WO2023008546A1

WO2023008546A1 - Method for separating amines by liquid chromatography

Info

Publication number: WO2023008546A1
Application number: PCT/JP2022/029231
Authority: WO
Inventors: 徹柴田; 聡新蔵
Original assignee: 株式会社ダイセル
Priority date: 2021-07-29
Filing date: 2022-07-29
Publication date: 2023-02-02

Abstract

This method for separating amines by liquid chromatography employs, as a stationary phase, a separating agent in which a ligand having a crown ether-like ring structure is supported on a carrier, and uses a mobile phase containing at least one acid additive selected from among perfluoroalkanoic acids having a carbon number of 3 to 8 inclusive, perfluoroalkanesulfonic acids having a carbon number of 1 to 3 inclusive, and hydrogen halide.

Description

Method for separating amines by liquid chromatography

The present disclosure relates to a method for separating amines by liquid chromatography.

A separating agent having a crown ether-like cyclic structure is widely used as a stationary phase for liquid chromatography to separate compounds having a primary amino group and similar substances. In particular, a separating agent having a crown ether-like cyclic structure bonded to a chiral structure is known to be useful for separating enantiomers.

Patent Literature 1 and Patent Literature 2 disclose, as an excellent separating agent for optical isomers, a separating agent in which a crown ether-like cyclic structure is bonded to an S- or R-binaphthyl structure. It is believed that these separating agents exert their separating ability in the following manner. That is, a primary ammonium group (—NH ₃ ⁺ ) produced by protonation of a primary amine forms a crown ether-like cyclic structure through a hydrogen bond between a hydrogen atom of the primary ammonium group and an oxygen atom of the crown ether-like cyclic structure. It is thought that separation becomes possible as a result of being subsumed in and retained by the separating agent. Therefore, when using such a stationary phase, it is known that a strongly acidic mobile phase is suitably used in order to obtain high separation performance. For example, Patent Document 2 discloses a mobile phase obtained by mixing an aqueous perchloric acid solution or an aqueous trifluoroacetic acid solution with an organic solvent such as methanol or acetonitrile. Thus, the strongly acidic mobile phase contains a strong acid, and among the strong acids, perchloric acid is known to retain amines best and improve separation performance.

However, perchloric acid is not only a strong acid, but also a strong oxidizing agent. Therefore, if mishandled, it may lead to an explosion or corrode metal parts of a liquid chromatography device. Further, when another strong acid is added to the mobile phase instead of perchloric acid, there is a problem that the retention of the amine by the stationary phase is insufficient and the amine cannot be separated satisfactorily.
On the other hand, in Patent Document 3, by adding a salt of a chaotropic anion and a salt of a hydrophobic organic acid to the mobile phase, it is possible to strengthen the retention of amines in the column, thereby It is disclosed that the use of strong acids and strong oxidizing acids can be avoided.

In recent years, in HPLC (high performance liquid chromatography), LC-MS (liquid chromatography-mass spectrometry) has rapidly spread in order to obtain information on molecular structure with high sensitivity, and the mobile phase for LC-MS is being actively developed. For example, Non-Patent Document 1 discloses that a mobile phase containing trifluoroacetic acid as an acid additive can be applied to LC-MS.

JP-A-2-69472 WO2012/050124 WO2020/251003

However, 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 in many cases satisfactory separation cannot be obtained. In addition, there is a problem that known separation methods including the method described in Patent Document 3 cannot be applied to LC-MS.

In the method for separating amines by liquid chromatography, high amine retention and high separation ability are required, and in order to apply the method to LC-MS, it is necessary to inhibit detection at least in MS. No adverse effects are required. Therefore, the present disclosure is a method for separating amines by liquid chromatography using, as a stationary phase, a separating agent in which a ligand having a crown ether-like cyclic structure is supported on a carrier, which enables good retention and separation of amines. It is an object of the present invention to provide a method that is present and does not adversely affect detection by MS.

As a result of intensive studies to solve the above problems, the present inventors have found that in liquid chromatography using a separating agent having a crown ether-like cyclic structure, a specific highly volatile acid is added to the mobile phase as an acid additive. It was found that the above problem can be solved by adding That is, the gist of the present disclosure is as follows.

[1]
Using as a stationary phase a separating agent in which a ligand having a crown ether-like cyclic structure is supported on a carrier,
Liquid chromatography using a mobile phase containing one or more acid additives selected from perfluoroalkanoic acids having 3 to 8 carbon atoms, perfluoroalkanesulfonic acids having 1 to 3 carbon atoms, and hydrogen halides Graphical separation of amines.
[2]
the acid additive is the perfluoroalkanoic acid having 3 to 8 carbon atoms,
The solvent contained in the mobile phase is a mixed solvent of water and an organic solvent,
The separation method according to [1], wherein in the mixed solvent, the ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is 50% or more.
[3]
The acid additive is the perfluoroalkanesulfonic acid having 1 to 3 carbon atoms,
The solvent contained in the mobile phase is a mixed solvent of water and an organic solvent,
The separation method according to [1], wherein in the mixed solvent, the ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is 50% or more.
[4]
the acid additive is the hydrogen halide,
The solvent contained in the mobile phase is a mixed solvent of water and an organic solvent,
The separation method according to [1], wherein the mixed solvent has a ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is 50% or less.
[5]
A method for the analysis of amines by liquid chromatography-mass spectrometry, comprising:
A method for analyzing amines, comprising a separation step of separating amines by the separation method according to any one of [1] to [4], and a mass spectrometry step of analyzing the amines separated in the separation step by mass spectrometry.

According to the present disclosure, a method for separating amines by liquid chromatography using, as a stationary phase, a separating agent in which a ligand having a crown ether-like cyclic structure is supported on a carrier, which enables good retention and separation of amines. It is possible to provide a method that is present and does not adversely affect detection by MS.

4 is a liquid chromatogram of dl-tryptophan in Comparative Example 1. FIG. 1 is a liquid chromatogram of dl-tryptophan in Example 1. FIG. 2 is a liquid chromatogram of dl-tryptophan in Example 2. FIG. 2 is a liquid chromatogram of dl-tryptophan in Example 3. FIG. 4 is a liquid chromatogram of dl-tryptophan in Example 4. FIG. 2 is a liquid chromatogram of dl-tryptophan in Example 5. FIG. 4 is a liquid chromatogram of dl-1-phenylethylamine in Comparative Example 2. FIG. 2 is a liquid chromatogram of dl-1-phenylethylamine in Example 6. FIG. 2 is a liquid chromatogram of dl-1-phenylethylamine in Example 7. FIG. 2 is a liquid chromatogram of dl-1-phenylethylamine in Example 8. FIG. 4 is a liquid chromatogram of dl-tyrosine in Comparative Example 3. FIG. FIG. 10 is a liquid chromatogram of dl-tyrosine in Example 9. FIG.

Specific embodiments of the present disclosure will be described below, but each configuration and combination thereof in each embodiment are examples, and can be configured as appropriate within the scope of the present disclosure. can be added, omitted, substituted, and otherwise modified. This disclosure is not limited by the embodiments, but only by the scope of the claims.
Also, each aspect disclosed in this specification may be combined with any other feature disclosed in this specification.

<1. Amine Separation Method>
One embodiment of the present disclosure is a method for separating amines by liquid chromatography, comprising a separating agent in which a ligand having a crown ether-like cyclic structure is supported on a carrier as a stationary phase (hereinafter referred to as "having a crown ether-like cyclic structure A solution containing a highly volatile specific acid is used as the mobile phase. In the separation method according to the present embodiment, the amine separation means may be liquid chromatography using the above-described stationary phase and mobile phase, and may appropriately include other configurations such as identification and quantification of the separated amine. good too.
Liquid chromatography can be performed using a commercially available liquid chromatography device. Column equilibration, flow rate and the like can be appropriately selected depending on the column size, sample capacity and the like.

<2. Amine>
In the separation method according to this embodiment, a separating agent in which a ligand having a crown ether-like ring structure is supported on a carrier is used as a stationary phase to separate amines. According to the separation method according to this 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. Among the former, the separation method according to the present embodiment is particularly effective in separating a mixture of amines having similar structures to each amine.

The amine is not particularly limited, and may be appropriately selected from primary amines, secondary amines, and tertiary amines. Specific amines include amino acids such as alanine, cysteine, glutamic acid, methionine, leucine, tyrosine and tryptophan; derivatives such as esters of the above amino acids; amino alcohols such as dimethylaminoethanol, propanolamine, methioninol and norephedrine; phenylethylamine. , aniline, methylaniline, chloroaniline, amino group-containing hydrocarbons such as aminobenzoic acid;

The separation method according to this embodiment is particularly suitable for separating a desired primary amine from a mixture containing primary amines. This is because the primary amine can be well retained and separated by the crown ether-like cyclic structure of the stationary phase.
Moreover, according to the separation method according to the present embodiment, since high separation performance is exhibited, a mixture of amines, which are difficult to separate due to their similar structures, can be separated into individual amines. Mixtures of amines having similar structures include mixtures of chain isomers, mixtures of positional isomers, mixtures of geometric isomers, mixtures of analogues, and the like. Furthermore, when the crown ether-like cyclic structure is an optically active substance, it is effective for separating a mixture of enantiomers into individual enantiomers. The separation method according to this embodiment is particularly useful in that a mixture of enantiomers of a compound containing an amino group can be separated by using a ligand having an optically active crown ether-like cyclic structure.

<3. Mobile Phase>
In this embodiment, the mobile phase used for liquid chromatography contains a highly volatile specific acid as an acid additive. Specific acids with high volatility include perfluoroalkanoic acids having 3 to 8 carbon atoms (hereinafter sometimes simply referred to as "perfluoroalkanoic acid"), perfluoroalkanesulfonic acids having 1 to 3 carbon atoms (hereinafter sometimes simply referred to as "perfluoroalkanesulfonic acid"), and hydrogen halide. These acid additives can promote the retention of amines on the stationary phase in LC, and have high volatility that does not interfere with the detection of amines in MS, so they are suitable for LC-MS. One type of acid additive may be used alone, or two or more types may be used in any combination and ratio.

(3-1. Perfluoroalkanoic acid)
The perfluoroalkanoic acid in the present embodiment is an acid represented by the following formula (A) (in formula (A), m is an integer of 2 or more and 7 or less), specifically pentafluoropropion acid, heptafluorobutyric acid, nonafluoropentanoic acid, undecafluorohexanoic acid, tridecafluoroheptanoic acid, and pentadecafluorooctanoic acid.
_CmF2m ₊ _1CO2H (A)

Trifluoroacetic acid in which m in formula (A) is replaced by 1 is often used in liquid chromatography using a stationary phase having a crown ether-like cyclic structure, and is also applied to LC-MS (for example, See Patent Document 1). However, as described above, trifluoroacetic acid has a weak ability to retain a sample, so there were cases where sufficient separation was not achieved depending on the type of amine.

On the other hand, the perfluoroalkanoic acid in this embodiment is a compound represented by formula (A), where m is 2 or more and 7 or less. By including such a perfluoroalkanoic acid in the mobile phase, high retention performance and high volatility are exhibited in amine separation by liquid chromatography using a stationary phase having a crown ether-like cyclic structure. The reason is explained below.

All of the perfluoroalkanoic acids in this embodiment are strong acids. However, the present inventors have found that as the number of carbon atoms in perfluoroalkanoic acid, that is, the number of fluorine atoms, increases, the ability to retain amines on a stationary phase having a crown ether-like cyclic structure increases. Surprisingly, such an effect is completely different from the effect expected from the mere fact that perfluoroalkanoic acid is a strong acid, and is dramatically improved as the number of fluorine atoms in the molecule increases. More specifically, the product of the molar concentration of the acid additive in the mobile phase and the number of fluorine atoms in one molecule thereof, that is, the number of carbon atoms is 3 or more and 8 or less so that the molar concentration of fluorine atoms in the mobile phase is the same. of perfluoroalkanoic acid and trifluoroacetic acid, a conventional acid additive, compared relative retention, which is an indicator of the ability of the stationary phase to retain amines. phase is significantly higher. For example, when the relative retention of the more strongly retained enantiomer such as dl-tryptophan is calculated from the column empty volume of 1.6 mL, it is 0.59 in Comparative Example 1 (20 mM trifluoroacetic acid) described later, but 3 (5 mM tridecafluoroheptanoic acid) is 1.76. Thus, the relative retention of the tryptophan enantiomer in the latter mobile phase was about three times higher than in the former, even though the molar concentration of fluorine atoms in both mobile phases was almost the same.

Incidentally, the relative retention is calculated by the following formula.
k=(V/V ₀ )−1
k: relative retention V: elution volume of amine V ₀ : column empty volume (elution volume of substances not retained)

Therefore, the lower limit of m in formula (A) is usually 2 or more, preferably 3 or more, more preferably 4 or more, from the viewpoint of improving retention performance. The upper limit of m in formula (A) is usually 7 or less, preferably 6 or less, from the viewpoint of ensuring volatility. Preferred ranges of m in formula (A) include, for example, 2 to 6, 3 to 7, and 4 to 6.

As the perfluoroalkanoic acid, a commercially available product may be used, or one manufactured according to a known method may be used. The perfluoroalkanoic acid is preferably selected from pentafluoropropionic acid, nonafluoropentanoic acid, and tridecafluoroheptanoic acid in comprehensive consideration of volatility, retention performance, and separation performance.

In addition, the perfluoroalkanoic acid in this embodiment is commercially available as an ion-pair reagent, and is known to be applicable to LC-MS using a stationary phase that does not have a crown ether-like cyclic structure (for example, Tokyo Chemical Industry Co., Ltd.工業株式会社、“イオン対試薬 for HPLC”、[online]、2019年9月13日、インターネット＜URL: https://www.tcichemicals.com/medias/Brochure-A1084-J.pdf?context=bWFzdGVyfGJyb2NodXJlLXBkZnN8NTIxNTc1fGFwcGxpY2F0aW9uL3BkZnxoZjMvaDM4LzkxMjY2OTE5OTU2NzgvQnJvY2h1cmVfQTEwODRfSi5wZGZ8Zjg3MDFmMzM4MGQwMDVkZWE3NWY4MDVjNGNiYzliZWI1M2YxYTJkYjVhNmYyZmQyMmQ2ODExYzNkNmJhNDA0OQ >). On the other hand, it has not been investigated whether the perfluoroalkanoic acid in this embodiment is applicable to LC-MS using a stationary phase having a crown ether-like ring structure.

It is presumed that the hydrophobicity of perfluoroalkyl groups is involved in the mechanism by which perfluoroalkanoic acid helps retain amines. More specifically, since the liquid chromatography in this embodiment is ion pair chromatography, in order for the amine to be retained on the stationary phase, the amine is protonated to an ammonium group, which forms an ion pair with the anion. It is presumed that the charge needs to be neutralized by Since the perfluoroalkanoic acid ion generated from perfluoroalkanesulfonic acid is essentially hydrophobic, it is likely to form an ion pair with an ammonium group through hydrophobic interaction in a solvent containing a large amount of water. Conceivable. Therefore, when perfluoroalkanoic acid is used as the acid additive, the solvent contained in the mobile phase is preferably a solvent mainly composed of water from the viewpoint of exhibiting high retention performance.

(3-2. Perfluoroalkanesulfonic acid)
Perfluoroalkanesulfonic acid in the present embodiment is an acid represented by the following formula (B) (in formula (B), p is an integer of 1 or more and 3 or less), specifically trifluoromethane It is selected from sulfonic acid, pentafluoroethanesulfonic acid, and heptafluoropropanesulfonic acid.
_CpF2p _+1SO3H ₍ B)

The sulfonic acid group has significantly higher retention performance than the carboxyl group. However, perfluoroalkanesulfonic acids have a lower vapor pressure, ie lower volatility, than perfluoroalkanoic acids having the same perfluoroalkyl group. Therefore, p in formula (B) is desirably not as large as m in formula (A), and is usually 3 or less, preferably 2 or less (that is, 1 or 2), more preferably 1.

As the perfluoroalkanesulfonic acid, a commercially available product may be used, or one manufactured according to a known method may be used. Perfluoroalkanesulfonic acid is preferably trifluoromethanesulfonic acid from the viewpoint of availability.

The mechanism by which perfluoroalkanesulfonic acid helps amine retention is presumed to involve the hydrophobicity of perfluoroalkyl groups, similar to the mechanism by which perfluoroalkanoic acid helps amine retention. Therefore, when perfluoroalkanesulfonic acid is used as the acid additive, the solvent contained in the mobile phase is preferably a solvent mainly composed of water from the viewpoint of exhibiting high retention performance.

(3-3. Hydrogen halide)
Hydrogen halides have strong acidity and high volatility and are effective for good retention and separation of amines. The hydrogen halide is not particularly limited, and examples thereof include hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide and the like. Among these, the hydrogen halide is preferably hydrogen chloride because of its low metal corrosiveness and/or toxicity.
Hydrogen halides exhibit higher retention performance in mobile phases containing more organic solvents, such as acetonitrile, than in mobile phases mainly composed of water. It is speculated that this is because hydrogen halides, unlike perfluoroalkanoic acids and perfluoroalkanesulfonic acids, produce halide ions that undergo strong hydration in the mobile phase. Halide ions are strongly hydrated in solvents with a high water content and are less likely to form ion pairs with ammonium groups, whereas they are weakly hydrated in solvents with a high organic solvent content. Therefore, it is thought that ion pairs with ammonium groups are likely to be formed. Therefore, when hydrogen halide is used as the acid additive, the solvent contained in the mobile phase is preferably a solvent mainly composed of an organic solvent.

The concentration of the acid additive in the mobile phase (the total concentration of each acid additive when two or more acid additives are used) is not particularly limited depending on the type of amine, and is usually 1 mM or more, preferably 2 mM or more. , more preferably 3 mM or more, and usually 200 mM or less, preferably 100 mM or less, more preferably 30 mM or less, and still more preferably 25 mM or less. Accordingly, preferred ranges for the concentration of the acid additive include, for example, 1 mM to 100 mM, 2 mM to 200 mM, 3 mM to 30 mM, and 3 mM to 25 mM. The relative retention of amines generally increases with the acid concentration, but there are cases where the effect does not appear at concentrations above a certain level. In addition, even within the above concentration range of the acid additive, contamination may occur due to the acid additive remaining in the stationary phase when chromatography is performed later by changing the mobile phase, and volatilization of the acid additive in the MS detector It is also preferable to select a lower acid additive concentration from the viewpoint of avoiding or reducing problems such as imperfections.

When the mobile phase containing these acid additives is actually applied to LC-MS, the method of ionization, the material and structure of the liquid chromatography device, and the subsequent separation due to the adsorption residue of the acid additive on the stationary phase It is desirable to select the type of acid additive, the concentration of the acid additive, etc. in consideration of the effects, the characteristics of each device, the purpose of use (for example, determination of separation accuracy, preliminary separation, protocol construction, etc.).

(3-4. Solvent)
The mobile phase in this embodiment is one in which an acid additive is dissolved in a solvent. The solvent is not particularly limited, and examples thereof include organic solvents, water, and mixed solvents thereof.

Organic solvents include, but are not limited to, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, or acetonitrile, which have high chemical stability, low viscosity, and allow detection of amines by ultraviolet absorption. is preferred, methanol or acetonitrile is more preferred, and acetonitrile is even more preferred.

When using a mixed solvent of water and an organic solvent as the solvent, the mixing ratio of water and the organic solvent is not particularly limited. However, as mentioned above, depending on the type of acid additive, using a mixed solvent with a high water content will exhibit high retention performance, or conversely, using a mixed solvent with a low water content will result in high retention. It may exhibit performance. In addition, even when a mixed solvent with a high water content is used, if the water content is too high, peak shape problems such as broadening of the peak width occur even if the retention performance is high. Sometimes. Therefore, in a mixed solvent with a high water content, the ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is preferably 95% or less.

Specifically, when using one or more selected from perfluoroalkanoic acid and perfluoroalkanesulfonic acid as an acid additive, before mixing with respect to the total volume of water before mixing and volume of organic solvent before mixing is preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, particularly preferably 80% or more, and preferably 95% or less. Preferred ranges of the ratio include, for example, 50% to 95%, 60% to 95%, 70% to 95%, and 80% to 95%.
Further, when hydrogen halide is used as the acid additive, the ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is usually more than 0%, preferably 5%. In addition, it is preferably 50% or less, more preferably 40% or less, still more preferably 30% or less, and particularly preferably 20% or less. Moreover, preferable ranges of the ratio include, for example, 0% to 50%, 5% to 40%, 0% to 30%, and 5% to 20%.
In addition to the type of acid additive described above, it is desirable to appropriately select the concentration of the acid additive and adjust the composition of the mobile phase according to the intended separation.

(3-2. Other components)
The mobile phase in this embodiment contains an acid additive and a solvent, preferably a mixture of the acid additive and solvent, but may contain other components as long as they do not interfere with the separation of the amine and detection by MS. you can stay Other ingredients include substances that can promote ionization, such as volatile pH adjusting reagents.

(3-5. Preparation of mobile phase)
A mobile phase can be prepared by mixing an acid additive, a solvent, and other components as necessary, and the order of mixing is not particularly limited. When a mixed solvent of water and an organic solvent is used as a solvent, it is preferable to prepare a mobile phase by mixing an aqueous solution of an acid additive, an organic solvent, and, if necessary, water.

<4. Stationary phase>
The stationary phase in this embodiment is a separating agent in which a ligand having a crown ether-like cyclic structure is supported on a carrier.

(4-1. Ligands)
As used herein, the term "ligand" means a compound that is carried on a carrier and that exhibits physical affinity and, if necessary, asymmetric recognition ability for a separation target. The ligand in this embodiment has a crown ether-like cyclic structure. That is, the ligand is a compound in which a crown ether skeleton represented by formula (I) is chemically bonded to an aliphatic, alicyclic or aromatic hydrocarbon to form a macrocyclic polyether structure. be.

—O(CH ₂ CH ₂ O) _n — (I)
In the formula, n can be appropriately selected from an integer of 4 to 6 depending on the hydrocarbon to which the amino group of the amine and the crown ether skeleton are bonded. For example, the ligand represented by formula (II) or (III), which will be described later, has n=5 and has a crown ether-like cyclic structure with a size suitable for encapsulating a primary ammonium group. is suitably used for the separation of
Further, the hydrogen atoms of the ethylene groups in the repeating units may be substituted with various functional groups, but are preferably unsubstituted.

When enantiomers are separated in this embodiment, a compound in which a crown ether-like ring structure is bound to a homochiral structure is used as the ligand. Examples of such ligands include, for example, ligands represented by formula (II) described in JP-A-2-69472 and WO 2012/050124, and formula (III) described in JP-A-2014-169259. ) and the like. Further, the phenyl groups at the 3-position and 3'-position of the 1,1'-binaphthyl structure in formula (II) are halogen atoms such as bromine atoms; alkyl groups such as methyl groups; substituted aromatic groups; heterocyclic groups; etc. (Peng Wu, et.al., Chin. J. Chem., 2017, 35, 1037-1042) can also be employed.

The ligand is used as a separating agent while being carried on a carrier. A known mode of carrying can be adopted, and for example, a mode in which the ligand is carried on the carrier by chemical bonding such as covalent bonding can be preferably adopted. A specific supporting method includes a method of introducing a reactive group into a ligand, a starting material of the ligand, or an intermediate of the ligand, and reacting this substituent with a reactive group present on the surface of the carrier. The reactive group present on the surface of the carrier may be a group present on the surface of an untreated carrier. It may be a group introduced onto the carrier surface by surface treatment with a silane coupling agent such as methoxysilane. In addition to the method of forming a bond between a ligand and a carrier, an insolubilized layer can also be formed on the carrier surface by forming a so-called cross-linked bond between atomic groups containing a ligand.

(4-2. Carrier)
The carrier is not particularly limited as long as the ligand can be immobilized by chemical bonding such as covalent bonding. Such a carrier may be an inorganic carrier or an organic carrier, but is preferably an inorganic carrier. Examples of inorganic carriers include silica gel, alumina, magnesia, glass, kaolin, titanium oxide, silicates, and hydroxyapatite. Examples of organic carriers include polystyrene, polyacrylamide, polyacrylate, polysaccharide and the like. These organic carriers are preferably insolubilized by cross-linking with a cross-linking agent.

The shape of the carrier is not particularly limited, and examples thereof include particles and porous cylindrical bodies (monoliths) liquid-tightly accommodated in column tubes. The carrier can also include the inner wall of a capillary.

In this embodiment, the carrier is preferably a porous body, more preferably a porous body having a BET specific surface area of 100 to 600 m ² /g, from the viewpoint of improving separation performance. Moreover, the porous body preferably has a pore diameter of 60 to 300 Å as measured by a mercury porosimetry method from the viewpoint of improving separation performance.
Moreover, in this embodiment, the carrier is preferably silica gel. This is because silica gel has the above-mentioned characteristics, that is, excellent separation ability, and is also hard and durable. As silica gel, in addition to fully porous silica gel, so-called core-shell type silica gel may be used.

<5. Amine Analysis Method>
Various analyzes such as quantitative analysis and qualitative analysis of amines can be performed by applying the method for separating amines according to this embodiment to LC-MS. The method for analyzing amines by LC-MS includes a separation step of separating amines by the method for separating amines according to this embodiment, and a mass spectrometry step of analyzing the amines separated in the separation step by mass spectrometry.

A known mass spectrometry method used in LC-MS can be adopted as the mass spectrometry of amines in the mass spectrometry step. For example, ionization in mass spectrometry includes atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electrospray method (ESI), fast atom bombardment method (FAB), thermospray method (TSP), amine can be appropriately selected according to the type of analysis, the purpose of analysis, and the like. Also, as a mass spectrometer, quadrupole mass spectrometer (Q-MS), ion trap mass spectrometer (IT-MS), time-of-flight mass spectrometer (TOF-MS), etc. It can be appropriately selected and used according to sensitivity, resolution, and the like.

Hereinafter, the present disclosure will be described more specifically by way of examples, but the present disclosure is not limited to the following examples as long as it does not deviate from the gist thereof.

[Preparation of mobile phase]
Taking as an example the preparation of a mobile phase containing hydrogen chloride at a concentration of 20 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as a solvent, the procedure for mobile phase preparation is shown.
First, 700.0 g of water and 383.1 g of acetonitrile were weighed and mixed to obtain a mixed solvent of water/acetonitrile=70/30 (v/v). Next, 5.00 mL of a commercially available 1.00 N hydrogen chloride aqueous solution was weighed out with a whole pipette and added to a 250 mL volumetric flask. After adding 1.65 g of acetonitrile (equivalent to 3/7 volume of water in the aqueous hydrogen chloride solution), a previously prepared water/acetonitrile = 70/30 (v/v) mixed solvent was used to The mobile phase was prepared by adding up.

Hereinafter, the mobile phases of each example and comparative example were prepared in the same manner. However, when trifluoromethanesulfonic acid or tridecafluoroheptanoic acid is used as an acid additive, these are not in a form containing a solvent such as an aqueous solution, and are commercially available by themselves. A mobile phase was prepared by adding to the flask and making up with the mixed solvent.

[Separation of amines by liquid chromatography]
The following comparative examples and examples relate to separation of various amines by liquid chromatography. The operation of separation analysis is as follows.
A column packed with a separating agent in which a ligand having a crown ether-like cyclic structure represented by formula (IV) is supported on silica gel ("CROWNPAK CR-I (-)" manufactured by Daicel Corporation, inner diameter 3 mm, length 150 mm), and a high-performance liquid chromatography device ("LC-20AD" manufactured by Shimadzu Corporation) was used as the liquid chromatography device. In addition, a photodiode array detector (manufactured by Shimadzu Corporation "SPD-M20A", detection wavelength 254 nm) was used as a detector, and the data obtained by the detector was analyzed by data analysis software (manufactured by Shimadzu Corporation "LCsolution"). The mobile phase was as described in each comparative example and example, and was sent to the column adjusted to 30°C at 0.43 mL/min. Each of the amines to be separated was dissolved in a mixed solvent of water/acetonitrile (1:1 (v/v)) to a concentration of about 0.1% w/v, and 2 μL of the resulting solution was sampled by an autosampler. injected onto the column.

<Comparative Example 1: Chiral separation of dl-tryptophan>
Chiral separation of dl-tryptophan was performed using a mobile phase containing trifluoroacetic acid (TFA) at a concentration of 20 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as solvent. The chromatogram obtained is shown in FIG. In FIG. 1, two peaks attributed to enantiomers were observed, but the separation was insufficient.

<Example 1: Chiral separation of dl-tryptophan>
Chiral separation of dl-tryptophan was performed using a mobile phase containing pentafluoropropionic acid at a concentration of 20 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 2, it can be seen that in Example 1, retention performance and separation performance are significantly higher than in Comparative Example 1 using TFA as an acid additive.

<Example 2: Chiral separation of dl-tryptophan>
Chiral separation of dl-tryptophan was performed using a mobile phase containing nonafluoropentanoic acid at a concentration of 20 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 3, it can be seen that in Example 2, the retention and separation performance are much higher than in Comparative Example 1 using TFA as an acid additive, and even higher than in Example 1.

<Example 3: Chiral separation of dl-tryptophan>
Chiral separation of dl-tryptophan was performed using a mobile phase containing tridecafluoroheptanoic acid at a concentration of 5 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 4, it can be seen that in Example 3, retention performance and separation performance are significantly higher than in Comparative Example 1 using TFA as an acid additive.

<Example 4: Chiral separation of dl-tryptophan>
Chiral separation of dl-tryptophan was performed using a mobile phase containing tridecafluoroheptanoic acid at a concentration of 20 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 5, it can be seen that in Example 4, retention performance and separation performance are significantly higher than in Comparative Example 1 using TFA as an acid additive.

<Example 5: Chiral separation of dl-tryptophan>
Chiral separation of dl-tryptophan was performed using a mobile phase containing trifluoromethanesulfonic acid at a concentration of 20 mM and a mixed solvent of water/acetonitrile (70/30 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 6, it can be seen that in Example 5, although the peaks of impurities overlap, the retention performance and resolution are slightly higher than those in Example 1.

<Comparative Example 2: Chiral separation of dl-1-phenylethylamine>
Chiral separation of dl-1-phenylethylamine was performed using a mobile phase containing trifluoroacetic acid at a concentration of 21 mM and water/acetonitrile (20/80 (v/v)) mixed solvent as solvent. The chromatogram obtained is shown in FIG. From FIG. 7, in Comparative Example 2, the amines were seemingly separated completely due to the negative peak, but the separation was actually insufficient.

<Example 6: Chiral separation of dl-1-phenylethylamine>
Chiral separation of dl-1-phenylethylamine was performed using a mobile phase containing hydrogen chloride at a concentration of 20 mM and a mixed solvent of water/acetonitrile (20/80 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 8, it can be seen that in Example 6, retention performance and separation performance are significantly higher than in Comparative Example 2 using TFA as an acid additive.

<Example 7: Chiral separation of dl-1-phenylethylamine>
Chiral separation of dl-1-phenylethylamine was performed using a mobile phase containing nonafluoropentanoic acid at a concentration of 5 mM and a mixed solvent of water/acetonitrile (90/10 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 9, it can be seen that Example 7 exhibits high resolution even at an acid additive concentration of 5 mM. It should be noted that this acid additive concentration (5 mM) is a general value as the ion-pairing reagent concentration in the mobile phase containing the ion-pairing reagent.

<Example 8: Chiral separation of dl-1-phenylethylamine>
Chiral separation of dl-1-phenylethylamine was performed using a mobile phase containing hydrogen chloride at a concentration of 5 mM and a mixed solvent of water/acetonitrile (10/90 (v/v)) as solvent. The chromatogram obtained is shown in FIG. From FIG. 10, it can be seen that in Example 8, the separation ability is extremely high.

<Comparative Example 3: Chiral separation of dl-tyrosine>
Chiral separation of dl-tyrosine was performed using a mobile phase containing trifluoroacetic acid at a concentration of 5 mM and a mixed solvent of water/acetonitrile (10/90 (v/v)) as solvent. The chromatogram obtained is shown in FIG. As can be seen from FIG. 11, in Comparative Example 3, the retention performance is low and the peaks are very close to each other.

<Example 9: Chiral separation of dl-tyrosine>
Chiral separation of dl-tyrosine was performed using a mobile phase containing hydrogen chloride at a concentration of 5 mM and a mixed solvent of water/acetonitrile (10/90 (v/v)) as solvent. The chromatogram obtained is shown in FIG. 12 and 11, it can be seen that Example 9 exhibits much higher resolution than Comparative Example 3, which uses a mobile phase prepared similarly except that trifluoroacetic acid is used as the acid additive.

According to the separation method according to at least some embodiments of the present disclosure, amines can be separated by liquid chromatography and can also be detected by MS. Therefore, such a separation method can be widely used in the fields of organic chemistry, medicine, pharmacy, etc. where analysis, purification, etc. are performed by various liquid chromatography.

Claims

Using as a stationary phase a separating agent in which a ligand having a crown ether-like cyclic structure is supported on a carrier,
Liquid chromatography using a mobile phase containing one or more acid additives selected from perfluoroalkanoic acids having 3 to 8 carbon atoms, perfluoroalkanesulfonic acids having 1 to 3 carbon atoms, and hydrogen halides Graphical separation of amines.
the acid additive is the perfluoroalkanoic acid having 3 to 8 carbon atoms,
The solvent contained in the mobile phase is a mixed solvent of water and an organic solvent,
2. The separation method according to claim 1, wherein the mixed solvent has a ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is 50% or more.
The acid additive is the perfluoroalkanesulfonic acid having 1 to 3 carbon atoms,
The solvent contained in the mobile phase is a mixed solvent of water and an organic solvent,
2. The separation method according to claim 1, wherein the mixed solvent has a ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is 50% or more.
the acid additive is the hydrogen halide,
The solvent contained in the mobile phase is a mixed solvent of water and an organic solvent,
2. The separation method according to claim 1, wherein the mixed solvent has a ratio of the volume of water before mixing to the sum of the volume of water before mixing and the volume of the organic solvent before mixing is 50% or less.
A method for the analysis of amines by liquid chromatography-mass spectrometry, comprising:
A method for analyzing amines, comprising a separation step of separating amines by the separation method according to any one of claims 1 to 4, and a mass spectrometry step of analyzing the amines separated in the separation step by mass spectrometry.