Process for separation of amines

Abstract

A method for separating an amine, said method comprising a separation step for separating an amine by chromatography using a stationary phase containing an oligosaccharide, a polysaccharide, and / or a derivative thereof, and a mobile phase containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent; when A is the value calculated by the following formula (1) for each specific acid contained in the mobile phase, SA is the total value of each A calculated by each specific acid, B is the value calculated by the following formula (2) for each base contained in the mobile phase, and SB is the total value of each B calculated by each base, SB / SA is 0.05-0.95. Formula (1): A = the number of elements of the specific acid * the molar concentration (mol / L) of the specific acid Formula (2): B = the number of elements of the base * the molar concentration (mol / L) of the base.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a separation method of amines. BACKGROUND

[0002] It is known that derivatives of polysaccharides as a separation agent of chiral selectors (hereinafter, also referred to as "polysaccharide-based CSPs") are extremely effective in the separation of many enantiomers, substances that are difficult to separate using a general separation agent due to structural similarity, such as structural isomers, and the like. In the case of these polysaccharide-based CSPs, the adsorbent is generally a neutral molecule, and therefore, in the case of analyzing a sample that is ionic under the analysis conditions, for some reason, the affinity with the separation agent can be lowered due to the ionization of the sample, the adsorption of ionic molecules can be electrostatically repelled, the loading amount can be reduced, and a small amount of ionic or polar atomic groups (silanol derived from silica gel used as a support is problematic in many cases.) can strongly interact with each other, and the like, so that the sample is hardly held, or a peak having a very large width or a distorted shape is exhibited, and the desired analysis cannot be performed.

[0003] In Non-Patent Literature 1, a method for solving the above problem by adding an appropriate salt to the mobile phase is disclosed. Specifically, it is considered to be a so-called ion pair chromatography in which the cation of the sample and the appropriate anion are simultaneously held to the stationary phase.

[0004] PRIOR ART DOCUMENTS NON-PATENT LITERATURE Non-Patent Literature 1: A. Ishikawa, T. Shibata, "Cellulosic Chiral Stationary Phase Under Reversed-Phase Condition", Journal of Liquid Chromatography, 1993, Vol. 16, 859-878 SUMMARY

[0005] PROBLEMS TO BE SOLVED BY THE INVENTION However, in the separation operation using the mobile phase disclosed in Non-Patent Literature 1, the separation performance of the separation target substance is not sufficient, and there is room for improvement.

[0006] The present disclosure relates to a separation method of amines.

[0007] MEANS FOR SOLVING THE PROBLEMS As a result of diligent studies by the inventors of the present application, it has been found that the above problems can be solved by using a stationary phase containing at least any one of an oligosaccharide derivative and a polysaccharide derivative, and a mobile phase containing a specific acid and an amine at a specific concentration, thereby completing the invention relating to the present disclosure. That is, the gist of the present disclosure is as described below.

[0008] [1] A separation method of an amine, comprising a separation process of: separating an amine by chromatography using a stationary phase containing at least any one of an oligosaccharide, a polysaccharide, and a derivative thereof, and a mobile phase containing 1 or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, 1 or more bases, and a solvent; when a value calculated from the following formula (1) with respect to each of the specific acids contained in the aforementioned mobile phase is set as A, a total value of each A calculated from each of the specific acids is set as SA, a value calculated from the following formula (2) with respect to each of the bases contained in the aforementioned mobile phase is set as B, and a total value of each B calculated from each of the bases is set as SB, SB / SA is 0.05 or more and 0.95 or less.

[0009] Formula (1): A = number of moles of specific acid × molar concentration of specific acid (mol / L) Formula (2): B = number of moles of base × molar concentration of base (mol / L) [2] The separation method of an amine according to [1], further comprising an analysis process of analyzing the amine separated by the aforementioned separation process, in the aforementioned analysis process, analysis using at least a mass spectrometer is performed.

[0010] [3] The separation method of an amine according to [1] or [2], wherein the base is 1 or more selected from the group consisting of ammonia, a primary amine, a secondary amine, and a tertiary amine.

[0011] [4] The separation method of an amine according to [3], wherein the base is 1 or more selected from the group consisting of ammonia, ethylamine, n-propylamine, 2-propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyrrolidine, and piperidine.

[0012] [5] The separation method of an amine according to any one of [1] to [4], wherein the mobile phase contains at least oxalic acid as the aforementioned specific acid.

[0013] [6] The separation method of an amine according to any one of [1] to [5], wherein at least any one of the aforementioned oligosaccharide, polysaccharide, and derivative thereof has an optically active site.

[0014] [7] The method for separating amines as described in any one of [1] to [6], wherein the solvent is one or more selected from the group consisting of water, acetonitrile, ethanol, methanol, 2-propanol, hexane, heptane, THF and liquid carbon dioxide.

[0015] Invention Effects According to this disclosure, a method for separating amines with excellent separation performance can be provided. Attached Figure Description

[0016] [ Figure 1 [This is a liquid chromatogram obtained by analyzing the amine in Example 1.]

[0017] [ Figure 2 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 1.]

[0018] [ Figure 3 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 2.]

[0019] [ Figure 4 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 3.]

[0020] [ Figure 5 [This is a liquid chromatogram obtained by analyzing amines in Example 2.]

[0021] [ Figure 6 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 4.]

[0022] [ Figure 7 [This is a liquid chromatogram obtained by analyzing amines in Example 3.]

[0023] [ Figure 8 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 5.]

[0024] [ Figure 9 [This is a liquid chromatogram obtained by analyzing amines in Example 4.]

[0025] [ Figure 10 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 6.]

[0026] [ Figure 11 [This is a liquid chromatogram obtained by analyzing amines in Example 5.]

[0027] [ Figure 12 [This is a liquid chromatogram obtained by analyzing the amine in Comparative Example 7.]

[0028] [ Figure 13 [This is a liquid chromatogram obtained by analyzing amines in Example 6.]

[0029] [ Figure 14 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 8.

[0030] [ Figure 15 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 9.

[0031] [ Figure 16 ] is a liquid chromatogram obtained by analysis of the amine in Example 7.

[0032] [ Figure 17 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 10.

[0033] [ Figure 18 ] is a liquid chromatogram obtained by analysis of the amine in Example 8.

[0034] [ Figure 19 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 11.

[0035] [ Figure 20 ] is a liquid chromatogram obtained by analysis of the amine in Example 9.

[0036] [ Figure 21 ] is a liquid chromatogram obtained by analysis of the amine in Example 10.

[0037] [ Figure 22 ] is a liquid chromatogram obtained by analysis of the amine in Example 11.

[0038] [ Figure 23 ] is a liquid chromatogram obtained by analysis of the amine in Example 12.

[0039] [ Figure 24 ] is a liquid chromatogram obtained by analysis of the amine in Example 13.

[0040] [ Figure 25 ] is a liquid chromatogram obtained by analysis of the amine in Example 14.

[0041] [ Figure 26 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 12.

[0042] [ Figure 27 ] is a liquid chromatogram obtained by analysis of the amine in Example 15.

[0043] [ Figure 28 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 13.

[0044] [Figure 29 ] is a liquid chromatogram obtained by analysis of the amine in Example 16.

[0045] [ Figure 30 ] is a liquid chromatogram obtained by analysis of the amine in Comparative Example 14.

[0046] [ Figure 31 ] is a liquid chromatogram obtained by analysis of the amine in Example 17.

[0047] [ Figure 32 ] is a liquid chromatogram obtained by analysis of the amine in Example 18.

[0048] [ Figure 33 ] is a liquid chromatogram obtained by analysis of the amine in Example 19.

[0049] [ Figure 34 ] is a mass spectrum obtained by mass spectrometric analysis of the amine in Example 20.

[0050] [ Figure 35 ] is a mass spectrum obtained by mass spectrometric analysis of the amine in Example 21.

[0051] [ Figure 36 ] is a mass spectrum obtained by mass spectrometric analysis of the amine in Example 22.

[0052] [ Figure 37 ] is a mass spectrum obtained by mass spectrometric analysis of the amine in Example 23.

[0053] [ Figure 38 ] is a mass spectrum obtained by mass spectrometric analysis of the amine in Example 24. DETAILED DESCRIPTION

[0054] Hereinafter, the present disclosure will be described with specific embodiments, but each configuration and their combinations and the like in each embodiment are one example, and addition, omission, substitution, and other changes of the configuration can be appropriately made within the scope of the gist of the present disclosure. The present disclosure is not limited by the embodiments, but is defined only by the scope of the claims.

[0055] In addition, each of the modes disclosed in the present disclosure can be combined with any of the other features disclosed in the present disclosure.

[0056] Note that in the present disclosure, in a case where a lower limit value and an upper limit value of a numerical range are described separately, the aforementioned numerical range can be a range combining any of the lower limit values and any of the upper limit values. In addition, in the present disclosure, in a case where "~" is used to express a range in which a numerical value or a physical property value is inserted before and after it, it is used as a range including the values of the lower limit and the upper limit as end points. In addition, in a case where a numerical range represented by "A ~ B" or "A or more and B or less" is described in stages (for example, in order of more preference), the upper limit and the lower limit of each numerical range can be combined arbitrarily.

[0057] In addition, in the present disclosure, the expression "A or B" can be replaced with "at least one selected from the group consisting of A and B".

[0058] <Method for separating amine> One embodiment of the present disclosure relates to a method for separating amine (hereinafter, also referred to simply as "the method for separating amine.") which is a method for separating amine including a separation process described below: The amine is separated by chromatography using a stationary phase containing at least any one of an oligosaccharide, a polysaccharide, and a derivative thereof, and a mobile phase containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent; In a case where a value calculated from the following formula (1) with respect to each of the specific acids contained in the aforementioned mobile phase is set as A, a total value of each A calculated from each of the specific acids is set as SA, a value calculated from the following formula (2) with respect to each of the bases contained in the aforementioned mobile phase is set as B, and a total value of each B calculated from each of the bases is set as SB, SB / SA is 0.05 or more and 0.95 or less.

[0059] Formula (1): A = number of moles of specific acid × molar concentration of specific acid (mol / L) Formula (2): B = number of moles of base × molar concentration of base (mol / L) The aforementioned method for separating amine can include a process other than the aforementioned separation process, such as an analysis process of analyzing (for example, identifying) the separated amine, a quantification process of quantifying the separated amine, a process of inferring the structure of the separated amine, or a recovery process of recovering the separated amine.

[0060] In addition, the aforementioned embodiment relates to a mobile phase before the amine becomes a separation object, and the "content ratio of amine in the aforementioned mobile phase" is the content ratio of amine in the mobile phase before the amine becomes a separation object.

[0061] In addition, in the present disclosure, an oligosaccharide is a sugar having 5 or more sugar residues.

[0062] In addition, regarding the distinction between oligosaccharides and polysaccharides in the present disclosure, the distinction can be made in terms of molecular weight, and sugars having a molecular weight of 3000 or less are regarded as oligosaccharides, and sugars having a molecular weight of more than 3000 are regarded as polysaccharides.

[0063] In addition, in the present embodiment, the specific acid includes both the free state and the state in which an acid anion is formed, and the base includes both the free state and the state in which a cation is formed. In addition, the acid anion and the cation can form a salt with each other or with other ions.

[0064] The inventors of the present application found that, by using a stationary phase including at least any one of oligosaccharides, polysaccharides, and derivatives thereof (hereinafter, also collectively referred to as "oligosaccharide-polysaccharide derivatives"), and a mobile phase including one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent, and further adjusting the content of these acids and bases, the separation performance of amines is improved. Regarding the reason therefor, the inventors of the present application conjecture as follows.

[0065] In an environment in which the mobile phase is acid-excessive (an environment in which the acid is more than the base), while the acid anion is generated, the analysis object (amine) is protonated to generate an ammonium cation, and an ion pair is generated from these acid anion and ammonium cation. The polarity of this ion pair is weak, and thus, finally, the oligosaccharide-polysaccharide derivative, which has weak polarity, is adsorbed, and the separation of the analysis object can be achieved.

[0066] The inventors of the present application conducted intensive research, and as a result, found that, by adding a base to the mobile phase at a molar concentration lower than that of the acid (content), a part of the acid is neutralized to be converted into a salt, and thus, even if the acid is a weak acid, the concentration of the counter anion in the mobile phase can be adjusted to an ideal level.

[0067] In addition, based on the above-described mechanism discovered by the inventors of the present application, the inventors of the present application repeatedly conducted research on the kind of acid, and the content of the acid and amine, and the like, and as a result, found that, by the above-described configuration according to the present embodiment, the separation performance of amines can be improved, and specifically, the retention time of the analysis object in chromatography can be made sufficient.

[0068] In addition, the inventors of the present application further conducted intensive research, and as a result, found that, in a case where the stationary phase has acidity, and non-specific adsorption of the sample amine is caused by this, for example, in a case where the stationary phase includes silica gel as a support, a phenomenon called peak tailing, which is considered to be caused by the silanol group thereof, often occurs. It was found that, in such a case, according to the above-described mode according to the present embodiment, the separation performance of amines can be improved, and specifically, the peak of the chromatogram obtained by analysis can be made sharp.

[0069] In the case where an ionic interaction occurs between the cation and the conjugate anion of the dissociated silanol, the peak of the chromatogram obtained by the analysis becomes broad, but the inventors of the present application presume that in the mode involved in the present embodiment, the dissociation of the silanol is inhibited due to the presence of the excess acid, and thus the ionic interaction is inhibited.

[0070] Further, in the related art, there is a method of increasing the pH of the mobile phase so as to make the sample amine a neutral molecule and hold it to the non-ionic stationary phase. However, in the case where the stationary phase contains silica gel, the silica gel is easily dissolved under such conditions to cause deterioration of the column, and thus the following countermeasure is taken: a salt having a buffering action such as ammonium bicarbonate is added in a manner that the pH does not excessively increase, and a guard column that can be replaced more cheaply than the main column is inserted before the main column, and the mobile phase flowing into the main column is saturated with respect to the silica portion, whereby the elution of silica from the main column is inhibited.

[0071] On the other hand, in the mode involved in the present embodiment, the pH is lowered by the excess acid, and thus it is not necessary to take the countermeasure as described above, which is a great advantage.

[0072] Further, in the separation of amines, in the case where a separation operation using the mobile phase disclosed in Non-Patent Literature 1 is applied, since a high-boiling non-volatile salt is added to the mobile phase, the salt is easily left after the evaporation of the solvent in the mobile phase, and thus the separation performance of the amines is reduced.

[0073] On the other hand, in the mobile phase in the present embodiment described above, in the case where an amine is used as the base, a salt of an anion containing one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid and a cation of the amine is generated in the mobile phase, and these salts are easily volatilized or thermally decomposed, and thus even if the solvent is evaporated, a high-boiling non-volatile salt is not easily left.

[0074] [Ammonia] According to the separation method involved in the present embodiment, it is possible to separate a mixture of a plurality of amines into each amine, or separate an amine from a mixture containing an amine and a non-amine. In particular, the fact that it is possible to separate enantiomers (mirror isomers) of an amine is of great importance in science and technology.

[0075] The amine to be separated is not particularly limited as long as it is a compound containing an amino group, and can be any of primary amines, secondary amines, and tertiary amines. As specific amines, amino acids such as alanine, cysteine, glutamic acid, methionine, leucine, tyrosine, or tryptophan; amino acid derivatives such as esters and amides of the aforementioned amino acids; amino alcohols such as dimethylaminoethanol, propanolamine, colitranol, or phenylpropanolamine; phenylethylamine; cyclic amines such as 1-phenyl-1,2,3,4-tetrahydroisoquinoline; and other basic pharmaceuticals containing an amino group, particularly their enantiomers, can be given.

[0076] According to the separation method related to the present embodiment, high separation performance that distinguishes the shape of a molecule can be achieved, and thus, a mixture of amines that are difficult to separate because their structures are similar to each other can also be separated into each amine. As a mixture of amines whose structures are similar to each other, a mixture of chain isomers, a mixture of position isomers, a mixture of geometric isomers, and the like can be given. Furthermore, by using a chiral stationary phase containing an oligosaccharide / polysaccharide derivative having an optically active site as the stationary phase, a mixture of enantiomers can be separated into each enantiomer, which is a great feature.

[0077] [Stationary phase] The stationary phase contains at least any one of oligosaccharides, polysaccharides, and derivatives thereof (hereinafter, these will be collectively referred to as "oligosaccharide / polysaccharide derivatives"). As an adsorbent (also referred to as a "separation agent"), the manner of containing the oligosaccharide / polysaccharide derivative is not particularly limited. For example, as the adsorbent, a substance obtained by solidifying an oligosaccharide / polysaccharide derivative or a mixture of an oligosaccharide / polysaccharide derivative and other components, or a substance obtained by supporting an oligosaccharide / polysaccharide derivative or a mixture of an oligosaccharide / polysaccharide derivative and other components on a support can be used. In the support, a substance obtained by making the polysaccharide or oligosaccharide derivative insoluble by providing a chemical bond between the oligosaccharide / polysaccharide derivative and the support, providing a chemical bond between the oligosaccharide / polysaccharide derivatives, or providing a layer of a high-molecular non-permeable layer on the surface of the oligosaccharide / polysaccharide derivative is particularly useful.

[0078] The shape of the stationary phase is not particularly limited, and for example, can be crushed to be amorphous or the like, but is preferably spherical. In this case, column chromatography in which the particles are packed into a column can be used. The oligosaccharide / polysaccharide derivative itself can be granulated, but from the viewpoint of being excellent in efficiency, pressure resistance, and ease of use, it is preferable to be supported on a porous silica gel or the like. In the case of being porous, it can be a substance in which only the surface is made porous, and is made in a manner in which the oligosaccharide / polysaccharide derivative is distributed near the surface when supported (various terms such as core-shell, surface porosity, or peripheral).

[0079] When the shape of the stationary phase is spherical, the diameter thereof is not particularly limited, but is preferably from 0.5 to 100 μm, more preferably from 0.5 to 50 μm, and further preferably from 1 to 20 μm (from the viewpoint of suitability in the chromatographic apparatus in the current state). The diameter is preferably small from the viewpoint of separation of amines, but in the case of a large-scale apparatus for fractionation, it is preferably large in order to reduce the flow resistance. In the present disclosure, "spherical" also includes a substantially spherical shape.

[0080] The kind of the oligosaccharide / polysaccharide derivative is not particularly limited, and for example, it can be an oligosaccharide / polysaccharide in which a part or all of the hydroxyl groups or the amino groups are substituted with a substituent. The polysaccharide derivative is, for example, a substance obtained by reacting a part or all of the hydroxyl groups or the amino groups of the oligosaccharide / polysaccharide with another compound, and one example is a substance obtained by making a part or all of the hydroxyl groups of the oligosaccharide / polysaccharide into an ester of an aliphatic carboxylic acid, benzoic acid, or phenylcarbamic acid which can further contain one or more substituents. Among them, from the viewpoint of separation performance, an oligosaccharide / polysaccharide obtained by making a part or all of the hydrogen atoms contained in the benzene ring into an ester of benzoic acid or phenylcarbamic acid in which each of the hydrogen atoms is independently substituted with a methyl group or a halogen atom is preferable.

[0081] Note that the oligosaccharide / polysaccharide derivative can be used alone or in combination of two or more.

[0082] The kind of the oligosaccharide / polysaccharide which is the parent of the oligosaccharide / polysaccharide derivative is not particularly limited.

[0083] The number of sugar residues of the oligosaccharide / polysaccharide is not particularly limited as long as it is 5 or more, and for example, it can be 10 or more, 20 or more, 1000 or less, 500 or less, 100 or less, 75 or less, or 50 or less.

[0084] The kind of the monosaccharide constituting the oligosaccharide / polysaccharide is not particularly limited, and for example, alditols such as erythritol or threitol; aldopentoses such as ribose, lyxose, xylose, or arabinose; aldohexoses such as allose, talose, gulose, glucose, altrose, mannose, galactose, or idose; ketotetrose such as erythrulose; ketopentoses such as xylulose or ribulose; or ketohexoses such as psicose, fructose, sorbose, or tagatose; and the like can be given. Among them, as a substance which is easily obtained as a raw material in high purity, glucose, fructose, or glucosamine is preferable.

[0085] Note that these monosaccharides can be used alone or in combination of two or more.

[0086] More specifically, as the polysaccharide, for example, amylose or amylopectin, glycogen, cellulose, curdlan, paramylum, chitin, chitosan, dextran, nigeran, agarose, carrageenan, heparin, alginic acid, hyaluronic acid, pectin, xyloglucan, xylan, glucomannan, or fructan can be mentioned. Among them, from the viewpoint of excellent separation performance and easy availability in high purity, cellulose, amylose, chitin, chitosan, curdlan, schizophyllan, agarose, and the like are preferable. Note that amylose and amylopectin can each be used as a monomer, or can be used as a mixture containing both, such as starch.

[0087] In addition, as described above, from the viewpoint of being able to separate a mixture of enantiomers into each enantiomer, an oligosaccharide / polysaccharide derivative having an optically active site is preferable.

[0088] The amount of the oligosaccharide / polysaccharide derivative in the stationary phase is not particularly limited, and can be appropriately set in accordance with known methods using an oligosaccharide / polysaccharide derivative as a separation agent for the stationary phase. For example, the content ratio of the oligosaccharide / polysaccharide derivative in the stationary phase can be 100% by mass, and in the case of using the above-described support or the like, it is preferable to be 5% by mass or more and 50% by mass or less, more preferable to be 5% by mass or more and 30% by mass or less, and further preferable to be 10% by mass or more and 30% by mass or less.

[0089] As described above, the form of the oligosaccharide / polysaccharide derivative is not particularly limited, and can be an oligosaccharide / polysaccharide derivative or a substance obtained by, as needed, particleizing, porous particleizing, or porous monolithizing the oligosaccharide / polysaccharide derivative itself, can use a substance obtained by solidifying an oligosaccharide / polysaccharide derivative or a mixture of an oligosaccharide / polysaccharide derivative and other components, or can use a substance obtained by supporting an oligosaccharide / polysaccharide derivative or a mixture of an oligosaccharide / polysaccharide derivative and other components on a support, but it is preferable to be a substance obtained by supporting an oligosaccharide / polysaccharide derivative or a mixture of an oligosaccharide / polysaccharide derivative and other components on a support, and more preferable to be a substance obtained by supporting an oligosaccharide / polysaccharide derivative on a support.

[0090] As the above-mentioned supporting method, a known method can be employed, and the supporting can be physical supporting or chemical supporting. As the chemical supporting, a method of supporting the oligosaccharide / polysaccharide derivative to the support by a chemical bond such as a covalent bond is preferable. Specifically, a method of introducing a reactive group into the oligosaccharide / polysaccharide derivative, a raw material of the oligosaccharide / polysaccharide derivative, or an intermediate of the oligosaccharide / polysaccharide derivative, and reacting the substituent with a reactive group present on the surface of the support can be mentioned. At this time, a process of providing a chemical bond between the oligosaccharide / polysaccharide derivatives can also be performed. Note that the reactive group present on the surface of the support can be a group present on the surface of an untreated support, or a group introduced to the surface of the support by surface treatment of the support with a surface treatment agent such as a silane coupling agent such as 3-aminopropyltriethoxysilane and 3-glycidyloxypropyltrimethoxysilane. In addition to these, a method of bonding a reaction pair of a so-called click reaction to the stationary phase and the oligosaccharide / polysaccharide derivative, respectively, and reacting them with each other can be mentioned. Alternatively, not only a method of forming a bond between the oligosaccharide / polysaccharide derivative and the support, but also by forming a so-called cross-linking bond between atomic groups including the oligosaccharide / polysaccharide derivative, it is possible to form a layer insolubilized on the surface of the support. Furthermore, as the physical supporting, for example, a method of physically adsorbing (e.g., coating) the oligosaccharide / polysaccharide derivative onto the support by impregnating a solution of the oligosaccharide / polysaccharide derivative in silica gel and evaporating it can be mentioned, and the oligosaccharide / polysaccharide derivative is supported to the support.

[0091] Among these methods, from the viewpoint of being able to inhibit the dissolution of the oligosaccharide / polysaccharide derivative into a solvent, it is preferable to achieve insolubilization by a method of forming a certain chemical bond between the silica gel and the skeleton of the oligosaccharide / polysaccharide derivative, or a method of forming a certain chemical bond between the oligosaccharide / polysaccharide derivatives on the support. Note that in order to inhibit the dissolution of the oligosaccharide / polysaccharide derivative into a solvent, even in the case where no support is used, it is preferable to provide a chemical bond between the oligosaccharide / polysaccharide derivatives.

[0092] As the support, it is not particularly limited as long as the oligosaccharide / polysaccharide derivative can be fixed to the support. The support can be an inorganic support or an organic support, but an inorganic support is preferable. As the inorganic support, for example, silica gel, alumina, magnesia, glass, kaolin, titania, silicate, zirconia, or hydroxyapatite can be mentioned. As the organic support, for example, cross-linked polystyrene, cross-linked poly(meth)acrylamide, cross-linked poly(meth)acrylate, or polysaccharide can be mentioned. In addition, a hybrid silica gel obtained by copolymerizing a polyfunctional silane coupling agent at the time of forming silica gel can also be used. These organic supports are preferably insolubilized by cross-linking using a cross-linking agent.

[0093] The shape of the carrier is not particularly limited, and examples include particles, and a porous cylinder (monolith) that is housed in a column tube in a liquid-tight manner. In addition, the inner wall of a capillary tube can also be given as an example of a carrier. In the case where the separation factor is sufficiently large, the carrier can be used in a membrane sheet type that is spread in the thickness direction, or can be spread in the plane direction as a thin layer chromatography.

[0094] In addition, in the present embodiment, the carrier is preferably silica gel. This is because the aforementioned properties of silica gel, i.e., excellent separation performance, in addition to being hard and robust. As the silica gel, in addition to a fully porous silica gel, a so-called core-shell type silica gel can also be used, and a silica gel whose surface is chemically modified can also be used.

[0095] [Mobile phase] The mobile phase contains one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, one or more bases, and a solvent.

[0096] The mobile phase can also contain components other than the above components within a range that does not hinder the separation performance. In addition, the mobile phase is preferably a homogeneous system (single phase system) at least under the conditions in which the analysis is performed.

[0097] Note that, in the present disclosure, the "mobile phase" refers to a liquid that is injected into a stationary phase together with the separation object in chromatography such as liquid chromatography and supercritical fluid chromatography, and moves together with the separation object, and then elutes the separation object from the stationary phase.

[0098] The content of the salt contained in the mobile phase is not particularly limited, and from the viewpoint of ensuring the desired separation performance, it is preferably 0.5 mmol / L or more and 1000 mmol / L or less, more preferably 1 mmol / L or more and 200 mmol / L or less, further preferably 1 mmol / L or more and 50 mmol / L or less, and particularly preferably 1 mmol / L or more and 20 mmol / L. As described above, in the case where mass spectrometry (MS) is used, the content of the salt with a high boiling point is preferably low. As the salt with a high boiling point, for example, a salt containing a metal ion as a cation, or a salt containing a quaternary ammonium ion as a cation can be given, and specifically, the content of the salt is preferably 5 mM or less, and more preferably substantially does not contain (is lower than the detection limit).

[0099] (Specific acid) The mobile phase contains one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid. Among them, from the viewpoint of the higher acidity, it is preferable to contain at least one or more acids selected from the group consisting of oxalic acid and trifluoroacetic acid. Further, it is particularly preferable to contain at least oxalic acid as the specific acid, since oxalic acid does not have the tendency to cause contamination in other analyses compared to trifluoroacetic acid, which is easily adsorbed to the components of liquid chromatography and the like, and needs attention to prevent fluorine pollution of the environment after disposal, and is easily decomposed into carbon monoxide, carbon dioxide, and water, which are low molecular weight, at the temperature of the MS detector, and thus does not easily cause adverse effects on MS analysis, and can be obtained inexpensively as a two-water crystal that is easy to handle and does not have hygroscopicity and the like. These acids can be one alone, or any two or more can be combined in any ratio. In addition, other acids can also be included within a range in which the effects of the present disclosure can be obtained.

[0100] Note that oxalic acid also has the advantage that, in the case where the eluate is fractionated, it can be used for precipitation separation with calcium ions.

[0101] As for the specific acid contained in the mobile phase, it is preferable that a part thereof becomes a counter ion that promotes the retention of the sample ammonium onto the stationary phase in ion pair chromatography, and a part thereof exists in the form of an acid, which reliably converts the sample amine into ammonium ions. The sample amine is rare under the chromatographic conditions, and thus even if the mobile phase is substantially neutral, the probability of existing in the form of ammonium ions is high, but by making the acid excessive, it can be reliably performed.

[0102] From the viewpoint of improving the separation performance of amines, the content rate of the specific acid contained in the mobile phase (the total content rate in the case of using two or more) is preferably 1 mmol / L or more, and in addition, it is preferably 1000 mmol / L or less, more preferably 200 mmol / L or less, further preferably 50 mmol / L or less, and particularly preferably 20 mmol / L or less.

[0103] If the above content rate is above the lower limit of the above range, the effect of retaining the sample can be substantially obtained (no matter how rare, a large or small effect can be obtained, but a certain degree of concentration is required to obtain a practical effect). In addition, if the above content rate is below the upper limit of the above range, there is no adverse effect on detection, especially MS-based detection. That is, as the preferable range of the content rate contained in the mobile phase, for example, the range of 1 to 1000 mmol / L, 1 to 200 mmol / L, 1 to 50 mmol / L, 1 to 20 mmol / L can be cited.

[0104] As the acid other than the specific acid, for example, methanesulfonic acid, nitric acid, phosphoric acid, or hydroxyacetic acid, etc. can be given, from the viewpoint of not easily forming a non-volatile salt of high boiling point, an organic acid such as nitric acid or hydroxyacetic acid is preferred, from the viewpoint of less corrosion to the device, a sulfonic acid, phosphoric acid, or an organic acid is preferred. These acids other than the specific acid can be one alone, or any two or more can be combined in any ratio.

[0105] (bases) The mobile phase contains a base. In order to reliably perform the retention as the ion pair chromatography that makes a part of the above specific acid a conjugate anion, a base is added in a molar concentration smaller than the specific acid, and a part of the specific acid is neutralized. As the base, for example, a hydroxide or a carbonate of a metal or a quaternary ammonium, etc. can be given. The salt can be a salt of a weaker acid than the aforementioned specific acid, and an amine is preferred. In the case of an amine, it is dissociated into the specific acid and the amine by heat and volatilized, and thus does not hinder the detection based on mass spectrometry (MS). Note that since the mobile phase becomes an environment in which the acid is in excess, the base is present in the mobile phase substantially in the form of a salt.

[0106] Regarding the content rate of the base contained in the mobile phase (the total content rate in the case of using two or more), from the viewpoint of improving the separation performance of the amine, the base is contained in a manner satisfying the following condition: in the case where the value calculated from the following formula (1) with respect to each specific acid contained in the mobile phase is set as A, the total value of each A calculated from each specific acid is set as SA, the value calculated from the following formula (2) with respect to the base contained in the mobile phase is set as B, and the total value of each B calculated from each base is set as SB, SB / SA is 0.05 or more and 0.95 or less.

[0107] Formula (1): A = number of moles of specific acid x molar concentration of specific acid (mol / L) Formula (2): B = number of moles of base x molar concentration of base (mol / L) Note that the number of moles of acetic acid is 1, the number of moles of formic acid is 1, and the number of moles of trifluoroacetic acid is 1. The number of moles of oxalic acid can be 1 or 2, but there is a difference of 10 to the third power in the dissociation constant between the first step and the second step, and thus it is inferred that, under the condition of the present disclosure in which the amount of the base is less than the number of moles of the acid (<0.095), the second step dissociation occurs only to a negligible extent, and thus is treated as 1 in the present disclosure.

[0108] Note that, as described above, the number of moles of the specific acid is 1, and thus the above formula (1) can also be represented by the following formula (1)'.

[0109] Formula (1)': A = molar concentration of specific acid (mol / L) The SB / SA described above is not particularly limited as long as it is 0.05 or greater and 0.95 or less, but from the viewpoint of being able to improve the separation performance of amines, it is preferably 0.1 or greater, more preferably 0.2 or greater, and further preferably 0.4 or greater, and from the viewpoint of the possibility of not being able to completely ionize the sample amine due to a decrease in the acidity of the system, it is preferably 0.9 or less, and more preferably 0.7 or less. That is, as a preferable range of the content ratio contained in the mobile phase, for example, in the case of a monobasic acid, ranges of 0.1 to 0.9, 0.2 to 0.7, and 0.4 to 0.7 can be given.

[0110] For example, in the case where X1 mol / L of acetic acid (basicity: 1) is contained in the mobile phase as the specific acid, and Y1 mol / L of triethylamine (basicity: 1) is contained as the base, A calculated from the above-described formula (1) becomes X1 mol / L (= 1 x X1), and B calculated from the above-described formula (2) becomes Y1 mol / L (= 1 x Y1). In addition, the number of types of acids is 1, and the number of types of bases is also 1, and thus SA = X1, SB = Y1, and therefore SB / SA = Y1 / X1.

[0111] Note that an acid having a smaller degree of dissociation than the specific acid can be contained at an arbitrary concentration, but in the case where an acid having a larger degree of dissociation than the specific acid is contained, the acid preferentially consumes the "base", and thus it is also possible to calculate the above-described SB / SA by regarding (the above-described SB) - (the number of basicity of the acid having a larger degree of dissociation than the specific acid contained in the mobile phase) x (the concentration (mol / L) of the acid having a larger degree of dissociation than the specific acid contained in the mobile phase) as SB. In this calculation, in the case where the acid having a larger degree of dissociation than the specific acid is a polybasic acid, the second term is multiplied by the number of charges up to the stage at which the degree of dissociation is larger than the "specific acid".

[0112] Note that the degree of dissociation described above is the degree of dissociation in water at 25°C. This degree of dissociation is described in, for example, "pKa Data Compiled by R. Williams" issued by the American Chemical Society.

[0113] In addition, other embodiments of the present disclosure are described below: the above-described SB / SA is set by considering only the concentrations of the specific acid and the base. In this mode, the above-described formula (1) and formula (2) are replaced by the following formula (1)" and formula (2)", respectively. In addition to the replacement of the formulas, the conditions of the above-described embodiments can be applied as is.

[0114] Formula (1)": A = molar concentration of the specific acid (mol / L) Formula (2)": B = molar concentration of the base (mol / L) Further, another other embodiment of the present disclosure is a manner in which the number of members of oxalic acid is treated as 1 or 2. In this manner, in a case where oxalic acid exists as a monobasic acid, the number of members is treated as 1, and in a case where oxalic acid exists as a dibasic acid, the number of members is treated as 2. Except for this, the conditions of the above-described embodiments can be applied as they are.

[0115] These two other embodiments, although different from the above-described embodiments in terms of the number of members of the acid and / or the base, can achieve the same effects as those achieved by the above-described embodiments.

[0116] Hereinafter, amines as preferred manners of the base are described in detail.

[0117] The kind of the amine is not particularly limited, and examples include ammonia, primary amines, secondary amines, tertiary amines, and the like, and one or more selected from the group consisting of ammonia, primary amines, secondary amines, and tertiary amines is preferred. Note that the amine can be one alone, or any two or more can be combined at any ratio.

[0118] As the primary amine, for example, a primary amine having an alkyl group can be given. Among them, a monoalkylamine such as methylamine, ethylamine, n-propylamine, 2-propylamine, or butylamine can be preferably exemplified.

[0119] As the secondary amine, for example, a secondary amine each independently having two alkyl groups can be given. Among them, a non-cyclic dialkylamine such as dimethylamine, diethylamine, diisopropylamine, or dibutylamine; or a cyclic amine such as N-unsubstituted pyrrolidine or N-unsubstituted piperidine can be preferably exemplified.

[0120] As the tertiary amine, for example, a tertiary amine each independently having three alkyl groups can be given. Among them, a non-cyclic trialkylamine such as trimethylamine, triethylamine, diisopropylethylamine, or tributylamine; or a cyclic trialkylamine such as N-substituted pyrrolidine, N-substituted piperidine, or 1,4-diazabicyclo[2.2.2]octane can be preferably exemplified.

[0121] The number of carbon atoms of the alkyl group bonded to each of the amines described above is not particularly limited, but amines having a small molecular weight have a high volatility and there is a problem in that handling is difficult, and if the molecular weight is large, it easily becomes a cause of noise in MS detection, and thus can be 1 or more, can be 2 or more, can be 3 or more, and in addition, can be 10 or less, can be 8 or less, can be 6 or less, and particularly preferably 1 to 4, and more preferably 1 to 2. In this way, the molecular weight of the primary to tertiary ammonium ion having a small number of carbon atoms of the alkyl group is small, and thus in the case where chromatography is performed using LC-MS, ions having a mass close to the amine that is the separation target are less likely to be generated, and noise is less. However, in the analysis of such a sample having a molecular weight of more than 200, triethylamine is convenient when handling easiness is also taken into consideration. On the other hand, in the case where ammonia is used as the base, the solubility of the salt thereof in an organic solvent is generally low, and thus an amine having an alkyl substituent is sometimes more advantageous.

[0122] In addition, the substituents bonded to the N-substituted pyrrolidine, the N-substituted piperidine, or the N,N-disubstituted pyrrolidine, the N,N-disubstituted piperidine are not particularly limited, and each is preferably an alkyl group, and the conditions for the alkyl group can be the same as those described for the alkyl group bonded to each of the amines described above. This is because, as described above, the molecular weight of the ammonium ion is preferably small in LC-MS.

[0123] The amine is preferably one or more selected from the group consisting of ammonia, ethylamine, n-propylamine, 2-propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyrrolidine, and piperidine, and more preferably one or more selected from the group consisting of ammonia, diethylamine, triethylamine, and 2-propylamine, for the reasons of low boiling point, ease of availability, suitability for UV detection, volatility, and small molecular weight, and the like, and less likely to be an obstacle to MS detection.

[0124] Note that a low-boiling amine represented by ammonia has a high vapor pressure, and thus it is sometimes difficult to quantitatively add. In such a case, a carbonate or bicarbonate of the amine can be used, and the acid contained in the salt reacts with a small amount of water to generate carbon dioxide, and thus an additive substantially the same as in the case where ammonia monomer is added is obtained.

[0125] In addition, in the case where a metal is used as the base, for example, an alkali metal such as lithium, sodium, or potassium, or another metal can be used.

[0126] Note that the method of using a specific acid and a base that partially neutralizes the acid is the most convenient method of preparing the mobile phase involved in the present embodiment, but an equivalent mobile phase can be obtained by adding a salt of the specific acid and the acid, and the method of preparation is not limited to this method. In addition, the specific acid added here does not necessarily have to be a specific acid corresponding to the salt, and can be an arbitrary acid selected from one or more of the group consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid.

[0127] (solvent) The solvent contained in the mobile phase in the present embodiment is not particularly limited as long as it can dissolve various components such as the amine to be separated and the specific acid, and water or a liquid other than water (hereinafter, sometimes referred to as "non-aqueous solvent") or the like can be used.

[0128] As the non-aqueous solvent, for example, an organic solvent (water content: 0 vol%) or subcritical or supercritical carbon dioxide or the like can be mentioned.

[0129] As the solvent contained in the mobile phase, for example, water alone, a non-aqueous solvent alone, a mixed solvent of water and a non-aqueous solvent, a mixed solvent containing pressurized carbon dioxide and a non-aqueous solvent, a trace amount of water as needed, or the like can be mentioned. In the case where the solvent contained in the mobile phase is a mixed solvent formed from two or more solvents, the aforementioned two or more solvents are preferably a combination of solvents that do not undergo phase separation under the separation conditions of the amine. Specifically, for example, a solvent having a low polarity, so-called normal phase solvent, a solvent obtained by mixing an alcohol, an ester, an ether, a nitrile, or a halogenated hydrocarbon, which has a higher polarity than a hydrocarbon such as hexane or heptane, or further mixing water thereto within a range in which they are uniformly dissolved, or a solvent obtained by mixing water into a pressurized carbon dioxide, which is a solvent called subcritical or supercritical carbon dioxide, can be used. More specifically, for example, the following solvents can be used as the solvent: a solvent having an organic solvent as a basic component with a high polarity, which has acetonitrile as a base and contains a small amount (1 to 20 vol%) of water; a solvent having water as a base and further containing a small amount (usually 1 to 40 vol%) of acetonitrile or methanol as a high-polarity reverse phase solvent; or a pressurized carbon dioxide, which is a solvent called subcritical or supercritical carbon dioxide, as a solvent generally referred to as supercritical fluid chromatography.

[0130] Note that, in the technical field, generally, "supercritical" is not limited to the case where it is strictly in a supercritical state under all conditions of composition and temperature, and the term "supercritical" is often used in the case where a solvent mainly using carbon dioxide is used. In the present disclosure, the term "supercritical" is also treated in the same manner.

[0131] As the organic solvent, there is no particular limitation, and it is preferable to be an organic solvent capable of dissolving the amine as the separation target. As the preferable organic solvent, for example, acetonitrile, acetone, tetrahydrofuran (THF), t-butyl methyl ether, methanol, ethanol, n-propanol, 2-propanol, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or dimethylacetamide (DMAc), and the like can be exemplified. In addition, as long as the amine as the separation target, and various components selected from one or more of the specific acids consisting of oxalic acid, acetic acid, formic acid, and trifluoroacetic acid, and the like can be dissolved, the organic solvent can also include hexane, or hexane, or heptane, and the like hydrocarbon; or dichloromethane, and the like halogenated hydrocarbon; and the like. Among them, from the aspect that the chemical stability is high, the organic solvent preferably includes one or more selected from the group consisting of acetonitrile, acetone, methanol, ethanol, 2-propanol, hexane, heptane, THF, and t-butyl methyl ether, and more preferably acetonitrile. The solvent included in the mobile phase particularly preferably includes acetonitrile of 70% by volume or more. The aforementioned organic solvent can be a single one, or any two or more can be combined in any ratio.

[0132] The organic solvent can also be a mixed solution of a hydrocarbon having 5 or more and 8 or less carbon atoms and an alcohol commonly used in chromatography. As such a mixed solution, for example, a hexane / ethanol mixed solution that is miscible with water can be exemplified. If the organic solvent is a hexane / ethanol mixed solution having a mixing ratio of, for example, 100 / 100 (v / v), a mixed solvent having a water content of about 10% by volume can be prepared, and the addition of water allows the peak number to be slightly reduced, but the peak number to be increased, and the time efficiency of the analysis to be improved. In addition, hexane / ethanol / methanol also provides a good peak number within the compatible range.

[0133] The mixed solution of a hydrocarbon having 5 or more and 8 or less carbon atoms and an alcohol has a low viscosity, and therefore, in chromatography, a high flow rate can be achieved at a low pressure, and, in addition, the ultraviolet light absorption is small, and therefore, the detection of the amine based on the ultraviolet light absorption can be achieved.

[0134] In the case where the non-aqueous solvent includes subcritical or supercritical carbon dioxide, the chromatography is performed using a supercritical fluid chromatograph. In this case, it is preferable to use a mixed solvent of water and an organic solvent, or an organic solvent (water content of 0% by volume) as an auxiliary solvent in subcritical or supercritical carbon dioxide in a manner that the target component can be dissolved in the mobile phase at a target concentration. The organic solvent referred to here is synonymous with the organic solvent exemplified as the non-aqueous solvent. As the mixed solvent of water and an organic solvent, and the organic solvent as the auxiliary solvent, a mixed solvent of water and methanol, or methanol can be exemplified, respectively.

[0135] Note that in the case where the solvent included in the mobile phase includes subcritical or supercritical carbon dioxide, for convenience, the volumes thereof are calculated based on the density of liquid carbon dioxide at -20°C, 1.030 g / mL.

[0136] The kind of solvent is described, but from the viewpoint of low viscosity, transparency with respect to ultraviolet rays (the most commonly used case of ultraviolet detection), no strong toxicity, danger, inexpensive, and the tendency that a chiral stationary phase formed from a polysaccharide derivative enables good separation, it is preferable to be one or more selected from the group consisting of water, acetonitrile, ethanol, methanol, 2-propanol, hexane, heptane, THF, and liquid carbon dioxide.

[0137] The content ratio of the solvent in the mobile phase (the total content ratio in the case of containing two or more) is not particularly limited, and can be appropriately set according to the use, for example, can be 90% by mass or more, can be 95% by mass or more, can be 98% by mass or more, can be 99% by mass or more, and in addition, can be less than 100% by mass, can be 99.9% by mass or less, and can be 99.5% by mass or less. In addition, in the mobile phase, the remaining component other than the above-mentioned specific acid and base can also be present. Note that, the following describes an example of the mixing ratio of each component in the case of a 3-component mixture of the solvent. As a reverse phase solvent, a mixture of acetonitrile, alcohol, and THF containing 50% by volume or more of water can be exemplified, and in order to achieve good separation, the water content is preferably 70% by volume or more. In addition, as a normal phase solvent, a mixture containing 40% by volume or more of hexane and / or heptane, the remaining being 2-propanol or ethanol, further containing a small amount of water can be exemplified.

[0138] (Other components) The mobile phase can also contain a component other than the above-mentioned components (other components) within a range in which the effects of the present disclosure can be obtained. However, in the case of performing detection based on mass spectrometry (MS), a component that can supply a non-volatile salt is not preferable.

[0139] (Method for preparing a mobile phase) Regarding the method for preparing the mobile phase, it can be performed by a known solution preparation method or a method based on a known solution preparation method. For example, a method of mixing each of the above-mentioned components can be exemplified.

[0140] As a known method, a method of forming a salt from an acid and a base and using it to prepare a mobile phase can be given. In the present embodiment, the expression including an acid and a base is adopted, but it is also possible to dissolve both of them in an appropriate amount in a mobile phase solvent as such. On the other hand, even if the acid and the salt formed from the acid and the base are dissolved in a mobile phase solvent, substantially the same mobile phase can be obtained. Thus, a method of preparing a mobile phase by contacting an acid and a base in a solvent without directly contacting them is preferable in terms of convenience and in terms of avoiding problems caused by heat due to neutralization, such as vaporization of a part of the components. In the case of using an amine such as ammonia having high volatility as a base, a solvent such as water can also be used, and the composition of the mobile phase solvent is corrected as necessary. In addition, as described above, a salt of a weak acid such as a carbonate or a bicarbonate can also be used as a precursor. Here, a weak acid generated as a byproduct does not have a large impact on the preservation, whether it remains directly in the mobile phase or is dispersed by vaporization.

[0141] A salt of a cation and an acid anion is commercially available, or, in the case where it can be easily obtained, the aforementioned commercially available product can be dissolved in a solvent, mixed with an acid for adjusting the acidity as necessary, and thereby a mobile phase can be prepared. The necessity of adjusting the acidity arises in the case where the preservation of the sample amine is insufficient or the shape of the peak is not ideal.

[0142] [Chromatography] The chromatography method employed in the present embodiment is not particularly limited, and for example, liquid chromatography, supercritical fluid chromatography, or the like can be given. In addition, the mode of the chromatography method is also not particularly limited, and a known mode can be employed, and not only column chromatography, but also thin layer chromatography, a chromatography method in which a layer provided with a stack of adsorbents in a fiber shape is used, or a capillary or microchannel chromatography method based on a thin layer in which an adsorbent layer is provided in some form on the inner wall of a fine tube, or the like can be employed.

[0143] The chromatography method can be performed using a commercially available chromatograph, such as a liquid chromatograph and a supercritical fluid chromatograph, or the like. The equilibration conditions of the column and the flow rate and the like of various conditions can be appropriately selected depending on the column size, the sample capacity, or the kind of the mobile phase, or the like.

[0144] The amine separation method related to the present embodiment can further include an analysis process of analyzing the amine separated by the separation process.

[0145] In the chromatography method, the method of detecting the amine is not particularly limited, but in the method related to the present embodiment, a method using a mass spectrometer can be given. In the present embodiment, the formation of a salt that promotes volatility can be facilitated by appropriately selecting an additive, and thus a mass spectrometer can be applied as an analyzer, which is a great advantage.

[0146] The separation and analysis of the amines can be performed using the same apparatus or separate apparatuses. For example, in the case where liquid chromatography is used as the chromatography, liquid chromatography-mass spectrometry (LC-MS) combining liquid chromatography with mass spectrometry (MS) can be used to continuously perform the separation of the amines and the mass spectrometric analysis of the separated amines. Note that the mass spectrometric analysis of the amines can be quantitative analysis or qualitative analysis of the separated amines. In addition, a UV detector alone can be used or a mass spectrometer can be used in combination to obtain more information.

[0147] As the mass spectrometric analysis, known mass spectrometry used in LC-MS can be used. For example, the ionization method in the mass spectrometric analysis can be appropriately selected from atmospheric pressure chemical ionization (APCI), atmospheric pressure photo ionization (APPI), electrospray ionization (ESI), fast atom bombardment (FAB), or thermal spray (TSP), or the like, depending on the kind of salt, the kind of acid, the kind of amine, and the purpose of the analysis. In addition, the mass detector can be appropriately selected and used from a quadrupole mass spectrometer (Q-MS), an ion trap mass spectrometer (IT-MS), or a time-of-flight mass spectrometer (TOF-MS), or the like, depending on the required sensitivity and resolution, and the like.

[0148] Examples Hereinafter, the present disclosure will be more specifically described using examples, but the present disclosure is not limited to the following examples as long as the gist thereof is not deviated.

[0149] <Experiment I-1> <Example 1> [Preparation of mobile phase] Oxalic acid dihydrate 1260 mg was weighed and transferred to a 1000 mL volumetric flask. To the volumetric flask, about 20 mL of a mixed solvent (80 / 20 in terms of volume ratio) prepared by mixing water and acetonitrile weighed at a ratio of 800.0 g and 156.6 g, respectively, was charged, followed by charging triethylamine 506 mg. Then, the volumetric flask was filled with the above-described mixed solvent of water / acetonitrile (80 / 20 (v / v)), and stirring was performed until the undissolved matter disappeared, thereby obtaining a mobile phase. The composition of the solvent in the mobile phase was water / acetonitrile = 80 / 20 (v / v), and the content of oxalic acid (OxA) and triethylamine (TEA) in the mobile phase was 10.0 mmol / L and 5.0 mmol / L, respectively. Note that the amount of water contained in the oxalic acid dihydrate was small and thus had no influence on the solvent composition. In addition, 100 μL of the mobile phase was dropped onto a stainless steel plate heated to 200°C, but no residue was observed. This result was the same in the other examples described below.

[0150] Note that the preparation of the following mobile phases is also the same, the solvent is weighed to the order of 0.1 g, the additive is weighed to the order of 1 mg, and the concentration is calculated based on this.

[0151] In addition, the density of water is 1.00 g / mL, and the density of acetonitrile is 0.783 g / mL.

[0152] [Separation and analysis of amines] Using a column filled with a chiral stationary phase obtained by supporting cellulose tris (3,5-dimethylphenylcarbamate) on silica gel (CHIRALPAK IB-N (manufactured by Daicel Corporation, registered trademark), average filler diameter 5 μm, length 250 mm, inner diameter 4.6 mm ) as the column, a high-performance liquid chromatograph was used as the liquid chromatograph, and separation of amines was performed. At this time, an amine represented by the following formula (3) (manufactured by USP, "Pindolol") as the separation object was dissolved in a water / acetonitrile (1 / 1 (v / v)) mixed solvent so as to have a concentration of about 1 mg / mL, and 2 μL of the obtained solution was injected into the column using an autosampler. In addition, the mobile phase was sent to the column at 0.43 mL / min after adjusting the temperature to 30°C.

[0153] Details of the separation method and the analysis method of amines are shown below.

[0154] (Separation method and analysis method) Using an SPD-M20A (manufactured by Shimadzu Corporation, UV-Vis detector of diode array) as the detector of the high-performance liquid chromatograph (HPLC), after sending the mobile phase to the column at 1.0 mL / min for 1 hour or more, 5 μL of a sample solution obtained by dissolving a sample in the mobile phase at about 1 mg / mL was injected using an autosampler, the column temperature was set to 30°C at the same flow rate, and separation and analysis of amines were performed.

[0155] Details of the apparatus used in the HPLC and the data processing apparatus are shown below. The apparatuses below are all manufactured by Shimadzu Corporation.

[0156] • HPLC apparatus: system controller CBM-20A, pump LC-20AD, autosampler SIL-20AC, column oven SPD-M20A • Data processing apparatus: LabSolutions Version 5.97 The results are shown in Figure 1 .

[0157] [Chemical formula 1] <Comparative Example 1> To the column after separation and analysis in Example 1, using a mixed solvent of water / acetonitrile (80 / 20 (v / v)) as the mobile phase, the liquid was passed for 1 hour or more without injecting a sample, and otherwise, the separation and analysis of the amine represented by Formula (3) above were performed again using the same method as in Example 1. Note that in the mobile phase used, no acid or amine was added, and thus the content of the acid and amine in the mobile phase was 0 mmol / L and 0 mmol / L, respectively.

[0158] The results are shown in Figure 2 .

[0159] <Comparative Example 2> Using oxalic acid dihydrate, instead of triethylamine, ammonium bicarbonate (AHC) and diethylamine (DEA) were used, and otherwise, the same operation as in Example 1 was performed to prepare a mobile phase in which the content of AHC and DEA was 10.0 mmol / L and 15.5 mmol / L, respectively.

[0160] Using the mobile phase above as the mobile phase, and inserting the guard column noted below Table 1 (only this comparative example) before the column of CHIRALPAK IB-N, the separation and analysis of the amine represented by Formula (3) above were performed using the same method as in Example 1.

[0161] The results are shown in Figure 3 .

[0162] <Comparative Example 3> To the column after separation and analysis in Comparative Example 2, using a mixed solvent of water / acetonitrile (80 / 20 (v / v)) as the mobile phase, the liquid was passed for 1 hour or more without injecting a sample, and otherwise, the separation and analysis of the amine represented by Formula (3) above were performed using the same method as in Example 1. Note that in the mobile phase used, no acid or amine was added, and thus the content of the acid and amine in the mobile phase was 0 mmol / L and 0 mmol / L, respectively.

[0163] The results are shown in Figure 4 .

[0164] <Example 2> Changing the solvents from water and acetonitrile to hexane and ethanol (hexane / ethanol = 50 / 50 (v / v)), and otherwise, the same operation as in Example 1 was performed to prepare a mobile phase in which the content of OxA and TEA was 10.0 mmol / L and 5.0 mmol / L, respectively.

[0165] Using the mobile phase above as the mobile phase, and otherwise, the separation and analysis of the amine represented by Formula (3) above were performed using the same method as in Example 1.

[0166] The results are shown in Table 2. Figure 5 .

[0167] <Comparative Example 4> The same operation as in Example 2 was performed, except that diethylamine ("DEA" in Table 1) was used instead of triethylamine, and no oxalic acid dihydrate was used, to prepare a mobile phase containing 10.0 mmol / L of DEA.

[0168] The separation and analysis of the amine represented by the above-described formula (3) were performed by the same method as in Example 1, except that the above-described mobile phase was used as the mobile phase.

[0169] The results are shown in Table 2. Figure 6 .

[0170] <Example 3> A column packed with a chiral stationary phase obtained by supporting cellulose tris (3-chloro-4-methylphenylcarbamate) on silica gel (CHIRALPAK IM (manufactured by Daicel Corporation, registered trademark), average packing diameter: 5 μm, length: 250 mm, inner diameter: 4.6 mm ) was used as the column, and the amine represented by the following formula (4) was used as the analysis object, and the separation and analysis of the amine as the analysis object were performed by the same method as in Example 2.

[0171] The results are shown in Table 2. Figure 7 .

[0172] [Chemical Formula 2] <Comparative Example 5> The separation and analysis of the amine represented by the above-described formula (4) were performed by the same method as in Comparative Example 4, except that the column used in Example 3 was used as the column.

[0173] The results are shown in Table 2. Figure 8 .

[0174] <Example 4> The separation and analysis of the amine as the analysis object were performed by the same method as in Example 2, except that the amide of phenylglycine represented by the following formula (5) was used as the analysis object.

[0175] C6H5-CH (NH2) -CONH2 (manufactured by Tokyo Chemical Industry Co., Ltd.) (5) The results are shown in Table 2. Figure 9 .

[0176] <Comparative Example 6> The analysis object was changed to the amide of the phenylglycine represented by the above formula (5), and otherwise, the separation and analysis of the amine as the analysis object were performed in the same manner as in Comparative Example 5.

[0177] The results are shown in Table 2. Figure 10 .

[0178] <Example 5> The solvent was changed from hexane and ethanol to hexane, ethanol, and methanol (hexane / ethanol / methanol = 70 / 20 / 19 (v / v / v)), and otherwise, the same operation as in Example 3 was performed to prepare a mobile phase in which the contents of OxA and TEA were 10.0 mmol / L and 5.0 mmol / L, respectively.

[0179] The above mobile phase was used as the mobile phase, and a column (CHIRALPAK ID (manufactured by Daicel Corporation, registered trademark), average filler diameter 5 μm, length 250 mm, inner diameter 4.6 mm ) packed with a chiral stationary phase obtained by supporting amylose tris (3-chlorophenylcarbamate) on silica gel was used as the column, and otherwise, the separation and analysis of the amine represented by the above formula (4) were performed in the same manner as in Example 3.

[0180] The results are shown in Table 2. Figure 11 .

[0181] <Comparative Example 7> The addition of oxalic acid dihydrate and triethylamine was not performed, and otherwise, the same operation as in Example 5 was performed to prepare a mobile phase in which the contents of OxA and TEA were 0 mmol / L and 0 mmol / L, respectively.

[0182] The above mobile phase was used as the mobile phase, and otherwise, the separation and analysis of the amine represented by the above formula (4) were performed in the same manner as in Example 5.

[0183] The results are shown in Table 2. Figure 12 .

[0184] <Example 6> A column (CHIRALPAK IE (manufactured by Daicel Corporation, registered trademark), average filler diameter 5 μm, length 250 mm, inner diameter 4.6 mm ) packed with a chiral stationary phase obtained by supporting amylose tris (3,5-dichlorophenylcarbamate) on silica gel was used as the column, and otherwise, the separation and analysis of the amine represented by the above formula (4) were performed in the same manner as in Example 5.

[0185] The separation and analysis of the amine represented by the above formula (4) were performed using the above mobile phase as the mobile phase, except that, by the same method as in Example 5.

[0186] The results are shown in Figure 13 .

[0187] <Comparative Example 8> The same operation as in Example 6 was performed, except that no oxalic acid dihydrate and triethylamine were added, to prepare a mobile phase in which the contents of OxA and TEA were 0 mmol / L and 0 mmol / L, respectively.

[0188] The separation and analysis of the amine represented by the above formula (4) were performed using the above mobile phase as the mobile phase, except that, by the same method as in Example 6.

[0189] The results are shown in Figure 14 .

[0190] The separation conditions of Examples 1 to 4 and Comparative Examples 1 to 6 are summarized in Table 1 below.

[0191] (Resolution) The resolution in each of the examples and comparative examples was the USP resolution automatically calculated by the chromatogram data analysis program LabSolutions Version 5.97 of Shimadzu Corporation. The results are shown in Table 1.

[0192] In each of the tables below, "AN" indicates acetonitrile, "OxA" indicates oxalic acid, "TEA" indicates triethylamine, "AHC" indicates ammonium bicarbonate, and "DEA" indicates diethylamine.

[0193] The above SB / SA is shown in Tables 1 to 5 below. Note that, if calculated from the dissociation degree, most of the oxalic acid exists in the form of monobasic acid.

[0194] [Table 1] By comparing Example 1 with Comparative Examples 1 and 3, Example 5 with Comparative Example 7, and Example 6 with Comparative Example 8, it was found that, even if the same solvent was used, in the case where nothing was added, the peak tailing was generally severe and the resolution was low, or the obtained chromatogram was unstable due to the history of the column before and after (Comparative Examples 1 and 3 used the same mobile phase, but sandwiched Example 1 and Comparative Example 2, respectively), but using the mobile phase involved in the embodiments of the present disclosure, stable retention and separation could be obtained.

[0195] In addition, in comparison with the mobile phase of Comparative Example 9 involving the addition of only an acid, in Example 2 in which a partially neutralized mobile phase was used, good separation was able to be achieved. Specifically, Comparative Example 9, which was based on separation using a mobile phase to which only oxalic acid was added, was significantly inferior to Example 2 in terms of both peak shape and separation.

[0196] In addition, in chromatography of amine samples, a method of adding a low-molecular-weight amine to the mobile phase is often used. It is thought that the chromatographic packing material, or silica gel, which is used as a support therefor in many cases, has an acidic atomic group called silanol, which forms a salt with the sample, thus causing significant peak tailing. In this case, if an amine is added, the salt formation of the sample with the silanol is inhibited, thus inhibiting the abnormal retention of the sample. Comparative Examples 4, 5, and 6 are cases in which diethylamine, which is often used for this purpose, was added. Comparative Examples 4, 5, and 6 correspond to Examples 2, 3, and 4, respectively. With respect to Comparative Examples 4 and 5, it was found that although little peak tailing occurred, the separation degree was less than that of Examples 2 and 3, respectively.

[0197] In addition, a method of making the mobile phase slightly basic in a mobile phase mainly composed of water to inhibit the protonation of an amine sample and retain it as a neutral molecule on the stationary phase has also been used in the past. Comparative Example 2 is one of such conditions, and is a basic mobile phase. In this case, the peak width was large, and the time efficiency of the separation was poor, compared with Example 1. Furthermore, when a slightly basic mobile phase is used with respect to a stationary phase including silica gel, it is necessary to prevent the deterioration of the column by inserting a guard column (precolumn) including silica gel or the like, and the same method was used in this comparative example. However, in Comparative Example 3, which was analyzed after this, the peak shape became abnormal, and it was thus determined that there was a history remaining in the column.

[0198] <Experiment I-2> <Comparative Example 9> The solvent was changed from a mixed solution of water / acetonitrile = 80 / 20 (v / v) to a mixed solution of water / acetonitrile = 2.5 / 97.5 (v / v), TFA was used instead of OxA at a content rate of 10.0 mol / L in the mobile phase, TEA was not used, and the same operation as in Example 1 was performed, except for this, to prepare a mobile phase.

[0199] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except for this, by the same method as in Example 1.

[0200] The results are shown in Figure 15 .

[0201] <Example 7> The solvent was changed from a mixed solution of water / acetonitrile = 50 / 50 (v / v) to a mixed solution of water / acetonitrile = 2.5 / 97.5 (v / v), and TFA was used instead of OxA in a manner that the content ratio of TFA in the mobile phase was 10.0 mol / L, and the same operation as in Example 1 was performed, except for this, to prepare the mobile phase (the content ratio of TEA was 5.0 mol / L).

[0202] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except for this, by the same method as in Example 1.

[0203] The results are shown in Table 1. Figure 16 .

[0204] <Comparative Example 10> The solvent was changed from a mixed solution of water / acetonitrile = 2.5 / 97.5 (v / v) to a mixed solution of hexane / ethanol = 50 / 50 (v / v), and the same operation as in Comparative Example 9 was performed, except for this, to prepare the mobile phase.

[0205] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except for this, by the same method as in Example 1.

[0206] The results are shown in Table 1. Figure 17 .

[0207] <Example 8> The solvent was changed from a mixed solution of water / acetonitrile = 2.5 / 97.5 (v / v) to a mixed solution of hexane / ethanol = 50 / 50 (v / v), and the same operation as in Example 7 was performed, except for this, to prepare the mobile phase.

[0208] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except for this, by the same method as in Example 1.

[0209] The results are shown in Table 1. Figure 18 .

[0210] The separation conditions of Examples 7 to 8 and Comparative Examples 9 to 10 are summarized in Table 2 below.

[0211] (Resolution) The resolution in each of the examples and comparative examples was the USP resolution automatically calculated by the chromatogram data analysis program LabSolutions Version 5.97 of Shimadzu Corporation. The results are shown in Table 1.

[0212] In each of the tables below, "TFA" indicates trifluoroacetic acid.

[0213] [Table 2] By comparing Example 7 with Comparative Example 9, and Example 8 with Comparative Example 10, it was found that the resolution was improved when SB / SA was 0.05 or more and 0.95 or less.

[0214] <Experiment I-3> <Comparative Example 11> The same operation as in Example 1 was performed, except that AcOH was used instead of OxA so that the concentration of AcOH in the mobile phase was 10.0 mol / L, and TEA was not used, to prepare the mobile phase.

[0215] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except that the same operation as in Example 1 was performed.

[0216] The results are shown in Figure 19 .

[0217] <Example 9> The same operation as in Example 1 was performed, except that AcOH was used instead of OxA so that the concentration of AcOH in the mobile phase was 10.0 mol / L, to prepare the mobile phase (the concentration of TEA was 5.0 mol / L).

[0218] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except that the same operation as in Example 1 was performed.

[0219] The results are shown in Figure 20 .

[0220] <Example 10> The same operation as in Example 9 was performed, except that NH4OAc was used instead of TEA so that the concentration of NH4OAc in the mobile phase was 5.0 mol / L, to prepare the mobile phase.

[0221] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except that the same operation as in Example 1 was performed.

[0222] The results are shown in Figure 21 .

[0223] <Example 11> The same operation as in Example 9 was performed, except that the solvent was changed from a mixed solution of water / acetonitrile = 80 / 20 (v / v) to a mixed solution of hexane / ethanol = 50 / 50 (v / v), to prepare the mobile phase.

[0224] Using the above mobile phase as the mobile phase, otherwise, the separation and analysis of the above amine represented by Formula (3) were performed by the same method as in Example 1.

[0225] The results are shown in Figure 22 .

[0226] <Example 12> Using AcOH in such a manner that the content ratio thereof in the mobile phase becomes 10.0 mol / L, and using NH4OAc in such a manner that the content ratio thereof in the mobile phase becomes 10.0 mol / L, otherwise, the same operation as in Example 12 was performed to prepare the mobile phase.

[0227] Using the above mobile phase as the mobile phase, otherwise, the separation and analysis of the above amine represented by Formula (3) were performed by the same method as in Example 1.

[0228] The results are shown in Figure 23 .

[0229] <Example 13> Using the above mobile phase as the mobile phase, otherwise, the separation and analysis of the above amine represented by Formula (3) were performed by the same method as in Example 1.

[0230] The results are shown in Figure 24 .

[0231] [Chemical Formula 3] <Example 14> Using AcOH in such a manner that the content ratio thereof in the mobile phase becomes 10.0 mol / L, and using NH4OAc in such a manner that the content ratio thereof in the mobile phase becomes 10.0 mol / L, otherwise, the same operation as in Example 12 was performed to prepare the mobile phase.

[0232] Using the above mobile phase as the mobile phase, otherwise, the separation and analysis of the above amine represented by Formula (3) were performed by the same method as in Example 1.

[0233] The results are shown in Figure 25 .

[0234] The separation conditions of Examples 9 to 14 and Comparative Example 11 are summarized in Table 3 below.

[0235] (Resolution) The resolution in each of the examples and the comparative example is the USP resolution automatically calculated by the chromatogram data analysis program LabSolutions Version 5.97 of Shimadzu Corporation. The results are shown in Table 3.

[0236] In each of the following tables, "AcOH" represents acetic acid, "NH4OAc" represents ammonium acetate, and "Ph-β-isoq" represents phenyl-β-isoquinoline.

[0237] Note that, in Examples 10, 12, 13, and 14, ammonium acetate was added instead of the operation of adding ammonia as a base. Therefore, in the mobile phase, in addition to acetic acid added as an acid, acetic acid generated by dissociation of ammonium acetate was present. Therefore, in the case of use in such a manner that the ratio of acetic acid:ammonium acetate is 1:1 in terms of molar ratio, the ratio of acetic acid:ammonia in the mobile phase becomes 2:1 in terms of molar ratio, and SB / SA becomes 0.5.

[0238] [Table 3] By comparing Examples 9 to 14 with Comparative Example 11, it was found that, in the case where SB / SA is 0.05 or more and 0.95 or less, the resolution is improved.

[0239] <Experiment I-4> <Comparative Example 12> The solvent was changed from a mixed solution of water / acetonitrile = 80 / 20 (v / v) to a mixed solution of hexane / ethanol = 50 / 50 (v / v), FA was used instead of OxA in such a manner that the content of formic acid (FA) in the mobile phase was 10.1 mol / L, TEA was not used, and otherwise the same operation as in Example 1 was performed to prepare a mobile phase.

[0240] Using the above mobile phase as the mobile phase, otherwise the same method as in Example 1 was used to separate and analyze the above amine represented by formula (3).

[0241] The results are shown in Figure 26 .

[0242] <Example 15> The solvent was changed from a mixed solution of water / acetonitrile = 80 / 20 (v / v) to a mixed solution of hexane / ethanol = 50 / 50 (v / v), FA was used instead of OxA in such a manner that the content of formic acid (FA) in the mobile phase was 10.0 mol / L, and otherwise the same operation as in Example 1 was performed to prepare a mobile phase (the content of TEA was 5.0 mol / L).

[0243] Using the above mobile phase as the mobile phase, otherwise the same method as in Example 1 was used to separate and analyze the above amine represented by formula (3).

[0244] The results are shown in Figure 27 .

[0245] <Comparative Example 13> The analysis object was changed to the amine (phenyl-β-isoquinoline) represented by the above formula (6), and otherwise, separation and analysis of the amine as the analysis object were performed by the same method as in Comparative Example 12.

[0246] The results are shown in Table 4. Figure 28 .

[0247] <Example 16> The analysis object was changed to the amine (phenyl-β-isoquinoline) represented by the above formula (6), and otherwise, separation and analysis of the amine as the analysis object were performed by the same method as in Example 15.

[0248] The results are shown in Table 4. Figure 29 .

[0249] The separation conditions of Examples 15 to 16 and Comparative Examples 12 to 13 are summarized in Table 4 below.

[0250] (Resolution) The resolution in each of the examples and comparative examples was the USP resolution automatically calculated by the chromatogram data analysis program LabSolutions Version 5.97 of Shimadzu Corporation. The results are shown in Table 4.

[0251] In each of the tables below, "FA" means formic acid.

[0252] [Table 4] By comparing Example 15 with Comparative Example 12 and Example 16 with Comparative Example 13, it was found that the resolution was improved when SB / SA was 0.05 or more and 0.95 or less.

[0253] <Experiment I-5> <Comparative Example 14> The same operation as in Example 2 was performed except that TEA was not added, and a mobile phase containing 10.0 mmol / L of OxA was prepared.

[0254] Separation and analysis of the amine represented by the above formula (3) were performed by the same method as in Example 2 using the above mobile phase as the mobile phase.

[0255] The results are shown in Table 4. Figure 30 .

[0256] <Example 17> The same operation as in Example 2 was performed, except that OxA was used so that the concentration thereof in the mobile phase became 5.0 mol / L and TEA was used so that the concentration thereof in the mobile phase became 2.5 mol / L, to prepare the mobile phase.

[0257] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except that the same operation as in Example 1 was performed.

[0258] The results are shown in Table 2. Figure 31 .

[0259] <Example 18> The same operation as in Example 2 was performed, except that OxA was used so that the concentration thereof in the mobile phase became 5.0 mol / L and TEA was used so that the concentration thereof in the mobile phase became 2.5 mol / L, to prepare the mobile phase.

[0260] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except that the same operation as in Example 1 was performed.

[0261] The results are shown in Table 2. Figure 32 .

[0262] <Example 19> The same operation as in Example 1 was performed, except that the solvent was changed from a mixed solution of water and acetonitrile to a mixed solution of hexane, ethanol and water (hexane / ethanol / water = 50 / 50 / 2 (v / v / v)) and the concentrations of OxA and TEA were set to 10.0 mmol / L and 5.0 mmol / L, respectively.

[0263] The separation and analysis of the amine represented by the above formula (3) were performed using the above mobile phase as the mobile phase, except that the same operation as in Example 1 was performed.

[0264] The results are shown in Table 2. Figure 33 .

[0265] The separation conditions of Examples 17 to 19 and Comparative Example 14 are summarized in Table 5 below.

[0266] (Resolution) The resolution in each of the examples and comparative examples was the USP resolution automatically calculated by the chromatogram data analysis program LabSolutions Version 5.97 manufactured by Shimadzu Corporation. The results are shown in Table 5.

[0267] [Table 5] By comparing Example 17 to 18 with Comparative Example 14, it was found that the resolution was improved when SB / SA was 0.05 or more and 0.95 or less.

[0268] <Experiment II> <Example 20> [Preparation of mobile phase] The mobile phase was prepared by the same method as in Example 1.

[0269] [Separation of amine] For the separation and analysis of the amine, the separation and analysis of the amine represented by Formula (3) were performed by the same method as in Example 1, using the method shown below, except that the mobile phase was used.

[0270] (Separation method · analysis method) As a detector of a high-performance liquid chromatograph (HPLC), an ACQUITY Qda (manufactured by Nihon Waters K.K., mass detector) was used, and after the mobile phase was sent to the column at 0.75 mL / min for 1 hour, 1 μL of a sample solution obtained by dissolving a sample in the mobile phase in the form of about 1 to 2 mg / mL solution (filtration was completed using a filter) was injected using an autosampler, and the separation and analysis of the amine were performed at the same flow rate, with the column temperature set to room temperature (about 25°C).

[0271] Details of the device used in the HPLC are shown below.

[0272] • HPLC device: ACQUITY UPLC H-Class PLUS • Ionization method: ESI • Measurement mode: Positive • Probe temperature: 400°C • Source temperature: 120°C The results are shown in Figure 34 . The vertical axis of the graph in the figure sets the maximum value of the highest peak to 100%. This is also the same in the Figures 35-38 below.

[0273] In addition, the detected mass m / z (a value obtained by dividing the relative mass of the ion by the valence) obtained by the above analysis is shown in Table 6 below.

[0274] <Example 21> The mobile phase was prepared by the same method as in Example 1.

[0275] For separation and analysis of the amine, the method shown in Example 20 described above was used, and, except for this, separation and analysis of the amine as the object of analysis were performed by the same method as Example 20, using the amine (1-phenyl-1,2,3,4-tetrahydroisoquinoline) represented by the above-described formula (6) as the object of analysis.

[0276] The results are shown in Table 6. Figure 35 and Table 6.

[0277] <Example 22> The mobile phase was prepared by the same method as Example 2 described above.

[0278] For separation and analysis of the amine, the method shown in Example 20 described above was used, and, except for this, separation and analysis of the amine represented by the above-described formula (3) were performed by the same method as Example 2.

[0279] The results are shown in Table 6. Figure 36 and Table 6.

[0280] <Example 23> The mobile phase was prepared by the same method as Example 3 described above.

[0281] For separation and analysis of the amine, the method shown in Example 20 described above was used, and, except for this, separation and analysis of the amine represented by the above-described formula (3) were performed by the same method as Example 3.

[0282] The results are shown in Table 6. Figure 37 and Table 6.

[0283] <Example 24> The mobile phase was prepared by the same method as Example 3 described above.

[0284] For separation and analysis of the amine, the method shown in Example 20 described above was used, and, except for this, separation and analysis of the amine represented by the above-described formula (3) were performed by the same method as Example 3.

[0285] The results are shown in Table 6. Figure 38 and Table 6.

[0286] [Chemical Formula 4] The above-described SB / SA is shown in Table 2 below. In the calculation of the SB / SA, since most of the oxalic acid exists in the form of monobasic acid, the basicity of the oxalic acid was calculated as 1.

[0287] [Table 6] From the above Figures 34-38As is apparent from Table 6, a good quality chromatogram (chromatogram obtained using a mass detector) can be obtained. This indicates that the method according to the embodiment of the present disclosure is not only effective for the separation of amines, particularly, enantiomers thereof, but also suitably used for a detection method such as LC-MS. In the case of LC-MS, even if a plurality of elution peaks overlap in the chromatography, they can be distinguished based on the mass, and thus the method is being expanded in use in recent years, and it is significant in industry that the method can be applied.

[0288] Industrial applicability As is apparent from the above, the separation method of amines according to the embodiment of the present disclosure can improve the separation performance of amines. In addition, it is considered that by appropriately selecting the components contained in the mobile phase from components that can avoid or alleviate adverse effects on the detection based on a mass spectrometer, the method can also be applied to LC-MS.

[0289] Therefore, the separation method of amines can be widely applied to analysis and purification based on various liquid chromatography, and the like, and is expected to be expanded in the fields of organic chemistry, medicine, and pharmacy, and the like.

Claims

1. A method for separating amines, comprising the following separation steps: Amines are separated by chromatography using a stationary phase comprising at least one of oligosaccharides, polysaccharides and their derivatives, and a mobile phase comprising one or more specific acids selected from the group consisting of oxalic acid, acetic acid, formic acid and trifluoroacetic acid, one or more bases, and a solvent. Let A be the value calculated by the following formula (1) for each specific acid contained in the mobile phase, let SA be the sum of the values ​​of each A calculated for each specific acid, let B be the value calculated by the following formula (2) for each base contained in the mobile phase, and let SB be the sum of the values ​​of each B calculated for each base, where SB / SA is 0.05 or more and 0.95 or less. Equation (1): A = number of specific acids × molar concentration of specific acids (mol / L). Equation (2): B = number of base elements × molar concentration of base (mol / L).

2. The amine separation method according to claim 1, further comprising an analytical step of analyzing the amine separated by the separation step. In the analytical procedure, analysis was performed using at least a mass spectrometer.

3. The method for separating amines as described in claim 1 or 2, wherein, The base is selected from one or more of the group consisting of ammonia, primary amines, secondary amines and tertiary amines.

4. The method for separating amines as described in claim 3, wherein, The base is selected from one or more of the group consisting of ammonia, ethylamine, n-propylamine, 2-propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, pyrrolidine, and piperidine.

5. The method for separating amines as described in claim 1 or 2, wherein, The mobile phase contains at least oxalic acid as the specific acid.

6. The method for separating amines as described in claim 1 or 2, wherein, At least one of the oligosaccharides, polysaccharides, and their derivatives has an optically active site.

7. The method for separating amines as described in claim 1 or 2, wherein, The solvent is selected from one or more of the group consisting of water, acetonitrile, ethanol, methanol, 2-propanol, hexane, heptane, THF and liquid carbon dioxide.

Images