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, which is formed by supporting a ligand having a crown ether-like cyclic structure on a support, and a mobile phase, which contains a salt of a cation and an acid anion at a concentration of 0.2 mM to 50.0 mM (inclusive) and which contains a solvent having a water content of 50 vol% or less.

Description

Technical Field

[0001] The present disclosure relates to methods for separating amines. Background Art

[0002] Separating agents having a crown ether-like cyclic structure are widely used as stationary phases for liquid chromatography to separate compounds having primary amino groups and their analogs. In particular, separating agents in which a crown ether-like cyclic structure is bonded to a chiral structure are known to be useful for the separation of enantiomers and are also effective for the separation of a mixture of multiple compounds having similar structures (e.g., a mixture of positional isomers).

[0003] Patent Documents 1 and 2 disclose separating agents formed by bonding a crown ether-like cyclic structure to an S- or R-type binaphthyl structure as excellent separating agents for optical isomers. It is believed that these separating agents exert separation performance as described below. That is, it is believed that the primary ammonium group (-NH3 + ) is included by the crown ether sample ring structure through the hydrogen bond of the hydrogen atom of the aforementioned primary ammonium group and the oxygen atom of the crown ether sample ring structure, and is kept by the separating agent, and as a result, separation can be achieved. Therefore, it is known that in the case of using such a stationary phase, in order to obtain high separation performance, it is preferred to use a strongly acidic mobile phase. For example, in patent documentation 2, a mobile phase formed by mixing an aqueous perchloric acid solution or an aqueous trifluoroacetic acid solution with organic solvents such as methanol and acetonitrile is disclosed. In this way, the strongly acidic mobile phase is a mobile phase containing a strong acid, and among the strong acids, it is known that perchloric acid is the acid that best keeps amines and thus improves separation performance.

[0004] However, perchloric acid is not only a strong acid but also a strong oxidant, so there is a problem of difficulty in handling. In addition, when other strong acids are added to the mobile phase instead of perchloric acid, there is a problem that the stationary phase has insufficient retention of amines and cannot separate amines well.

[0005] In contrast, Patent Document 3 discloses a mobile phase as a mobile phase combined with a stationary phase having a crown ether-like cyclic structure, which is a mobile phase of an aqueous solution containing one or more hydrophobic anion salts selected from the group consisting of salts of solubilizing anions and salts of hydrophobic organic acids. By using such an aqueous mobile phase, the retention of amines in the column can be enhanced, and the use of strong acids such as perchloric acid and highly oxidizing acids can be avoided.

[0006] In addition, a mobile phase containing trifluoroacetic acid at a high concentration is disclosed in Non-Patent Document 1. This mobile phase can be suitable for liquid chromatography-mass spectrometry (LC-MS).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Laid-Open No. 2-69472

[0010] Patent Document 2: International Publication No. 2012 / 050124

[0011] Patent Document 3: International Publication No. 2020 / 251003

[0012] Non-patent literature

[0013] Non-patent document 1: Konya Y, Taniguchi M, Furuno M, Nakano Y, Tanaka N, Fukusaki E, Journal of Chromatography A, 2018, Vol. 1578, 35-44 Summary of the invention

[0014] Problems to be solved by the invention

[0015] The trifluoroacetic acid contained in the mobile phase described in Non-Patent Document 1 generally has a weak ability to retain amines on the stationary phase, and satisfactory separation cannot be obtained in many cases. In addition, usually after using high concentrations of trifluoroacetic acid, even if a mobile phase not containing it is used, so-called ghost peaks of trifluoroacetic acid adsorbed to certain components of the liquid chromatogram will appear, and therefore its use is sometimes avoided.

[0016] In the method described in Patent Document 3, although it is not necessary to add a strong acid or a strong oxidizing acid to the mobile phase containing a salt of a hydrophobic anion, the solvent contained in the mobile phase is a solvent mainly composed of water. On the other hand, even if a mobile phase with a low water content contains a salt of a hydrophobic anion, it is not necessarily possible to effectively retain amines in the stationary phase.

[0017] However, depending on the type of amine to be separated or the purpose of separation, a mobile phase with a low water content may be preferred. Therefore, in order to expand the range of selection of analytical conditions in chromatography, it is required to achieve high amine separation performance even when a mobile phase with a low water content is used.

[0018] An object of the present disclosure is to provide a method capable of achieving high separation performance for separating amines by chromatography using a stationary phase having a crown ether-like cyclic structure and a mobile phase having a low water content.

[0019] Means for solving problems

[0020] The inventors of the present application have conducted intensive research to solve the above problems and have found that by adding a specific salt to a mobile phase mainly composed of a non-aqueous solvent, the retention of amines on a stationary phase can be promoted, thereby achieving efficient separation of amines. That is, the gist of the present disclosure is as follows.

[0021] [1]

[0022] A method for separating amines comprises the following separation step: using a stationary phase formed by supporting a ligand having a crown ether-like cyclic structure on a carrier, and a mobile phase containing a salt of a cation and an acid anion at a concentration of 0.2 mM to 50.0 mM and containing a solvent with a water content of 50% by volume or less, to separate the amines by chromatography.

[0023] [2]

[0024] The method for separating amines according to [1], wherein the mobile phase contains an acid at a concentration of more than 0 mM and not more than 50.0 mM.

[0025] 〔3〕

[0026] The method for separating amines according to [1] or [2], wherein the content of water in the solvent is 20% by volume or less.

[0027] [4]

[0028] The method for separating amines according to any one of [1] to [3], wherein the liquid other than water in the solvent is at least one selected from the group consisting of an organic solvent and subcritical or supercritical carbon dioxide.

[0029] 〔5〕

[0030] The method for separating amines as described in [4], wherein the organic solvent is one or more selected from the group consisting of acetonitrile, acetone, methanol, ethanol, 2-propanol, hexane, tetrahydrofuran and tert-butyl methyl ether.

[0031] [6]

[0032] The method for separating an amine according to any one of [1] to [3], wherein the solvent contains 70% by volume or more of acetonitrile.

[0033] 〔7〕

[0034] The method for separating an amine according to any one of [1] to [6], wherein the acid serving as a source of the acid anion has a pKa in water at 25°C of n (n is the valence of the acid anion.) is -2.0 or more and 6.5 or less.

[0035] 〔8〕

[0036] The method for separating amines as described in any one of [1] to [6], wherein the acid anion is one or more selected from the group consisting of nitrate ion, trifluoroacetate ion, oxalate ion, hydrogen oxalate ion, dihydrogen phosphate ion, glycolate ion, formate ion, acetate ion and bicarbonate ion.

[0037] 〔9〕

[0038] The method for separating amines according to any one of [1] to [6], wherein the acid anion is one or more selected from the group consisting of oxalate ions, formate ions, and acetate ions.

[0039] 〔10〕

[0040] The method for separating amines according to any one of [1] to [9], wherein the cation is one or more selected from the group consisting of tetrahydroammonium ions, secondary ammonium ions, and tertiary ammonium ions.

[0041]

[11]

[0042] The method for separating amines as described in [2], wherein the value calculated by the following formula (1) in the mobile phase is 0.01 or more and 0.90 or less.

[0043] [Mathematical formula 1]

[0044]

[0045] (Where,

[0046] X is the concentration of the aforementioned acid in the aforementioned mobile phase (mM);

[0047] Y is the concentration of the aforementioned salt in the aforementioned mobile phase (mM);

[0048] q is the number of the aforementioned acid anions per molecule of the aforementioned salt.)

[0049]

[12]

[0050] The method for separating amines according to any one of [1] to

[11] , wherein the chromatography is performed by liquid chromatography-mass spectrometry.

[0051] Effects of the Invention

[0052] According to the present disclosure, a method capable of achieving high separation performance for separating amines by chromatography using a stationary phase having a crown ether-like cyclic structure and a mobile phase having a low water content can be provided.

[0053] In addition, in a preferred embodiment of the present disclosure, a method for separating amines that is applicable to LC-MS can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] [ Figure 1 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 1.

[0055] [ Figure 2 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 2.

[0056] [ Figure 3 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 3.

[0057] [ Figure 4 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 4.

[0058] [ Figure 5 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 5.

[0059] [ Figure 6 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 6.

[0060] [ Figure 7 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 7.

[0061] [ Figure 8 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 8.

[0062] [ Fig. 9 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 1.

[0063] [ Fig.10 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 2.

[0064] [ Fig.11 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 3.

[0065] [ Fig.12 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 4.

[0066] [ Fig.13 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 5.

[0067] [ Fig.14 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 6.

[0068] [ Fig.15 ] is the liquid chromatogram of dl-1-phenylethylamine in Example 9.

[0069] [ Fig.16 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 7.

[0070] [ Fig.17 ] is the liquid chromatogram of dl-1-phenylethylamine in Comparative Example 8.

[0071] [ Fig.18 ] is a liquid chromatogram of dl-tryptophan in Example 10.

[0072] [ Fig.19 ] is a liquid chromatogram of dl-tryptophan in Example 11.

[0073] [ Fig. 20 ] is a liquid chromatogram of dl-tryptophan in Example 12.

[0074] [ Fig.21 ] is a liquid chromatogram of dl-tryptophan in Comparative Example 9.

[0075] [ Fig. 22 ] is a liquid chromatogram of dl-tryptophan in Comparative Example 10.

[0076] [ Fig.23 ] is the liquid chromatogram of dl-tryptophan in Example 13.

[0077] [ Fig.24 ] is a graph showing the relationship between the salt concentration and acid concentration in the mobile phase used in Examples 14 to 16 and the retention time. DETAILED DESCRIPTION

[0078] Hereinafter, the present disclosure is described by giving specific embodiments, but each configuration and combination thereof in each embodiment is an example, and addition, omission, substitution and other changes of the configuration may be appropriately made within the scope of the gist of the present disclosure. The present disclosure is not limited to the embodiments, but only to the scope of the claims.

[0079] In addition, each aspect disclosed in this specification can be combined with any other feature disclosed in this specification.

[0080] In addition, in this specification, when the lower limit value and the upper limit value of a numerical range are described separately, the said numerical range may be a range combining any lower limit value and any upper limit value therein.

[0081] [Method for separation of amines]

[0082] One embodiment of the present disclosure is a method for separating amines, which includes the following separation steps: using a stationary phase formed by supporting a ligand having a crown ether-like cyclic structure on a carrier (hereinafter sometimes referred to as a "stationary phase having a crown ether-like cyclic structure"), and a mobile phase containing a salt of cations and acid anions at a concentration of not less than 0.2 mM and not more than 50.0 mM (hereinafter sometimes referred to as a "salt") and a solvent having a water content of not more than 50 volume %, to separate the amines by chromatography.

[0083] The method for separating amines according to the present embodiment may further include other steps such as an identification step of identifying the separated amine, a quantification step of quantifying the separated amine, and a step of recovering the separated amine in addition to the above-mentioned separation step.

[0084] First, the significance of the present embodiment is explained. The inventor of the present application speculates that when the amino group of the amine to be separated is protonated and the ammonium group generated is enclosed (retained) by the crown ether-like cyclic structure of the stationary phase, the anion present in the mobile phase becomes the counter ion of the ammonium group and exists in the vicinity of the ammonium group. This is a certain adsorption equilibrium reaction, therefore, in order to make the ammonium group fully retained by the crown ether-like cyclic structure, it is necessary to have an anion (hereinafter sometimes referred to as a "counter anion") that becomes a counter ion in a certain concentration in the mobile phase.

[0085] However, with regard to the dissociation of acid, except for special alkaline organic solvents, it is usually difficult to occur in organic solvents with low polarity. Therefore, if a solvent with low water content is used as the solvent for the mobile phase, it is difficult to supply the necessary amount of counter anions to the mobile phase. Especially in acids, weak acids are more difficult to dissociate, so when the acid contained in the mobile phase is a weak acid, it is more difficult to supply sufficient concentration of counter anions to the mobile phase.

[0086] Therefore, it is considered that when using a stationary phase having a crown ether-like cyclic structure to separate amines, it is inappropriate to use a solvent with a low water content in a mobile phase containing an acid. However, as described above, depending on the type of amine to be separated or the purpose of separation, a mobile phase with a low water content is sometimes preferred.

[0087] Therefore, the inventor of the present application has studied a method for achieving sufficient retention of amines on the stationary phase even when using a stationary phase with a crown ether-like cyclic structure and a mobile phase with a low water content. As a result, the inventor of the present application has found that by adding a base to a mobile phase containing a solvent with a low water content and an acid, a part or all of the acid is neutralized and converted into a salt, so that even if the acid is a weak acid, the counter anion concentration in the mobile phase can be adjusted to an ideal level. It is believed that by supplying a sufficient amount of counter anions in the mobile phase, the ammonium group generated by the amine as the separation object forms an ion pair with the aforementioned counter anion and is included in the crown ether-like cyclic structure of the stationary phase. Moreover, it is inferred that if the inclusion of the ammonium group in the crown ether-like cyclic structure, that is, the retention of the amine on the stationary phase is promoted, the separation performance of the amine is improved.

[0088] In summary, the solvent included in the mobile phase used in the separation process in the present embodiment is a solvent having a water content of less than 50% by volume, which means that in the separation process, a mobile phase with a low water content is used in the chromatography. In addition, the mobile phase contains a salt of a cation and an acid anion with a concentration of more than 0.2mM and less than 50.0mM, which means that the mobile phase contains a salt for supplying an appropriate amount of anion (that is, an acid anion), in other words, in the mobile phase, there is fully an anion that forms a pair with the ammonium group.

[0089] In this embodiment, by using a mobile phase in which a part or all of the acid is neutralized and converted into a salt, or a mobile phase equivalent thereto, it is possible to achieve an improvement in the separation performance of amines, and therefore, the acid is not an essential component. The mobile phase in which the acid concentration is 0 mM is equivalent to a mode in which the acid is completely neutralized, and on the other hand, the mobile phase in which the acid concentration is greater than 0 mM is equivalent to a mode in which a part of the acid is neutralized.

[0090] In addition, in the present embodiment, a mobile phase obtained by neutralizing part or all of the acid and converting it into a salt or a mobile phase equivalent to this, that is, a mobile phase containing a salt of a cation and an acid anion and an acid used as needed, promotes the retention of amines on the stationary phase to improve the separation performance of amines. At this time, the method for preparing the mobile phase containing the above-mentioned components is not particularly limited. Regardless of the mobile phase prepared by any method, as long as it is a mobile phase containing the above-mentioned components, the separation performance of amines can be improved. Therefore, as described later, with respect to the mobile phase, it can be a mobile phase prepared by a method of neutralizing a liquid containing an acid, or it can be a method of dissolving a pre-prepared salt in a solvent and adding an acid as needed.

[0091] In the separation method according to the present embodiment, amines can be separated with high separation performance using a mobile phase containing a solvent with a low water content (i.e., a solvent with a water content of 50% by volume or less). Therefore, it is particularly useful for separating a separation object that cannot be effectively retained on a stationary phase when a mobile phase containing an aqueous solvent with a high water content is used.

[0092] Next, the separation step in this embodiment will be described in detail.

[0093] 1. Amine

[0094] According to the separation method according to the present embodiment, a mixture of a plurality of amines can be separated into individual amines, or amines can be separated from a mixture containing amines and non-amines.

[0095] Amines are not particularly limited as long as they are compounds containing an amino group, and may be any of primary, secondary, and tertiary amines. Specific examples of amines include amino acids such as alanine, cysteine, glutamic acid, methionine, leucine, tyrosine, and tryptophan; amino acid derivatives such as esters and amides of the aforementioned amino acids; amino alcohols such as dimethylaminoethanol, propanolamine, methioninol, and norephedrine; hydrocarbons containing an amino group such as phenylethylamine, aniline, methylaniline, chloroaniline, and aminobenzoic acid; cyclic amines such as 1-phenyl-1,2,3,4-tetrahydroisoquinoline; and the like.

[0096] The separation method according to the present embodiment is suitable for separating a desired primary amine from a mixture containing primary amines because the primary amines can be well retained by the crown ether-like cyclic structure of the stationary phase.

[0097] In addition, according to the separation method involved in this embodiment, high separation performance can be achieved, so a mixture of amines that is difficult to separate due to their similar structures can also be separated into each amine. As a mixture of amines with similar structures, a mixture of chain isomers, a mixture of positional isomers, a mixture of geometric isomers, and a mixture of the like can be cited. In addition, by using a chiral stationary phase having a crown ether-like cyclic structure introduced with an optically active site as a stationary phase, a mixture of enantiomers can also be separated into each enantiomer.

[0098] 2. Mobile phase

[0099] The mobile phase used in the separation step contains a salt of a cation and an acid anion at a concentration of 0.2 mM to 50.0 mM, and contains a solvent having a water content of 50% by volume or less.

[0100] The mobile phase may contain other components within a range that does not impair separation performance. In addition, the mobile phase is preferably a homogeneous system (single-phase system) at least under the conditions for performing the analysis.

[0101] It should be noted that in this specification, the so-called "mobile phase" refers to a liquid that is injected into a stationary phase together with the separation object and moves together with the separation object in chromatography such as liquid chromatography and supercritical fluid chromatography, and then the separation object is eluted from the stationary phase.

[0102] 2-1.Solvent

[0103] The solvent contained in the mobile phase in the present embodiment is not particularly limited as long as the water content is less than 50 volume %. That is, the liquid (hereinafter, sometimes referred to as "non-aqueous solvent") other than water in the solvent contained in the mobile phase is not particularly limited. The non-aqueous solvent is preferably selected from more than one of the group consisting of an organic solvent (water content is 0 volume %) and subcritical or supercritical carbon dioxide. Therefore, as the solvent contained in the mobile phase, for example, a mixed solvent of water and an organic solvent, an organic solvent (water content is 0 volume %), a mixed solvent of subcritical or supercritical carbon dioxide and an organic solvent, and a mixed solvent of subcritical or supercritical carbon dioxide and water and an organic solvent, etc. can be cited. In the case where the solvent contained in the mobile phase is a mixed solvent formed by more than two solvents, the aforementioned more than two solvents are preferably a combination of solvents that will not undergo phase separation under the separation conditions of amine.

[0104] As an organic solvent, there is no particular limitation, and it is preferably an organic solvent that can dissolve the amine as the separation object. As a preferred organic solvent, for example, acetonitrile, acetone, tetrahydrofuran (THF), tert-butyl methyl ether, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and dimethylacetamide (DMAc) can be cited. In addition, as long as the amine as the separation object, and various components such as salts of cations and acid anions can be dissolved, the organic solvent may also include hydrocarbons such as hexane; halogenated hydrocarbons such as dichloromethane; and so on. Among them, from the perspective of high chemical stability, the organic solvent preferably includes one or more selected from the group consisting of acetonitrile, acetone, methanol, ethanol, 2-propanol, hexane, THF and tert-butyl methyl ether, and more preferably acetonitrile. The solvent contained in the mobile phase particularly preferably contains 70% by volume or more of acetonitrile. The aforementioned organic solvent can be a single one, or any two or more can be combined in any ratio.

[0105] The organic solvent may also be a mixture of hydrocarbons having 5 or more and 8 or less carbon atoms and alcohols commonly used in chromatography. As such a mixture, for example, a hexane / ethanol mixture that is compatible with water may be cited. If the organic solvent has a hexane / ethanol mixing ratio of, for example, 100 / 100 (v / v), a mixed solvent having a water content of about 10% by volume can be prepared.

[0106] The mixed liquid of hydrocarbons having 5 to 8 carbon atoms and alcohols has low viscosity, and thus can achieve a high flow rate at a low pressure in chromatography. Furthermore, since ultraviolet absorption is low, detection of amines based on ultraviolet absorption is possible.

[0107] When nonaqueous solvent comprises subcritical or supercritical carbon dioxide, chromatography utilizes supercritical fluid chromatograph to carry out.In this case, the compatibility of components such as salt and subcritical or supercritical carbon dioxide is not necessarily good, therefore, preferably, the mode that target component can be dissolved in mobile phase with target concentration in subcritical or supercritical carbon dioxide and use mixed solvent of water and organic solvent or organic solvent (water content is 0 volume %) as auxiliary solvent.Here so-called organic solvent is synonymous with the organic solvent enumerated as nonaqueous solvent.About the mixed solvent of water and organic solvent and organic solvent as auxiliary solvent, respectively can preferably exemplify mixed solvent of water and methanol and methanol.

[0108] In addition, when the solvent contained in the mobile phase contains subcritical or supercritical carbon dioxide, the volume thereof is calculated based on the density of liquid carbon dioxide at -20°C, which is 1.030 g / mL.

[0109] The water content of the solvent included in the mobile phase is not particularly limited as long as it is below 50 volume %, but is preferably below 40 volume %, more preferably below 30 volume %, further preferably below 20 volume %, particularly preferably below 10 volume %.In addition, the lower limit of the water content in the solvent included in the mobile phase is not particularly limited, usually more than 0 volume %, preferably greater than 0 volume %, more than 0.5 volume % or more than 1.0 volume %.That is, as the preferred range of the water content of the solvent included in the mobile phase, for example, can enumerate 0 volume % above 50 volume %, 0 volume % above 40 volume %, greater than 0 volume % and be below 30 volume %, 0.5 volume % above 20 volume % and 1.0 volume % above 10 volume % below scope.

[0110] 2-2. Salts of cations and acid anions

[0111] The salt of a cation and an acid anion contained in the mobile phase used in the separation step is formed of an arbitrary cation and an arbitrary acid anion. The salt may be a single salt or a combination of two or more salts at an arbitrary ratio.

[0112] It should be noted that in this specification, since it has been found that the separation performance of amines can be improved by using a mobile phase obtained by neutralizing a part or all of the acid and converting it into a salt, or a mobile phase equivalent to this, the anion component that forms the salt is called "acid anion". As long as it is an anion of the same type, it does not necessarily have to be an anion generated by an acid, and for example, it can be an anion derived from cation exchange, etc.

[0113] As the cation that forms salt with acid anion bonding, tetrahydro ammonium ion, primary ammonium ion, secondary ammonium ion, tertiary ammonium ion and quaternary ammonium ion etc. can be preferably enumerated.But cation is not limited to this, except the ion of alkali metals such as lithium, sodium and potassium, can also be the ion of other metals.It should be noted that the cation that forms salt can be single 1 kind, also can be by any 2 kinds or more with any ratio combination.

[0114] Preferred examples of the primary ammonium ion include monoalkylammonium ions such as a methylammonium ion, an ethylammonium ion, a propylammonium ion, and a butylammonium ion.

[0115] Preferred examples of the secondary ammonium ion include dialkylammonium ions such as dimethylammonium ion, diethylammonium ion, diisopropylammonium ion and dibutylammonium ion; and cyclic secondary ammonium ions such as N-unsubstituted pyrrolidinium ion and N-unsubstituted piperidinium ion.

[0116] Preferred examples of tertiary ammonium ions include trialkylammonium ions such as trimethylammonium ion, triethylammonium ion, diisopropylethylammonium and tributylammonium ion; and cyclic tertiary ammonium ions such as N-substituted pyrrolidinium ion, N-substituted piperidinium ion and N-unsubstituted 1,4-diazabicyclo[2.2.2]octane-1,4-dionium ion; and the like.

[0117] As the quaternary ammonium ion, preferred examples include tetraalkylammonium ions such as tetramethylammonium ion, tetraethylammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion and octadecyltrimethylammonium ion; and cyclic quaternary ammonium ions such as N,N-disubstituted pyrrolidinium ion, N,N-disubstituted piperidinium ion and N,N'-disubstituted-1,4-diazabicyclo[2.2.2]octane-1,4-dionium ion; and the like.

[0118] The alkyl group bonded to the nitrogen atom of the monoalkylammonium ion, dialkylammonium ion, trialkylammonium ion and tetraalkylammonium ion is preferably an alkyl group having 1 to 20 carbon atoms, more preferably an alkyl group having 1 to 10 carbon atoms, further preferably an alkyl group having 1 to 4 carbon atoms, and particularly preferably an alkyl group having 1 or 2 carbon atoms. Such primary to tertiary ammonium ions having a small number of carbon atoms in the alkyl group have a small molecular weight, and therefore, when chromatography is performed by LC-MS, unnecessary peaks are less likely to be generated near the peak of the amine to be separated, and thermal dissociation and gasification are easily caused, so they are preferred.

[0119] In addition, the substituent bonded to the nitrogen atom of the N-substituted pyrrolidinium ion, N-substituted piperidinium ion, N,N-disubstituted pyrrolidinium ion, N,N-disubstituted piperidinium ion and N,N'-disubstituted-1,4-diazabicyclo[2.2.2]octane-1,4-dionium ion is not particularly limited, and each is preferably an alkyl group having 1 to 20 carbon atoms, more preferably an alkyl group having 1 to 10 carbon atoms, further preferably an alkyl group having 1 to 4 carbon atoms, and particularly preferably an alkyl group having 1 or 2 carbon atoms. This is because, as described above, in LC-MS, the molecular weight of the ammonium ion is preferably small.

[0120] The cation is preferably selected from one or more of the group consisting of tetrahydrogen ammonium ions, secondary ammonium ions and tertiary ammonium ions. That is, the salt is preferably selected from one or more of the group consisting of tetrahydrogen ammonium salts, secondary ammonium salts and tertiary ammonium salts. These ammonium salts are not easy to interact with the crown ether-like cyclic structure possessed by the stationary phase, and therefore, the risk of hindering the retention of the amine as the separation object is small, and the separation performance of the amine can be further improved.

[0121] As particularly preferred cations, more than one cation selected from the group consisting of secondary ammonium ions and tertiary ammonium ions can be cited. Cation is particularly preferably a cation with a small molecular weight among them, specifically dimethylammonium ions, diethylammonium ions, trimethylammonium ions and triethylammonium ions. That is, salt is more preferably more than one selected from the group consisting of secondary ammonium salts and tertiary ammonium salts, more preferably dimethylammonium salts, diethylammonium salts, trimethylammonium salts and triethylammonium salts with small molecular weights. Secondary ammonium salts and tertiary ammonium salts are prone to thermal dissociation and volatilization, and therefore, when a separation process is performed by LC-MS, there is little risk of precipitating in the detector and clogging the nozzle or contaminating the detector. In addition, if the molecular weight is small, as described above, when implementing LC-MS, the risk of producing unnecessary peaks near the peak of the amine as the separation object can be reduced.

[0122] The acid that becomes the source of acid anion is not particularly limited, and can be one or more known acids selected from organic acid and inorganic acid. The known acid is an acid that produces the aforementioned acid anion by dissociation, in other words, an electrically neutral acid that protonates the acid anion. The upper limit of the valence of the acid anion is not particularly limited, and is usually an integer below 6, preferably an integer below 4, and more preferably an integer below 2. An example of the relationship between the acid anion, the valence of the acid anion and the acid that becomes the source of the acid anion in the present embodiment is shown in Table 1 below. As shown in Table 1, multiple acid anions with different valences are sometimes produced by the same acid. It should be noted that the acid anion that forms the salt can be a single one, or any two or more can be combined in any ratio.

[0123] [Table 1]

[0124] Table 1

[0125]

[0126] The acid anion is preferably an anion derived from an acid having a specific pKa. Specifically, the pKa of the acid that is the source of the acid anion is n (n is the valence of the acid anion.) It is preferably -2.9 or more, more preferably -2.0 or more, further preferably -1.0 or more, particularly preferably 0.2 or more, and most preferably 1.0 or more. In addition, it is preferably 6.5 or less, more preferably 6.0 or less, further preferably 5.5 or less, particularly preferably 4.5 or less, and most preferably 3.5 or less. That is, the pKa of the acid that is the source of the acid anion is n The preferred range of is, for example, -2.9 to 6.5, -2.0 to 6.5, -1.0 to 6.0, 0.2 to 5.5, and 1.0 to 4.5. Alternatively, the pKa of the acid that is the source of the acid anion is n Preferred ranges include, for example, -2.9 to 6.5, -2.0 to 6.0, -1.0 to 5.5, 0.2 to 4.5, and 1.0 to 3.5.

[0127] The inventors of the present application speculate that the acid anion preferably originates from the pKa n The reason for being an acid anion of an acid with a pH of -2.9 or more.

[0128] It is believed that when the ammonium group is enclosed by the crown ether-like cyclic structure of the stationary phase, at least partial desolvation is required, but generally, in weakly polar solvents, the stabilization based on solvation of the acid anions derived from weak acids is insufficient. Therefore, it is speculated that the energy loss caused by the desolvation of the weak acid when the ammonium group is enclosed by the crown ether-like cyclic structure of the stationary phase is small. However, weak acids cannot generate conjugated anions of sufficient concentration in organic solvents. In contrast, in the present embodiment, it is speculated that by containing a conjugated anion derived from pKa in the mobile phase n The salt of the anion and cation of a weak acid having a pH of -2.9 or more can promote the retention of the amine on the stationary phase.

[0129] In addition, in this specification, unless otherwise specified, the acid dissociation constant "pKa" of an acid means the acid dissociation constant of the acid in water at 25°C.

[0130] As having the above pKa nThe acid preferably includes methanesulfonic acid (pKa1=-2.6), nitric acid (pKa1=-1.4), trifluoroacetic acid (pKa1=0.23), oxalic acid (pKa1=1.27, pKa2=4.27), phosphoric acid (pKa1=2.12), glycolic acid (pKa1=3.1), formic acid (pKa1=3.75), acetic acid (pKa1=4.76) and carbonic acid (pKa1=3.60). Among them, from the perspective of less noise in the detection by the mass spectrometer when performing the separation process by LC-MS, the acid is more preferably one or more selected from the group consisting of formic acid, oxalic acid and acetic acid, and oxalic acid is further preferred. In addition, it is also preferred that the acid is not any one or both of hydrochloric acid (pKa1=-3.7) and sulfuric acid.

[0131] In other words, the acid anion is preferably a methanesulfonate ion (CH3SO3 - ), nitrate ion (NO3 - ), trifluoroacetate ion (CF3COO - ), oxalate ion (C2O4 2- ), hydrogen oxalate ion (HC2O4 - ), dihydrogen phosphate ion (H2PO4 - ), hydroxyacetate ion (HOCH2COO - ), formate ion (HCOO - ), acetate ion (CH3COO - ) and bicarbonate ions (HCO3 - Among them, the acid anion is more preferably selected from the group consisting of formate ion (HCOO), from the perspective of less noise in the detection using a mass spectrometer when performing the separation process by LC-MS. - ), oxalate ion (C2O4 2- ) and acetate ion (CH3COO - ) is one or more, more preferably oxalate ion (C2O4 2- In addition, it is also preferred that the acid anion is not a chloride ion (Cl - ) and hydrogen sulfate ion (HSO4 - ) or both.

[0132] It should be noted that in this specification, the salt of a cation and an acid anion is sometimes expressed as a salt of a base that is the source of the aforementioned cation and an acid that is the source of the aforementioned acid anion. For example, a salt formed by an ammonium ion and an oxalate ion is sometimes expressed as 2NH3·(COOH)2, a salt formed by a triethylammonium ion and a formate ion is expressed as Et3N·HCOOH, a salt formed by a triethylammonium ion and a dihydrogen phosphate ion is expressed as Et3N·H3PO4, a salt formed by a trimethylammonium ion and a hydrochloride ion is expressed as Me3N·HCl, etc. It should be noted that, when the above-mentioned "base that is the source of the cation" is an amine, the above-mentioned amine is preferably not an amine to be separated.

[0133] The concentration of the salt in the mobile phase (in the case where the mobile phase comprises more than two kinds of salts, it refers to the total concentration of the salt.) It is preferably set in a manner within a specific range by multiplying the concentration of the salt by the number of acid anions in each molecular salt (equivalent to the value of qY in the formula (1) described later). Specifically, the value obtained by multiplying the concentration of the salt by the number of acid anions in each molecular salt is usually set to more than 0.2mM, preferably more than 0.4mM, more preferably more than 1.0mM, more preferably more than 1.5mM, particularly preferably more than 2.0mM, in addition, usually set to less than 50.0mM, preferably less than 30.0mM, more preferably less than 20.0mM, more preferably less than 10.0mM. That is, as the preferred range of salt concentration, for example, the value obtained by multiplying the concentration of salt by the number of acid anions in each molecule of salt is 0.2mM to 30.0mM, 0.4mM to 50.0mM, 1.0mM to 30.0mM, 1.5mM to 20.0mM, and 2.0mM to 10.0mM. By making the concentration of salt within the above range, sufficient counter anions can be supplied to the mobile phase, and adverse effects on chromatographs, detectors, etc. can be avoided.

[0134] 2-3.Acid

[0135] In the present embodiment, since the mobile phase contains a salt of a cation and an acid anion, the requirement that the amine to be separated is retained by the stationary phase is satisfied, but it is desirable to further protonate the amino group of the amine so that it exists in the form of an ammonium group. Moreover, for this reason, it is desirable to contain an acid in the mobile phase so as to adjust the acidity. The acid may be a single acid or any two or more acids may be combined in any ratio.

[0136] As the acid for adjusting the acidity of the mobile phase, it is preferred to cite an acid that releases one or more anions and protons selected from the anions cited as the above-mentioned acid anions forming the salt by dissociation. That is, as the acid for adjusting the acidity of the mobile phase, it is preferred to cite an acid that is the source of the acid anions recorded in the item "2-2. Salt of cations and acid anions". It is believed that the effect of improving the separation performance of amines achieved by the present embodiment is based on the above-mentioned principle, and therefore, the acid for adjusting the acidity of the mobile phase and the acid that is the source of the acid anions forming the salt coexisting in the mobile phase can be the same or different. For example, the mobile phase can contain a salt of ammonium ions and hydrogen oxalate ions as a salt, and contain oxalic acid as an acid. In addition, for example, the mobile phase can also contain a salt of ammonium ions and hydrogen oxalate ions as a salt, and contain formic acid as an acid. In addition, it is also preferred that the acid for adjusting the acidity of the mobile phase is an acid whose acidity is weaker than the acid that is the source of the acid anions forming the salt.

[0137] It should be noted that, it is believed that when the acidity of the acid used to adjust the acidity of the mobile phase is higher than the acidity of the acid that becomes the source of the acid anion forming the salt, proton exchange will occur, and the anion released by the acid used to adjust the acidity will replace the acid anion forming the salt. In this case, the anion released by the acid used to adjust the acidity of the mobile phase achieves the same effect as the acid anion forming the salt.

[0138] The acid used to adjust the acidity of the mobile phase is basically the same as the acid that serves as the source of the acid anion described in the item "2-2. Salt of cation and acid anion", but the following points should be noted regarding the acid dissociation constant of the acid that serves as the source of the acid anion.

[0139] pKa of the acid used to adjust the acidity of the mobile phase n (n is the number of protons released.) It is preferably -2.9 or more, more preferably -2.0 or more, further preferably -1.0 or more, particularly preferably 0.2 or more, and most preferably 1.0 or more. In addition, it is preferably 6.5 or less, more preferably 6.0 or less, further preferably 5.5 or less, particularly preferably 4.5 or less, and most preferably 3.5 or less. That is, the pKa of the acid that is the source of the acid anion is n The preferred range of is, for example, -2.9 to 6.5, -2.0 to 6.5, -1.0 to 6.0, 0.2 to 5.5, and 1.0 to 4.5. Alternatively, the pKa of the acid that is the source of the acid anion is n Preferred ranges include, for example, -2.9 to 6.5, -2.0 to 6.0, -1.0 to 5.5, 0.2 to 4.5, and 1.0 to 3.5.

[0140] Since the mobile phase contains salts and has a pKa value above the above lower limit n Therefore, the effect of improving the separation performance of amines is further improved. In addition, if the pKa of the acid contained in the mobile phase together with the salt is n When the concentration is not more than the upper limit, when the separation target is amino acids, almost all of the amino acids can be protonated.

[0141] It should be noted that the upper limit of n is pKa n The upper limit of n varies depending on the type of acid, but is generally an integer of 6 or less, preferably an integer of 4 or less, and more preferably an integer of 2 or less. n The upper limit of n is 6.5, and a specific example is given to illustrate. For phosphoric acid (H3PO4), pKa1=2.12, pKa2=7.21, and pKa3=12.67, so the upper limit of n is 1. In addition, for oxalic acid ((COOH)2), pKa1=1.27, and pKa2=4.27, so the upper limit of n is 2.

[0142] As having the above pKa n Acids such as methanesulfonic acid (pKa1=-2.6), nitric acid (pKa1=-1.4), trifluoroacetic acid (pKa1=0.23), oxalic acid (pKa1=1.27, pKa2=4.27), phosphoric acid (pKa1=2.12), hydroxyacetic acid (pKa1=3.1), formic acid (pKa1=3.75), acetic acid (pKa1=4.76) and carbonic acid (pKa1=3.60) are preferably mentioned.

[0143] When the mobile phase contains an acid, the concentration of the acid (when the mobile phase contains two or more acids, it refers to the total concentration of the acid.) is usually greater than 0mM, preferably more than 0.5mM, more preferably more than 1.0mM, more preferably more than 2.0mM, particularly preferably more than 5.0mM, and preferably less than 50.0mM, more preferably less than 40.0mM, more preferably less than 30.0mM, and particularly preferably less than 20.0mM. That is, as a preferred range of the concentration of the acid in the mobile phase, for example, a range greater than 0mM and less than 50.0mM, more than 0.5mM and less than 50.0mM, more than 1.0mM and less than 40.0mM, more than 2.0mM and less than 30.0mM, and more than 5.0mM and less than 20.0mM can be cited. By making the concentration of the acid within the above range, it is possible to ensure the appropriate maintenance of the amine and avoid the adverse effects of excess acid on the chromatograph, detector, etc.

[0144] 2-4. Relationship between the amount of salt and acid in the mobile phase

[0145] In the mobile phase used in the present embodiment, the salt and the acid as an arbitrary component are preferably in a specific quantitative relationship. The quantitative relationship of these components can be represented by the following formula (1). The following formula (1) represents the relationship between the concentration of the acid and the concentration of the salt. In addition, if it is recorded as other expressions, it can also be said that formula (1) represents the degree of neutralization of the aforementioned acid when the mobile phase is prepared by neutralizing the acid with a base.

[0146] The value calculated by the following formula (1) in the mobile phase is preferably 0.01 or more, more preferably 0.05 or more, further preferably 0.10 or more, particularly preferably 0.20 or more, and most preferably 0.30 or more. In addition, it is preferably 0.90 or less, more preferably 0.80 or less, further preferably 0.70 or less, and particularly preferably 0.60 or less. That is, as a preferred range of the value calculated by formula (1), for example, the range of 0.01 or more and 0.90 or less, 0.05 or more and 0.80 or less, 0.10 or more and 0.70 or less, 0.20 or more and 0.60 or less can be cited. By making the value calculated by formula (1) within the above range, sufficient counter anions can be supplied to the mobile phase, and the amino group of the amine to be separated can be efficiently converted into an ammonium group by using the proton generated by the acid. It should be noted that the case where the value calculated by formula (1) is 1 means that the mobile phase does not contain an acid.

[0147] [Mathematical formula 2]

[0148]

[0149] X is the concentration (mM) of the aforementioned acid in the mobile phase, as described above.

[0150] Y is the concentration of the salt in the mobile phase (mM), as described above.

[0151] q is the number of acid anions in one molecule of the salt. More specifically, for the salt of ammonium ion and oxalate ion (2NH3·(COOH)2), q=1. For the salt of triethylammonium ion and trifluoroacetate ion (Et3N·CF3COOH), q=1. In addition, for the salt formed by ammonium ion and formate ion of tetramethylethylenediamine (TMEDA·2HCOOH), q=2.

[0152] 2-5. Preparation of mobile phase

[0153] The mobile phase can be prepared by a known solution preparation method or a method based on a known solution preparation method.

[0154] As known method, can enumerate the method for forming salt by acid and alkali and using it to prepare mobile phase.For example, when using more than one salt selected from the group consisting of tetrahydrogen ammonium salt, primary ammonium salt, secondary ammonium salt and tertiary ammonium salt as salt, can be as shown in the embodiment described later, make the acid corresponding to the acid anion of target and the alkali corresponding to the target cation (for example, amine and ammoniacal liquor etc.) contact in the solvent of mobile phase, after a part or all of the aforementioned acid is converted into salt, utilize identical solvent constant volume to be specified capacity, thus, easily prepare the mobile phase containing desired composition.Like this, do not make acid and alkali directly contact and make it contact in solvent to prepare the method for mobile phase from the aspect of simple and convenient and can avoid the aspect of the problem that a part of component gasification etc. causes because of neutralization heat to consider to be preferred.

[0155] Salts of cations and acid anions are commercially available, or when readily available, the commercially available salts may be dissolved in a solvent and mixed with an acid for adjusting the acidity as required to prepare the mobile phase.

[0156] 3. Stationary phase

[0157] The stationary phase in the present embodiment is a separating medium in which a ligand having a crown ether-like cyclic structure is supported on a support.

[0158] In this specification, the so-called ligand refers to a compound that can be supported by a carrier and exhibits physical affinity and chiral recognition ability as required for the separation object. The ligand having a crown ether-like cyclic structure is a compound in which a polyoxyethylene chain represented by formula (I) is chemically bonded to an aliphatic, alicyclic or aromatic hydrocarbon to form a macrocyclic structure.

[0159] *-O(CH2CH2O) n -*(I)

[0160] In the formula, n can be appropriately selected from integers of 4 to 6 depending on the hydrocarbon bonded to the amino group of the amine and the crown ether skeleton. For example, the ligand represented by the formula (II) or (III) described below has n of 5 and has a crown ether-like cyclic structure of a size suitable for inclusion of the primary ammonium group, and therefore can be preferably used for the separation of primary amines. The hydrogen atoms of the ethylene group in the repeating unit may be substituted with various functional groups, but are preferably unsubstituted.

[0161] In the present embodiment, in the case of separation of enantiomers, a compound obtained by bonding a crown ether-like cyclic structure to a homochiral structure is used as a ligand. As such a ligand, for example, a ligand represented by formula (II) described in Japanese Patent Laid-Open No. 2-69472 and International Publication No. 2012 / 050124 and a ligand represented by formula (III) described in Japanese Patent Laid-Open No. 2014-169259 can be cited. In addition, the 3-position and 3'-position phenyls of the 1,1'-binaphthyl structure in formula (II) can also be replaced with halogen atoms such as bromine atoms; alkyl groups such as methyl; substituted aromatic groups; ligands obtained by heterocyclic groups, etc. (Pen g Wu, et.al., Chinese Journal of Chemistry, 2017, 35, 1037-1042), but the ligand is not limited thereto. In addition, in many cases, a ligand that is effective for the separation of enantiomers is also effective for separating molecules similar to each other.

[0162] [Chemical formula 1]

[0163]

[0164] The ligand is used as a stationary phase in a state supported on a carrier. As a method of support, a known method can be used, and a method of supporting the ligand on a carrier by chemical bonds such as covalent bonds can be preferably used. Specifically, the following method can be cited: a reactive group is introduced into a ligand, a raw material of a ligand, or an intermediate of a ligand, so that the substituent reacts with a reactive group present on the surface of the carrier. It should be noted that the so-called reactive group present on the surface of the carrier can be a group present on the surface of an untreated carrier, or a group introduced to the surface of the carrier by surface treatment of the carrier by a silane coupling agent such as a surface treatment agent, such as 3-aminopropyltriethoxysilane and 3-glycidyloxypropyltrimethoxysilane. In addition, a method of bonding the reaction pair of the so-called click reaction to the stationary phase and the ligand to react with each other can also be cited. Alternatively, not only a method of forming a bond between a ligand and a carrier, but also a layer insoluble can be formed on the surface of the carrier by forming a so-called cross-linked bond between the atomic groups comprising the ligand. Alternatively, the ligand may be supported on the support by other known supporting methods, for example, a method in which the ligand is physically adsorbed (coated) on the support.

[0165] As a carrier, as long as the ligand can be fixed to the carrier, there is no particular restriction. The carrier can be an inorganic carrier, or an organic carrier, but is preferably an inorganic carrier. As an inorganic carrier, for example, silica gel, aluminum oxide, magnesium oxide, glass, kaolin, titanium oxide, silicate, zirconium oxide and hydroxyapatite etc. can be enumerated. As an organic carrier, for example, cross-linked polystyrene, cross-linked poly (methyl) acrylamide, cross-linked poly (methyl) acrylate and polysaccharide etc. can be enumerated. These organic carriers are preferably cross-linked by utilizing a cross-linking agent to realize insolubilization.

[0166] The shape of the support is not particularly limited, for example, particles and porous cylinders (monolith) contained in the column tube in a liquid-tight manner can be enumerated. In addition, the inner wall of a capillary can also be enumerated as a support. When the separation coefficient is sufficiently large, a diaphragm-type support can be used to develop in the thickness direction, or it can be developed in the surface direction as a thin layer chromatography.

[0167] In this embodiment, from the viewpoint of improving separation performance, the support is preferably a porous body, and more preferably a support having a BET specific surface area of ​​100 m 2 / g above 600m 2 In addition, in terms of the improvement of separation performance, the pore diameter measured by mercury intrusion porosimetry is preferably

[0168] In addition, in the present embodiment, the carrier is preferably silica gel. This is because the aforementioned characteristics of silica gel, i.e., excellent separation performance, and in addition, hard and strong. As silica gel, in addition to fully porous silica gel, so-called core-shell silica gel can also be used, and silica gel with chemical modification on the surface can also be used.

[0169] 4. Chromatography

[0170] The chromatography method used in the present embodiment is not particularly limited, and examples thereof include liquid chromatography and supercritical fluid chromatography. The chromatography method can be implemented using commercially available chromatographs, such as liquid chromatographs and supercritical fluid chromatographs. Various conditions such as column equilibration conditions and flow rates can be appropriately selected according to column size, sample volume, and the type of mobile phase.

[0171] In addition, in the present embodiment, when liquid chromatography is used as the chromatography method, liquid chromatography-mass spectrometry (LC-MS) which is a combination of liquid chromatography and mass spectrometry (MS) can be used to continuously perform amine separation and mass spectrometry analysis of the separated amines. The mass spectrometry analysis of the amines can be a quantitative analysis of the separated amines, or a qualitative analysis of the separated amines.

[0172] As mass spectrometry, the known mass spectrometry used in LC-MS can be adopted. For example, the ionization method in mass spectrometry can be suitably selected from atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electrospray ionization (ESI), fast atom bombardment (FAB) and thermal spray ionization (TSP) etc. according to the kind of salt, the kind of acid, the kind of amine and the purpose of analysis etc. In addition, mass spectrometer detector can be suitably selected from quadrupole type mass spectrometer (Q-MS), ion trap type mass spectrometer (IT-MS) and time-of-flight type mass spectrometer (TOF-MS) etc. according to the required sensitivity and resolution etc. and use.

[0173] Example

[0174] Hereinafter, the present disclosure will be described in more detail using examples, but the present disclosure is not limited to the following examples unless it departs from the gist of the present disclosure.

[0175] 〔Preparation of mobile phase〕

[0176] The following is a method for preparing a mobile phase C, which contains oxalic acid at a concentration of 7.5 mM, triethylamine oxalate (2Et3N·(COOH)2) at a concentration of 2.5 mM, and a water / acetonitrile (5 / 95 (v / v)) mixed solvent as a solvent. Other mobile phases were prepared by the method for preparing mobile phase C or a method based thereon in such a manner that salts, acids, and solvents are as shown in Table 2.

[0177] Weigh 316.9 mg of oxalic acid dihydrate and move to a 250 mL volumetric flask. Into this volumetric flask, load about 50 mL of water / acetonitrile (5 / 95 (v / v)) mixed solvent, then, into this volumetric flask, load 1336.4 mg of acetonitrile and 127.3 mg of triethylamine. Then, utilize water / acetonitrile (5 / 95 (v / v)) mixed solvent to carry out constant volume, stir until undissolved matter disappears, thus obtain mobile phase C.

[0178] It should be noted that, in the preparation of mobile phase, under the situation of using the aqueous solution of acid, first, weigh the prescribed amount of the concentration calculation recorded in the warranty of the manufacturer of the aqueous solution of acid. Next, for this aqueous solution of acid, acetonitrile is added in the aqueous solution of acid, obtain mixed solution. At this moment, acetonitrile has added such amount that the water / acetonitrile volume ratio in the mixed solution is identical with the water / acetonitrile volume ratio as the solvent of target. Then, in this mixed solution, add the water / acetonitrile mixed solvent that mixes with aforementioned volume ratio, carried out constant volume.

[0179] In addition, the specific gravity of water is 1.00 g / mL, and the specific gravity of acetonitrile is 0.783 g / mL.

[0180] 〔Separation of amines〕

[0181] A column filled with a chiral stationary phase obtained by supporting a ligand having a crown ether-like cyclic structure represented by formula (IV) on silica gel via a chemical bond ("CROWNPAK CR-I (-)" manufactured by Daicel Co., Ltd., inner diameter 3 mm, length 150 mm) was used as a column, and a high performance liquid chromatograph ("LC-20AD" manufactured by Shimadzu Corporation) was used as a liquid chromatograph to separate the amines. At this time, the amine to be separated was dissolved in a water / acetonitrile (1 / 1 (v / v)) mixed solvent in a manner to a concentration of about 0.1% w / v, and 2 μL of the obtained solution was injected into the column using an automatic sampler. In addition, the mobile phase was sent to the column at a temperature adjusted to 30°C at 0.43 mL / min.

[0182] A photodiode array detector ("SPD-M20A" manufactured by Shimadzu Corporation, detection wavelength: 254 nm) was used as a detector, and data acquired by the detector were analyzed using data analysis software ("LCsolution" manufactured by Shimadzu Corporation).

[0183] [Chemical formula 2]

[0184]

[0185] [Examples 1 to 9 and Comparative Examples 1 to 8]

[0186] Using the mobile phase shown in Table 2, chiral separation of dl-1-phenylethylamine was performed according to the above-mentioned [Separation of Amines]. The results are shown in Figures 1 to 17 .

[0187] [Examples 10 to 12 and Comparative Examples 9 to 10]

[0188] Using the mobile phase shown in Table 2, chiral separation of dl-tryptophan was performed according to the above-mentioned [Separation of Amines]. The results are shown in Figures 18 to 22 .

[0189] [Example 13]

[0190] Chiral separation of dl-tryptophan was performed in the same manner as in Example 1 except that the mobile phase shown in Table 2 was used and the following LC-MS apparatus was used in the above-mentioned [Separation of Amines] and the column temperature was set to 20°C. The results are shown in Fig.23 .

[0191] LC-MS apparatus

[0192] Liquid Chromatograph

[0193] Device: "waters ACQUITY UPLC H-Clas PLUS" manufactured by Waters Corporation

[0194] Mass spectrometer

[0195] Device: "waters ACQUITY QDa" manufactured by Waters Corporation

[0196] Ionization method: ESI

[0197] Measurement mode: Positive

[0198] Detection MS: m / z 205

[0199] Probe temperature: 400°C

[0200] Source temperature: 120℃

[0201] When comparing the difference in retention time between the peaks of d-1-phenylethylamine and l-1-phenylethylamine between Example 1 using a mobile phase containing nitric acid and triethylamine nitrate and Comparative Example 1 using a mobile phase containing nitric acid but not containing the aforementioned salts, the difference in retention time between the peaks of d-1-phenylethylamine and l-1-phenylethylamine was less than 2 minutes in Comparative Example 1, whereas it was approximately 3.5 minutes in Example 1.

[0202] When comparing the difference in retention time between the two peaks in Example 2 using a mobile phase containing trifluoroacetic acid and triethylamine trifluoroacetate, and Comparative Examples 2 and 3 using a mobile phase containing trifluoroacetic acid but not containing a salt, the difference in retention time between the two peaks was about 1 minute in Comparative Examples 2 and 3, whereas it was about 3 minutes in Example 2. In Comparative Example 3, even though the concentration of trifluoroacetic acid was 10 times that of Example 2 and Comparative Example 2, the separation performance was hardly improved.

[0203] In Example 3, which used a mobile phase containing oxalic acid and triethylamine hydrogen oxalate, the difference in retention time between the two peaks was about 7 minutes, showing very high separation performance. In contrast, in Comparative Example 4, which used a mobile phase containing oxalic acid but not containing the aforementioned salt, dl-1-phenylethylamine could not be separated into each enantiomer. It is also known that even in Examples 4 and 5, where the types of salts in the mobile phase are different from those in Example 3, the two peaks are fully separated, and the amine can be separated efficiently.

[0204] For the mobile phase containing oxalic acid, even when the separation object was changed to dl-tryptophan, the same tendency as above was observed. Specifically, in Comparative Example 9 using a mobile phase containing no cations and no salts of acid anions, the difference in retention time between the two peaks was as small as about 0.7 minutes, whereas in Examples 10 to 12 using a mobile phase containing dimethylamine or hydrogen oxalate of ammonia, the difference in retention time between the two peaks was about 1.5 minutes to about 12 minutes.

[0205] In addition, when Example 3 using a mobile phase containing a water / acetonitrile (5 / 95 (v / v)) mixed solvent and Comparative Example 8 containing a water / acetonitrile (95 / 5 (v / v)) mixed solvent were compared, in Comparative Example 8, dl-1-phenylethylamine could not be separated into each enantiomer, while in Example 3, a very high separation performance was shown. In Comparative Example 10 using the same mobile phase as Comparative Example 8, dl-tryptophan could not be separated into each enantiomer. From these results, it can be seen that the effect of improving the separation performance of amines by containing a salt of a cation and an acid anion in the mobile phase is significantly exerted in a mobile phase with a low water content.

[0206] When comparing the difference in retention time between the two peaks in Example 6 using a mobile phase comprising dihydrogen phosphate of phosphoric acid and triethylamine, and Comparative Example 5 using a mobile phase comprising phosphoric acid but not containing the aforementioned salt, the retention time difference in Example 6 was approximately 4.5 minutes, whereas it was approximately 1 minute in Comparative Example 5.

[0207] In Examples 7 and 8, in which a mobile phase containing formic acid and a formate salt of triethylamine was used, the difference in retention time between the two peaks was about 16 minutes and about 5 minutes, respectively, showing very high separation performance. In contrast, in Comparative Example 6, in which a mobile phase containing formic acid but not containing the formate salt was used, dl-1-phenylethylamine could not be separated into each enantiomer.

[0208] When comparing the difference in retention time between the two peaks in Example 9 using a mobile phase containing methanesulfonic acid and a methanesulfonate of triethylamine, and Comparative Example 7 using a mobile phase containing methanesulfonic acid but not containing the aforementioned salt, the retention time difference in Example 9 was approximately 2 minutes, whereas it was approximately 1.5 minutes in Comparative Example 7.

[0209] In addition, the results of Example 13 show that even when chromatography is performed by LC-MS, there are few unnecessary peaks in the liquid chromatogram and little noise in detection by a mass spectrometer. These results show that the present separation method can also be applied to LC-MS.

[0210] [Table 2]

[0211]

[0212] [Examples 14 to 16]

[0213] Using a mobile phase containing oxalic acid and triethylamine hydrogen oxalate, chiral separation of Phe-Gly-NH2 (Example 14), dl-tryptophan (Example 15), and dl-1-phenylethylamine (Example 16) was performed according to the above-mentioned [Separation of Amines]. The retention time of the two peaks plotted against the concentration of oxalic acid and triethylamine hydrogen oxalate in the mobile phase is shown in the graph. Fig.24 .

[0214] From the results of Examples 14 to 16, it is understood that when the value calculated from the formula (1) in the mobile phase is within the above-mentioned specific range, the separation performance of amines is improved.

[0215] Industrial Applicability

[0216] The amine separation method according to at least some embodiments of the present disclosure can use a mobile phase with a low water content and achieve high amine separation performance. It is also believed that the mobile phase can be applied to LC-MS by appropriately selecting components contained in the mobile phase from components that can avoid or reduce adverse effects on mass spectrometer-based detection.

[0217] Therefore, the method for separating amines can be widely applied to analysis and purification by various liquid chromatography methods, and is expected to be developed in the fields of organic chemistry, medicine, and pharmacy.

Claims

1. A method for separating amines, comprising the following separation step: separating the amines by chromatography using a stationary phase formed by supporting a ligand having a crown ether-like cyclic structure on a carrier, and a mobile phase containing a salt of a cation and an acid anion at a concentration of 0.2 mM to 50.0 mM and containing a solvent having a water content of 50% by volume or less.

2. The method for separating amines according to claim 1, wherein: The mobile phase contains an acid at a concentration of greater than 0 mM and 50.0 mM or less.

3. The method for separating amines according to claim 1 or 2, wherein: The water content in the solvent is 20% by volume or less.

4. The method for separating amines according to any one of claims 1 to 3, wherein The liquid other than water in the solvent is one or more selected from the group consisting of organic solvents and subcritical or supercritical carbon dioxide.

5. The method for separating amines according to claim 4, wherein: The organic solvent is one or more selected from the group consisting of acetonitrile, acetone, methanol, ethanol, 2-propanol, hexane, tetrahydrofuran, and tert-butyl methyl ether.

6. The method for separating amines according to any one of claims 1 to 3, wherein The solvent contains 70% by volume or more of acetonitrile.

7. The method for separating amines according to any one of claims 1 to 6, wherein The pKa of the acid that is the source of the acid anion in water at 25°C n It is not less than -2.0 and not more than 6.5, and n is the valence number of the acid anion.

8. The method for separating amines according to any one of claims 1 to 6, wherein The acid anion is one or more selected from the group consisting of nitrate ion, trifluoroacetate ion, oxalate ion, hydrogen oxalate ion, dihydrogen phosphate ion, glycolate ion, formate ion, acetate ion, and bicarbonate ion.

9. The method for separating amines according to any one of claims 1 to 6, wherein The acid anion is one or more selected from the group consisting of oxalate ions, formate ions, and acetate ions.

10. The method for separating amines according to any one of claims 1 to 9, wherein The cation is one or more selected from the group consisting of tetrahydroammonium ions, secondary ammonium ions, and tertiary ammonium ions.

11. The method for separating amines according to claim 2, wherein: The value calculated by the following formula (1) in the mobile phase is 0.01 or more and 0.90 or less, [Mathematical formula 1] In formula (1), X is the concentration of the acid in the mobile phase (mM); Y is the concentration of the salt in the mobile phase (mM); q is the number of anions of the acid per molecule of the salt.

12. The method for separating amines according to any one of claims 1 to 11, wherein The chromatography was performed by liquid chromatography-mass spectrometry.

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