group, a linear or branched alkyl group, an alkenyl group, an aryl group, an alkylaryl group, a polycyclic aryl group, etc. At least one hydrogen atom of each substituent may or may not be substituted with any other substituent. Y1 and Y2 may be the same or different.

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

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

[0029] The packing material of this disclosure comprises Y1 and Y2 as shown in chemical formula (I). For example, if at least one of Y1 and Y2 is a hydroxyl group, the proportion will be 100 mol% or more. That is, for example, if only one of Y1 and Y2 is a hydroxyl group, the proportion will be 100 mol%, and if both Y1 and Y2 contain hydroxyl groups, the proportion will exceed 100 mol%. If the proportion is 100 mol% or more, the silanol group of the packing material of this disclosure is modified with a hydrophilic hydroxyl group, and therefore, for example, when analyzing a mixture containing at least one hydrophilic or amphiphilic analyte, the affinity with the hydrophilic or amphiphilic analyte is improved, making it particularly effective when separating them.

[0030] The aforementioned percentage may have, for example, a lower limit of 100 mol% or more, 110 mol% or more, or 116 mol% or more, and an upper limit of 135 mol% or less, 130 mol% or less, or 125 mol% or less. The aforementioned percentage may be, for example, 100-135 mol%, 110-130 mol%, or 116-125 mol%.

[0031] The aforementioned ratio can be calculated, for example, as follows. Note that the calculation method differs depending on whether Y1 and Y2 are hydroxyl groups or whether only one of Y1 or Y2 is a hydroxyl group in the aforementioned chemical formula (I).

[0032] First, the case where Y1 and Y2 are hydroxyl groups will be described. When Y1 and Y2 are hydroxyl groups, for example, the above ratio can be calculated using a liquid chromatograph. First, the carbon content D (wt%) of the silica after the modification is calculated by the following mathematical formula (1). D (wt%) = Aα + B (1)

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

[0034] Also, α in the above mathematical formula (1) can be measured as follows. First, under isocratic conditions using a mixed solvent of water / methanol = 20 / 80 (v / v) as the mobile phase with a liquid chromatograph, amylbenzene and butylbenzene are eluted using a column containing the filler of the present disclosure to analyze the elution times (t R ). From the obtained individual elution times (t R ), and the column one - volume elution time (t0) of the solvent, the retention factors (k( R )) of amylbenzene and butylbenzene are obtained using the following mathematical formula (2). k( R ) = (t R - t0) / t0 (2)

[0035] Based on the retention factors (k( R )) of amylbenzene and butylbenzene respectively, α is calculated using the following mathematical formula (3). α = k( アミルベンゼン ) / k( ブチルベンゼン ) (3)

[0036] And the carbon content D is the number of moles of substituents bonded per 1 m 2 of the silica, that is, the density E (μmol / m 2Between ) the following equation (4) holds. For example, by plotting D and E as described in the above document, x in equation (4) is determined to be 0.1848 and y to be -0.3744. E(μmol / m 2 ) = xD + y (4)

[0037] Here, the amount of unmodified silanol groups on the silica surface is, for example, 8 μmol / m³ 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 substituents, the amount of silanol groups present on the modified silica surface is "8-E(μmol / m³)". 2 )”

[0038] Here, the silanol group newly formed by the bonding of substituents is defined as "2 × E (μmol / m³)" when both Y1 and Y2 in chemical formula (I) are silanol groups. 2 Therefore, the amount of silanol groups present when both Y1 and Y2 are hydroxyl groups is "(8-E)+(2×E)=8+E(μmol / m³)". 2 Therefore, when both Y1 and Y2 are silanol groups, the percentage F (mol%) of silanol groups remaining on the silica surface after the modification reaction can be obtained based on the following formula (5). F(mol%) = (8+E) / 8 × 100 (5)

[0039] On the other hand, if only one of Y1 and Y2 in the above chemical formula (I) is a hydroxyl group, the silanol group newly formed by the bonding of substituents is "E (μmol / m³) 2 Therefore, the amount of silanol groups present when only one of Y1 or Y2 is a hydroxyl group is "(8-E)+E=8(μmol / m³)". 2 Therefore, in this case, the percentage of silanol groups remaining on the silica surface after the modification reaction, F (mol%), is 100 (mol%), given by "F (mol%) = 8 / 8 × 100".

[0040] The above method is described as one example of how to calculate the aforementioned ratio, but it is not limited to this method. For example, the ratio may be calculated by various quantitative analyses. For instance, quantitative analysis of the silanol groups before and after the modification of 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 (SEM-EDS) equipped with an energy-dispersive X-ray analyzer, mass spectrometry (MS), infrared spectroscopy, Raman spectroscopy, etc., 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 chemical formula (II), R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with substituents, but it is preferable that they are not substituted. If substituted with substituents, it is preferable that the substituents are, for example, hydrophobic groups.

[0042] In the above 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) above, 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, and may be 29 or less, 27 or less, 25 or less, 23 or less, 21 or less, or 19 or less. For example, n may be between 0 and 29, between 0 and 3, or between 2 and 3.

[0044] In the above chemical formula (II), X is a methyl group, a linear or branched alkyl group, an amino group, an amide group, a cyano group, an aryl group, an alkylaryl group, a carboxyl group, or a carbamoyl group. One or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxyl group, or the carbamoyl group may or may not be further substituted with substituents. If substituted, these substituents may be, for example, non-aromatic hydrocarbon groups (which may be linear or branched, saturated or unsaturated, and which may or may not have a cyclic structure), aromatic groups (for example, aromatic groups that do not contain heteroatoms (aryl groups), heteroaromatic groups that contain heteroatoms (heteroaryl groups), which may be monocyclic or fused rings), halogens, amino groups, nitro groups, sulfo groups, or cyano groups, and at least one hydrogen atom of each substituent may or may not be further substituted with any other substituent.

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

[0046] In the above chemical formula (II), Y 11 and Y 12At least one of the substituents is a functional group capable of forming a silanol group. For example, at least one is a linear 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 substituents. If substituted, for example, it may be a non-aromatic hydrocarbon group (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 without heteroatoms (aryl group), a heteroaromatic group containing heteroatoms (heteroaryl group), and may be 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 substituent. For example, a linear or branched alkoxy group may have 1 or more carbon atoms, 3 or more carbon atoms, 5 or more carbon atoms, or 10 or less carbon atoms, 8 or less carbon atoms, or 6 or less carbon atoms. Examples of the linear or branched alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy groups. Examples of the halogen atoms include fluorine, chlorine, bromine, and iodine atoms. Examples of the hydrophilic groups include hydroxyl, carboxyl, amino, dimethylamino, and diethylamino groups, and ionized forms of these include, for example, -O - , -COO - , -NH3 + The hydrophobic group may be, for example, a methyl group, a linear or branched alkyl group, an alkenyl group, an aryl group, an alkylaryl group, a polycyclic aryl group, etc. At least one hydrogen atom of each substituent may or may not be substituted with another substituent. 11 and Y 12 They may be the same or different.

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

[0048] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0049] Further explanations can be found by referring to the explanation of chemical formula (I) mentioned above.

[0050] <Chromatography column> Next, the chromatography column and the method for manufacturing the chromatography column described herein will be explained.

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

[0052] If the column of this 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] If the column of this 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, or 5000 mm or less, 3000 mm or less, or 2000 mm or less. The inner diameter of the column may be, for example, 0.01 mm or more, 0.05 mm or more, or 0.1 mm or more, or 1 mm or less, 0.5 mm or less, or 0.2 mm or less. The shape of the capillary column may be an elongated tube, or for example, a groove of a size equivalent to a capillary tube formed on a glass plate by microfabrication 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, those in which the silanol groups have been modified, and the monolithic silica is, for example, those in which the silanol groups have not been modified.

[0055] The columns of this disclosure are, for example, chromatographic columns for the analysis of plant-derived components. The plants are not particularly limited, but include, for example, ginkgo, fennel, turmeric, Corydalis, scutellaria, coptis japonica, zedoary, licorice, cinnamon, magnolia, coriander, diatomaceous earth, rehmannia, purple, peony, cardamom, ginger, ginger, lilac, chuanxiong, gentian, atractylodes, perilla, rhubarb, jujube, alis, clove, citrus peel, angelica, eucommia, ginseng, pinaria, pomelosium, aconite, peony bark, lilac, ginseng, and other crude drugs listed in the Japanese Pharmacopoeia; ISO / TR 23022-2018 Traditional Chinese These include herbal medicines listed in the medical journal; tea leaves such as green tea, matcha, and black tea; and foods such as coffee beans, sesame seeds, onions, yuzu, mandarins, bell peppers, garland chrysanthemum, shiso, grapes, blueberries, spinach, and broccoli. The plant-derived components include, for example, at least one of a hydrophilic compound and an amphiphilic compound, and may also include, for example, a hydrophobic compound. The plant-derived components are not particularly limited, but include, for example, phenol and its glycosides, coumarin and its glycosides, flavonoids and their glycosides, chalcones and their glycosides, anthocyanidins and their glycosides, anthraquinones and their glycosides, indole and its glycosides, nitriles and their glycosides, steroids and their glycosides, alkaloids and their glycosides, and compounds having structures that include these.

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

[0057] The columns of this disclosure can be manufactured, for example, by the following method.

[0058] First, a column with silica already formed inside (hereinafter sometimes simply referred to as the "pre-modified column") is dried, or silica before being packed into the 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, vacuum drying, or vacuum heat drying. The vacuum conditions during drying may be, for example, 1 kPa or more, 5 kPa or more, or 10 kPa or more, or 100 kPa or less, 80 kPa or less, or 50 kPa or less. The heating temperature during drying may be, for example, 80°C or more, 100°C or more, or 120°C or more, or 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, or 24 hours or less, 18 hours or less, or 12 hours or less.

[0059] Next, if it is a capillary column, the dried column before modification is connected to a liquid delivery device and heated while delivering the reaction reagent (heating step). The silica is modified by the reaction reagent during the heating step. The heating temperature during the modification may be, for example, 60°C or higher, 80°C or higher, or 100°C or higher, or 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, or 48 hours or lower, 36 hours or lower, or 24 hours or lower.

[0060] The liquid delivery device is capable of delivering the reaction reagent into the column by pressurization, for example, and could be a syringe pump or a liquid chromatography pump. The liquid delivery may also be done by pressurizing a gas, for example.

[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 any amount in the range of 10% 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 for the heating step in the capillary column. After filtering and washing the modified silica gel by a known method, the silica gel is packed into a column tube to obtain the packed column.

[0062] As mentioned above, the reaction reagent that can be used is the reaction reagent represented by the following chemical formula (II).

[0063] [ka]

[0064] In the above 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) above, 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, and may be 29 or less, 27 or less, 25 or less, 23 or less, 21 or less, or 19 or less. For example, n may be between 0 and 29, between 0 and 3, or between 2 and 3.

[0066] In the above chemical formula (II), X is a methyl group, a linear or branched alkyl group, an amino group, an amide group, a cyano group, an aryl group, an alkylaryl group, a carboxyl group, or a carbamoyl group. One or more hydrogen atoms of the alkyl group, the amino group, the aryl group, the alkylaryl group, the carboxyl group, or the carbamoyl group may or may not be further substituted with substituents. If substituted, these substituents may be, for example, non-aromatic hydrocarbon groups (which may be linear or branched, saturated or unsaturated, and which may or may not have a cyclic structure), aromatic groups (for example, aromatic groups that do not contain heteroatoms (aryl groups), heteroaromatic groups that contain heteroatoms (heteroaryl groups), which may be monocyclic or fused rings), halogens, amino groups, nitro groups, sulfo groups, or cyano groups, and at least one hydrogen atom of each substituent may or may not be further substituted with any other substituent.

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

[0068] In the above chemical formula (II), Y 11 and Y 12 The functional group Y is one in which at least one of the functional groups can form a silanol group. 11 and Y 12For example, at least one of the substituents may be a functional group capable of forming a silanol group by a substitution step described later. For example, at least one may be a linear 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 substituents. If substituted, for example, it may be a non-aromatic hydrocarbon group (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 without heteroatoms (aryl group), a heteroaromatic group containing heteroatoms (heteroaryl group), and may be 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 substituent. For example, the linear or branched alkoxy group may have 1 or more carbon atoms, 3 or more carbon atoms, 5 or more carbon atoms, or 10 or less carbon atoms, 8 or less carbon atoms, or 6 or less carbon atoms. Examples of the linear or branched alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy groups. Examples of the halogen atoms include fluorine, chlorine, bromine, and iodine atoms. Examples of the hydrophilic groups include hydroxyl, carboxyl, amino, dimethylamino, and diethylamino groups, and ionized forms of these include, for example, -O - , -COO - , -NH3 + The hydrophobic group may be, for example, a methyl group, a linear or branched alkyl group, an alkenyl group, an aryl group, an alkylaryl group, a polycyclic aryl group, etc. At least one hydrogen atom of each substituent may or may not be substituted with another substituent. 11 and Y 12 They may be the same or different.

[0069] In the above chemical formula (II), R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with substituents, but it is preferable that they are not substituted. If substituted with substituents, it is preferable that the substituents are, for example, hydrophobic groups.

[0070] In the above chemical formula (II), R2 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 substituents. If substituted, for example, it may be a non-aromatic hydrocarbon group (which may be linear or branched, saturated or unsaturated, and which may or may not have a cyclic structure), an aromatic group (for example, an aromatic group that does not contain heteroatoms (aryl group), a heteroaromatic group that contains heteroatoms (heteroaryl group), which may be 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 any other substituent. Linear or branched alkyl groups are, for example, linear or branched alkyl groups having 1 to 12 carbon atoms, such as methyl, ethyl, propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl (lauryl) groups.

[0071] When modifying the silica according to the chemical formula (II), the modification is carried out, for example, by a condensation reaction between R2-O- in the chemical formula (II) and the silanol group of the silica.

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

[0073] The reaction reagent may or may not contain other components. Examples of these other components include solvents and catalysts. Examples of solvents include 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 catalysts include amine catalysts. Examples of the amine catalysts 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, the process may include, for example, a step of washing the unreacted reaction reagent (reaction reagent washing step). The reaction reagent washing step is, for example, a step of delivering a solvent into the column after the heating step. The solvent is, for example, the same as the solvent included as the other components. The delivery time is not particularly limited, as long as it is a time sufficient to wash the unreacted reaction reagent, 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] In the above chemical formula (II), Y 11 and Y 12However, if, for example, the silica contains groups other than hydrophilic groups, the process may further include a step of substituting the groups other than hydrophilic groups with hydrophilic groups (substitution step). The substitution step is, for example, a step of further hydrolyzing the modified silica, thereby merging the Y of chemical formula (II) with the modified silica. 11 and Y 12 This may be a step of substituting at least one of the groups with a hydroxyl group (i.e., a step of forming a silanol group).

[0076] The substitution step is, for example, a step of delivering 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 included as the other components. The acid may be, for example, an organic acid or an inorganic acid. The organic acid is not particularly limited, but examples include acetic acid, formic acid, difluoroacetic acid, trifluoroacetic acid, etc. Examples of the inorganic acid include sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, hypofluorite, hypochlorous acid, hypobromous acid, hypoiodic acid, hypofluorite, chlorous acid, bromous acid, iodic acid, fluorite, chloric acid, bromic acid, iodic acid, perfluorite, perchloric acid, perbromic acid, and periodic acid, etc. 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 delivery time in the liquid delivery step is not particularly limited, for example, as long as it is a time that allows for the substitution of groups other than hydrophilic groups with hydrophilic groups, and is 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.

[0078] The substitution step may be followed by a washing step (substitution reagent washing step). The substitution reagent washing step is, for example, a step of delivering a solvent into the column after the substitution step. The solvent is, for example, the same as the solvent included as the other components. The delivery time is not particularly limited as long as it is sufficient time to wash away the unreacted reaction reagent, 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 analyzer> Next, the chromatography analyzer (also called a "chromatograph") of this disclosure will be described.

[0080] Examples of the chromatographic analyzer include 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 to any location between the sample injection unit and the detection unit. The gradient elution unit is preferably connected 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 by considering, for example, the analytical conditions, and one column may be used, or two or more columns may be used.

[0082] The eluent is, for example, a three-liquid system 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 fewer, seven or fewer, or six or fewer. The number of liquid systems of the eluent changes depending on the number of solvents combined, but it is preferable to have three or more liquid systems. Examples of the water-soluble organic solvents include alcohol-based solvents and nitrile-based solvents. The water-soluble organic solvent may, for example, contain at least one of the alcohol-based solvent and the nitrile-based solvent. Examples of the alcohol-based solvents 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 solvents include acetonitrile.

[0083] The gradient elution unit performs gradient elution by changing the solvent composition using the eluent as the mobile phase. The solvent composition of the eluent may be changed to, for example, a flow rate of 20 nL to 100 mL / min, 100 nL to 1 mL / min, or 300 nL to 0.01 mL / min during gradient elution. Furthermore, if the eluent consists of three or more liquids, the solvent composition of at least one of the eluents may be changed from the start of elution to the end of elution, for example, by changing it from 0 to 100%, 10 to 90%, and 20 to 80%, during gradient elution.

[0084] The chromatography analyzer of this disclosure may further include other analytical instruments, such as a mass spectrometer. These other analytical instruments may be, for example, integrated with the analyzer of this disclosure, or they may be separate instruments connected to each other for use.

[0085] <Analysis method> Next, we will explain the analysis method of this disclosure.

[0086] As described above, the analytical method described herein is an analytical method using the analytical apparatus described herein.

[0087] The analytical methods of this disclosure include, for example, methods for analyzing an analyte containing at least one of a hydrophilic compound and an amphiphilic compound. The analytical methods of this disclosure include, for example, methods for analyzing plant-derived components. The plants and plant-derived components are, for example, the same as those described in the columns of this disclosure. [Examples]

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

[0089] <Quantitative determination of hydrophilic groups> [Example 1] As Example 1, a chromatography column 1 (hereinafter sometimes referred to as "Column 1") containing the chromatography column packing material of the present disclosure was manufactured.

[0090] First, a monolithic silica capillary (0.1 mm inner diameter, 750 mm length, manufactured by Shinwa Chemical Co., Ltd.) was dried under reduced pressure at 140°C for 6 hours. Next, the dried monolithic silica capillary was connected to a liquid delivery device (pump) including a heating furnace, and the reaction was carried out at 100°C while the reaction reagent was delivered to the monolithic silica capillary. The reaction reagent consisted 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 delivery time of the reaction reagent was 40 hours. After that, toluene was delivered at room temperature for 24 hours to wash away any unreacted reaction reagent. Next, at 60°C, the substitution reagent was delivered to the monolithic silica capillary after the reaction, and the unreacted leaving groups of the octadecyltriethoxysilane after the reaction were hydrolyzed to obtain silanol groups. The substitution reagent consisted of 80 mL of acetonitrile, 20 mL of ultrapure water, and 0.1 mL of formic acid, with a pH of approximately 2. The substitution reagent was pumped for 20 hours. Finally, to wash off the substitution reagent, methanol was pumped as an organic solvent at room temperature for 24 hours to prepare column 1. Column 1 was cut at the end 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 concentrations of 0.05 mg / mL, 0.5 μL / mL, and 0.5 μL / mL, respectively. The uracil peak was defined as t0, and the butylbenzene and amylbenzene peaks were defined as t, respectively. R Based on the aforementioned formulas (1) to (5), the α of column 1 and the percentage of silanol groups remaining on the silica surface after the modification reaction were calculated. The results are shown in Table 1 below.

[0092] (HPLC measurement conditions) Equipment: 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℃ Detection wavelength: 220 nm

[0093] [Examples 2-14 and Comparative Example 1] In this embodiment, chromatographic columns 2 to 14 (hereinafter sometimes referred to as "columns 2 to 14") and a comparative chromatographic column (hereinafter sometimes referred to as "column 15") containing the chromatography column packing material of this disclosure were manufactured as Examples 2 to 14 and Comparative Example 1. In Examples 2 to 14, columns 2 to 14 were manufactured 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, the column (column 15) was prepared in the same manner as in Example 1, except that a reaction reagent containing 9 mL of toluene and 1 mL of octadecyldimethyl(dimethylamino)silane was used instead of the reaction reagent used in Example 1.

[0095] [Table 1]

[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 substituents containing hydrophilic groups (silanol groups), and the percentage 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 substituents that did not contain hydrophilic groups (silanol groups), and the percentage of silanol groups remaining on the silica surface after the modification reaction was less than 100 mol%.

[0097] Here, Figure 1 is a plot of columns 1 to 15, with the methylene group selectivity α on the vertical axis and the reaction reagent delivery time (reaction time) on the horizontal axis. As shown in Figure 1, the value of α changes from 1.30 to 1.40 when the reaction reagent containing octadecyltriethoxysilane is delivered for 15 to 40 hours, and it was found that columns with an α value in the range of 1.40 to 1.48 can be stably produced when the delivery time exceeds 40 hours.

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

[0099] [Table 2]

[0100] Untreated silica was found to have an O / Si ratio of approximately 2, meaning it has an SiO2 structure. In contrast, the packing material for column 1 had an increased O / Si ratio compared to the untreated silica. This indicates that the amount of hydroxyl groups increased in column 1. On the other hand, the packing material for column 15 had a decreased O / Si ratio compared to the untreated silica. This indicates that the amount of hydroxyl groups decreased in column 15.

[0101] <Separation of components contained in black tea> Based on the following procedure and conditions, components contained in black tea (Darjeeling) were separated using columns 1 and 15.

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

[0103] (HPLC measurement conditions) Equipment: High-performance liquid chromatograph (Thermo Fisher Scientific Co., Ltd.) Mobile phase A: 0.2 vol% aqueous solution with added phosphoric acid Mobile phase B: 0.2 vol% methanol with phosphoric acid 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℃ Detection wavelength: 203 nm Sample concentration: 0.5 mg / mL Sample injection volume: 1 μL

[0104] Figure 2 shows chromatograms obtained by separating 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 of components contained in black tea were detected over a wide range. In other words, column 1, which contains the chromatography column packing material of this disclosure, was able to adsorb and separate a wide range of components of black tea. On the other hand, column 15 showed weak adsorption of components contained in black tea and eluted quickly. Thus, it was found that column 1, which has a large number of silanol groups remaining on the silica surface of the chromatography column packing material, is suitable for the retention and separation of plant components.

[0106] <Separation of components contained in various types of tea> Based on the following procedure and conditions, the components contained in the tea were separated using column 1.

[0107] (Preparation of analytical samples) Except for substituting commercially available tea bags (Earl Grey), green tea, roasted green tea, and sweet tea for the tea leaves in commercially available tea bags (Darjeeling), the analytical samples were prepared using the same procedure as described above for "separation of components contained in black tea," and analytical samples of 0.5 mg / mL were obtained for each of the five types of tea (Darjeeling, Earl Grey, green tea, roasted green tea, and sweet tea).

[0108] (HPLC measurement conditions) The same measurement conditions as those used for the "separation of components contained in black tea" described above were employed.

[0109] Figure 3 shows chromatograms obtained by separating components from five types of tea (Darjeeling, Earl Grey, green tea, hojicha, and amacha) 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, numerous peaks of varying sizes were detected in each analytical sample. Furthermore, it was shown that Darjeeling, Earl Grey, green tea, and hojicha, all made from the same tea leaves, had different component patterns. In addition, amacha, made from tea leaves different from the other four types of tea, showed a different component pattern. Thus, it was found that columns containing the chromatography column packing material of this disclosure can effectively adsorb and separate plant components and separate them precisely, making them useful for precise analysis of plant components and pattern analysis of plant-containing components.

[0111] <Differences in separation capabilities due to mixed mobile phases> Based on the following procedure and conditions, components contained in black tea (Darjeeling) were separated using column 1, and the change in separation capability when using a mixed mobile phase was confirmed. (Preparation of analytical samples) The analytical sample was prepared using the same procedure as described above for "separation of components contained in black tea," and an analytical sample of 0.5 mg / mL was obtained. (HPLC measurement conditions) Equipment: High-performance liquid chromatograph (Thermo Fisher Scientific Co., Ltd.) Mobile phase A: 0.2 vol% aqueous solution with added phosphoric acid Mobile phase B: Condition 1.0.2 Volume % methanol with added phosphoric acid Condition 2.0.2 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℃ Detection wavelength: 203 nm Sample concentration: 0.5 mg / mL Sample injection volume: 1 μL

[0112] Figure 4 is a chromatogram comparing the differences in separation ability based on the mobile phase when separating components contained in black tea (Darjeeling) 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 with a two-component solvent of water and methanol (Condition 1), which is widely used in general HPLC, resulted in 62 detected peaks. On the other hand, gradient elution with a three-component solvent of water-methanol-acetonitrile (Condition 2) resulted in 89 detected peaks. In other words, while a column containing the chromatography column packing material of this disclosure can effectively separate plant components by gradient elution with a two-component solvent of water and methanol, a greater number of components can be separated by gradient elution with a three-component solvent of water-methanol-acetonitrile. This indicates that a column containing the chromatography column packing material of this disclosure is useful in the analysis of plant components that contain a wide variety of components.

[0114] <Separation of components contained in asparagus> Based on the following procedure and conditions, the components contained in asparagus were separated using column 1.

[0115] (Preparation of analytical samples) Except for using commercially available asparagus dried under reduced pressure at 40°C instead of commercially available tea bag black tea (Darjeeling), the analytical sample was prepared using the same procedure as described above for "separation of components contained in black tea," and an analytical sample of 0.5 mg / mL was obtained from the dried asparagus extract.

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

[0117] (HPLC measurement conditions) Equipment: High-performance liquid chromatograph (Thermo Fisher Scientific Co., Ltd.) Mobile phase A: 0.2 vol% aqueous solution with added phosphoric acid Mobile phase B: 0.2 vol% 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℃ Detection wavelength: 203 nm Sample concentration: 0.5 mg / mL Sample injection volume: 1 μL

[0118] Figure 5 shows the chromatogram obtained when separating the component (rutin) contained in asparagus 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, analysis of the rutin standard allowed us to identify rutin, which is abundant in asparagus. Thus, it was found that the column containing the chromatography column packing material of this disclosure is useful not only for tea but also for the analysis of other plant components, including asparagus. Furthermore, it was found that plant components can be identified and quantitatively analyzed by using standards.

[0120] As described above, a column containing the chromatography column packing material of this disclosure enables more sensitive component analysis than conventional columns.

[0121] Furthermore, since plant-derived components differ from plant to plant and exist in many varieties, analysis using general liquid chromatography analyzers is difficult. Particle-based columns use extremely small particles for these analyses. Such columns using extremely small particles generate an extraordinary ultra-high pressure of approximately 1000 atmospheres during liquid delivery, requiring a special dedicated system capable of withstanding ultra-high pressure conditions. However, with columns containing the chromatography column packing material of this disclosure, separation and analysis of various components is possible even using general liquid chromatography at around 100 atmospheres. Therefore, depending on the application, for example, it is possible to use a general liquid chromatography analyzer using a column containing the chromatography column packing material of this disclosure in a complementary manner with an ultra-high pressure liquid chromatography analyzer.

[0122] Furthermore, columns containing the chromatography column packing material of this disclosure are suitable for connection to mass spectrometers that require sample vaporization during component analysis, for example, because less 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 this disclosure is more sensitive than a liquid chromatography-mass spectrometer using a conventional column.

[0123] <Note> The above embodiments and some or all of the examples may also be described as follows, but are not limited to these. (Note 1) The silanol groups of the silica contained in the filler are modified with a group represented by the following chemical formula (I), Chromatography column packing material. (Chemistry I) TIFF0007898220000028.tif61170 In the above chemical formula (I), R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with substituents. m is an integer greater than or equal to 1, n is 0 or a positive integer, X is a methyl group, a linear or branched alkyl group, an amino group, an amide group, a cyano group, an aryl group, an alkylaryl group, a carboxyl 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 carboxyl group, or the carbamoyl group may or may not be further substituted with substituents. Y1 and Y2 are hydrophilic groups, and at least one of them may be the same or different. * indicates the bond location. (Note 2) The filler according to Appendix 1, wherein the ratio of silanol groups (a) contained in the modified silica to silanol groups (b) contained in the silica before modification is 100 mol% or more. (Note 3) The packing agent according to Appendix 1 or 2, wherein in the chemical formula (I), either Y1 or Y2 is a hydrophobic group. (Note 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. A filler as described in any of the notes 1 to 3. (Note 5) The silica contains a reaction reagent represented by the following chemical formula (II). Chromatography column packing material. [ka] In the above chemical formula (II), R1 is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with substituents. R2 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 substituents. m is an integer greater than or equal to 1, n is 0 or a positive integer, X is a methyl group, a linear or branched alkyl group, an amino group, an amide group, a cyano group, an aryl group, an alkylaryl group, a carboxyl 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 carboxyl group, or the carbamoyl group may or may not be further substituted with substituents. Y 11 and Y 12 These are functional groups in which at least one of them can form a silanol group. (Note 6) A chromatography column packed with one of the packing materials described in any of the appendices 1 to 5. (Note 7) A chromatography column for the analysis of plant-derived components, as described in Appendix 6. (Note 8) Including a modification step and a filling step, The modification step is a step of modifying the silanol groups of silica gel particles contained in the filler, The packing step is the step of packing the modified silica into a chromatography column. A method for producing a chromatography column as described in Appendix 6 or 7. (Note 9) Including a modification step, a filling step, The modification step is a step of modifying the silanol groups of monolithic silica contained in the filler, The packing step is a step of packing the silica before modification into a chromatography column. A method for producing a chromatography column as described in Appendix 6 or 7. (Note 10) Includes a chromatograph, eluent, and analytical column. The analytical column is the chromatography column described in Appendix 6 or 7. Chromatography analyzer. (Note 11) The chromatograph includes a gradient elution section, The eluent is a three-component or more eluent system consisting of water and two or more water-soluble organic solvents. The gradient elution unit uses the eluent as the mobile phase and performs gradient elution by changing the solvent composition of the eluent at a flow rate of 20 nL / min to 100 mL / min. The analytical apparatus described in Appendix 10. (Note 12) The water-soluble organic solvent comprises at least one of an alcohol-based solvent and a nitrile-based solvent. The analytical apparatus described in Appendix 11. (Note 13) An analytical method using the analytical apparatus described in any of the appendices 10 to 12. (Note 14) A method for analyzing an analyte containing at least one of a hydrophilic compound and an amphiphilic compound, according to the analytical method described in Appendix 13. (Note 15) Method for analyzing plant-derived components according to the analytical method described in Appendix 14.

[0124] This application claims priority based on Japanese Patent Application No. 2023-029001, filed on 27 February 2023, and incorporates all of its disclosures herein. [Industrial applicability]

[0125] As described above, this disclosure provides a chromatography column packing material, a chromatography column, a method for manufacturing a chromatography column, a chromatography analyzer, and an analytical method suitable for separating mixtures containing various analytes. In particular, it is suitable for separating plant components that contain many hydrophilic and amphiphilic analytes. The applications of this disclosure are not particularly limited and are arbitrary; for example, they can be used in a wide range of applications such as the analysis, testing, and diagnosis of food, pharmaceuticals, chemicals, etc.

Claims

1. The silanol groups of the silica contained in the filler are modified with a group represented by the following chemical formula (I), Chromatography column packing material. [Chemistry I] In the above chemical formula (I), R １ is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with hydrophobic groups. m is an integer greater than or equal to 1, n is a positive integer between 1 and 29, X is a methyl group, a linear or branched alkyl group, a cyano group, an aryl group, or an alkylaryl group, and one or more hydrogen atoms of the alkyl group, the aryl group, or the alkylaryl group may or may not be further substituted with a non-aromatic hydrocarbon group, an aromatic group, or a heteroaromatic group containing a heteroatom. Y １ and Y ２ Y is a group in which at least one of the components is a hydrophilic group. １ and Y ２ They may be the same or different. * indicates the bonding position.

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

3. In the above chemical formula (I), Y １ and Y ２ The filler according to claim 1, wherein 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 according to claim 1.

5. The silica contains a reaction reagent represented by the following chemical formula (II). Chromatography column packing material. 【Chemistry II】 In the above chemical formula (II), R １ is a methylene group, and one or more hydrogen atoms of the methylene group may or may not be substituted with hydrophobic groups. 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 greater than or equal to 1, n is a positive integer between 1 and 29, X is a methyl group, a linear or branched alkyl group, a cyano group, an aryl group, or an alkylaryl group, and one or more hydrogen atoms of the alkyl group, the aryl group, or the alkylaryl group may or may not be further substituted with a non-aromatic hydrocarbon group, an aromatic group, or a heteroaromatic group containing a heteroatom. Y １１ and Y １２ These are functional groups in which at least one of them can form a silanol group.

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

7. A chromatography column according to claim 6 for the analysis of plant-derived components.

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

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

10. Includes a chromatograph, eluent, and analytical column. The analytical column is the chromatography column described in claim 6. Chromatography analyzer.

11. The chromatograph includes a gradient elution section, The eluent is a three-component or more eluent system consisting of water and two or more water-soluble organic solvents. The gradient elution unit uses the eluent as the mobile phase and performs gradient elution by changing the solvent composition of the eluent at a flow rate of 20 nL / min to 100 mL / min. The analytical apparatus according to claim 10.

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

13. An analytical method using the analytical apparatus described in claim 10.

14. A method for analyzing an analyte containing at least one of a hydrophilic compound and an amphiphilic compound, according to the analytical method described in claim 13.

15. A method for analyzing plant-derived components according to the analytical method described in claim 14.

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