Mobile phase and method for separating ionizable organic compound

Abstract

To provide a mobile phase with a low water content, capable of improving holding of a desired separation target on a stationary phase.SOLUTION: A mobile phase contains one or more anions selected from chloride ions, bromide ions, and thiocyanate ions at a concentration of 1 mM or more and 300 mM or less, where a solvent is one or more solvents selected from a mixed solvent of water and an organic solvent in which a water content is more than 0 vol% and not more than 50 vol%, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a mobile phase and a method for separating ionizable organic compounds. [Background technology]

[0002] Chromatography, such as liquid chromatography and supercritical fluid chromatography, has become indispensable as a simple and precise method for the separation and analysis of mixtures, and the targets of chromatographic separation and analysis cover a wide range of substances, including low-molecular-weight compounds, high-molecular-weight compounds, and biopolymers.

[0003] In these chromatographic separation analyses, it is important to appropriately select both the stationary phase and the mobile phase depending on the structure of the target substance. Even if the stationary phase is appropriately selected, if the mobile phase is inappropriate, problems may occur, such as the target substance being barely retained in the stationary phase and eluting as a mixture, requiring an extremely long analysis time, or remaining retained in the stationary phase. Therefore, in order to achieve separation with the desired accuracy within an acceptable time, it is necessary to select a mobile phase with an appropriate design.

[0004] For example, when an ionizable organic compound such as an amine is to be separated, unless the stationary phase and mobile phase are appropriately selected, the compound may not be sufficiently retained in the stationary phase, or even if it is retained in the stationary phase, the peak shape may be significantly distorted, making it impossible to perform analysis, purification, etc. with the desired accuracy. Combinations of stationary phases and mobile phases commonly used for such separations include a combination of an ion exchanger and an electrolyte solution, a combination of a hydrophobic stationary phase and a weakly basic mobile phase (ion suppression mode), and a combination of a relatively hydrophobic stationary phase and a neutral to acidic mobile phase containing a specific anion (ion pair mode).

[0005] As a stationary phase used in the ion pair mode, a stationary phase made of octadecylsilyl (ODS)-modified silica gel or its homologue is widely known (Non-Patent Document 1). In addition, it is also known that the ion pair mode is useful for stationary phases having polysaccharide derivatives and stationary phases having a crown ether-like cyclic structure (Non-Patent Document 2, Patent Document 1).

[0006] As a mobile phase to be combined with a stationary phase having a crown ether-like cyclic structure, a mobile phase mainly composed of water and containing an aqueous solution of one or more salts of hydrophobic anions selected from the group consisting of salts of chaotropic anions and salts of hydrophobic organic acids is known. The ionized separation target accompanies the above-mentioned anions as counter ions in the vicinity, and when the anions are retained on the hydrophobic stationary phase, they must be dehydrated from their hydrated state. Therefore, it is believed that the presence of hydrophobic anions with low hydration energy works advantageously to retain the separation target on the stationary phase (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2020 / 251003 [Non-patent literature]

[0008] [Non-Patent Document 1] Analytical Chemistry, 2002, Vol.74, Issue19, p.4927-4932 [Non-patent document 2] Journal of Liquid Chromatography, 1993, Vol.16, Issue4, p.859-878 Summary of the Invention [Problem to be solved by the invention]

[0009] On the other hand, when a mobile phase with a low water content is used, the anionic species described above are not necessarily effective in effectively retaining the separation target on the stationary phase. However, depending on the separation target or purpose, a mobile phase with a low water content may be preferable. Therefore, in order to expand the options for analytical conditions in chromatography, there is a need to develop a mobile phase with a low water content that can promote the retention of the desired separation target, particularly ionizable organic compounds, on the stationary phase.

[0010] An object of the present disclosure is to provide a mobile phase with a low water content that can enhance the retention of the desired separation targets on the stationary phase. Another object of the present disclosure is to provide a method for effectively separating ionizable organic compounds such as amines by chromatography using the above mobile phase and a nonionic stationary phase. [Means for solving the problem]

[0011] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by adding a specific anion to a mobile phase, even if the mobile phase has a low water content.

[0012] [1] containing one or more anions selected from chloride ions, bromide ions, and thiocyanate ions at a concentration of 1 mM or more and 300 mM or less; A mobile phase in which the solvent is one or more solvents selected from a mixed solvent of water and an organic solvent, in which the water content is more than 0% by volume and 50% by volume or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. [2] The mobile phase according to [1], wherein the anion does not form a salt. [3] The mobile phase according to [1], wherein the anion forms a salt with a counter cation. [4] The mobile phase according to [3], wherein the counter cation is one or more selected from the group consisting of a secondary ammonium ion, a tertiary ammonium ion, and a quaternary ammonium ion. [5] The mobile phase according to [3] or [4], further comprising an acid having a pKa in water at 25°C of -2.0 or more and 4.0 or less. [6] The mobile phase according to any one of [1] to [5], wherein the anion is a chloride ion. [7] The mobile phase according to any one of [1] to [6], which is used in liquid chromatography or supercritical fluid chromatography using a non-ionic stationary phase (excluding a size exclusion type stationary phase). [8] The mobile phase according to [7], wherein the nonionic stationary phase is a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a stationary phase in which a polysaccharide derivative is supported on a carrier. [9] a separation step of separating the ionizable organic compounds by liquid chromatography or supercritical fluid chromatography; The mobile phase used in the separation step contains chloride ions, bromide ions, and thiocyanate. ions at a concentration of 1 mM or more and 300 mM or less, and the solvent is one or more solvents selected from a mixed solvent of water and an organic solvent, the water content of which is more than 0 vol% and 50 vol% or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide; A method for separating an ionizable organic compound, wherein the stationary phase used in the separation step is a non-ionic stationary phase (excluding a size exclusion type stationary phase).

[10] The separation method according to [9], wherein the anion does not form a salt.

[11]

[12] The separation method according to [9], wherein the anion forms a salt with a counter cation. The separation method according to

[11] , wherein the counter cation is one or more selected from the group consisting of a secondary ammonium ion, a tertiary ammonium ion, and a quaternary ammonium ion.

[13] The separation method according to

[11] or

[12] , wherein the mobile phase further contains an acid having a pKa in water at 25°C of -2.0 or more and 4.0 or less.

[14] The separation method according to any one of [9] to

[13] , wherein the nonionic stationary phase is a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a stationary phase in which a polysaccharide derivative is supported on a carrier. [Effects of the Invention]

[0013] According to the present disclosure, it is possible to provide a mobile phase having a low water content that can enhance the retention of a desired separation target on a stationary phase. Furthermore, the present disclosure can provide a method for effectively separating ionizable organic compounds such as amines by chromatography using the above mobile phase and a nonionic stationary phase. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a liquid chromatogram of dl-tryptophan in Example 1. [Figure 2] 1 is a liquid chromatogram of dl-tryptophan in Example 2. [Figure 3] 1 is a liquid chromatogram of dl-tryptophan in Example 3. [Figure 4] 1 is a liquid chromatogram of dl-tryptophan in Comparative Example 1. [Figure 5] 1 is a liquid chromatogram of dl-tryptophan in Comparative Example 2. [Figure 6] 1 is a liquid chromatogram of dl-tryptophan in Comparative Example 3. [Figure 7] 1 is a liquid chromatogram of dl-tyrosine in Example 4. [Figure 8] 1 is a liquid chromatogram of dl-tyrosine in Comparative Example 4. [Figure 9] 1 is a liquid chromatogram of dl-tryptophan in Example 5. [Figure 10] 1 is a liquid chromatogram of dl-tryptophan in Comparative Example 5. [Figure 11] 1 is a liquid chromatogram of the racemic form of 1-phenyl-1,2,3,4-tetrahydroisoquinoline in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0015] While the present disclosure will be described below with reference to specific embodiments, the configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible as appropriate within the scope of the gist of the present disclosure. The present disclosure is not limited by the embodiments, but is limited only by the scope of the claims. Additionally, each feature disclosed herein can be combined with any other feature disclosed herein. In this specification, when the lower limit and upper limit of a numerical range are separately stated, the numerical range may be a combination of any of the lower limit and upper limit. can.

[0016] 1. Mobile phase A first embodiment of the present disclosure relates to a mobile phase containing one or more anions selected from chloride ions, bromide ions, and thiocyanate ions at a concentration of 1 mM to 300 mM. The solvent contained in this mobile phase is one or more solvents selected from a mixed solvent of water and an organic solvent, with a water content of more than 0% by volume and not more than 50% by volume, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. The mobile phase according to this embodiment may contain other components to the extent that the separation performance is not impaired. Furthermore, the mobile phase according to this embodiment is preferably a homogeneous (single-phase) system, at least under the conditions for performing the analysis. In chromatography, such as liquid chromatography and supercritical fluid chromatography, the mobile phase is a liquid that is poured into a stationary phase together with the separation target, moves with the separation target, and then elutes the separated product from the stationary phase.

[0017] Solutions containing chloride ions have been used as wash solutions to elute cations adsorbed on ion-exchange stationary phases, but their use as mobile phases has not been explored. Furthermore, while trace amounts of hydrogen chloride have been found in mobile phases, this was not intentional addition of hydrogen chloride to improve separation performance; rather, it was residual hydrochloric acid used to adjust the pH of buffer solutions. Therefore, the effects of adding specific concentrations of chloride ions to the mobile phase have not been explored.

[0018] The present inventors have conducted extensive research into the effect of chloride ions on separation and have found that a mobile phase containing chloride ions is significantly more effective in increasing sample retention than a mobile phase not containing chloride ions. Chloride ions are not chaotropic ions, but are classified as kosmotropic ions, which have properties opposite to those of chaotropic ions. In other words, chloride ions have a large hydration energy and exhibit strong interactions with water molecules. Therefore, chloride ions are considered to be unfavorable ions from the viewpoint of forming ion pairs with ionized analytes and adsorbing them to the stationary phase. Nevertheless, it is surprising that adding chloride ions to the mobile phase can increase the retention of ionized analytes (e.g., organic ammonium ions) on the stationary phase.

[0019] The present inventors speculate that the reason why chloride ions increase the retention of the separation target on the stationary phase and improve separation performance is as follows. The solvent of the mobile phase in this embodiment is one or more solvents selected from a water / organic solvent mixture primarily containing an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide. In such a solvent with a low water content (i.e., a solvent with a water content of 50% by volume or less), chloride ions are not sufficiently stabilized by hydration, so they can easily form ion pairs with the ionized target substance. Furthermore, the energy loss due to dehydration during incorporation into the hydrophobic stationary phase is small, so they can be easily dehydrated. As a result, the target substance is well retained in the stationary phase. In addition, chloride ions have a spherical shape with little steric hindrance and a high polarizability due to the outer electron orbitals specific to heavy atoms, which gives them a high affinity for organic compounds. Therefore, it is believed that adding chloride ions to the mobile phase can well retain the target substance in the stationary phase and improve separation performance. Furthermore, bromide ions, which are halide ions, and thiocyanate ions, which are pseudohalide ions, have similar chemical properties to chloride ions and therefore function similarly to chloride ions for similar reasons.

[0020] The mobile phase according to this embodiment will be described in more detail below.

[0021] 1-1. Anion species The mobile phase according to this embodiment contains an anion selected from chloride ions, bromide ions, and thiocyanate ions (hereinafter, sometimes referred to as "(pseudo)halide ions"). Of these, the (pseudo)halide ions are preferably chloride ions. The (pseudo)halide ions may be used alone or in any combination and ratio of two or more.

[0022] In the mobile phase, the (pseudo)halide ions may or may not form a salt with a counter cation, but it is preferable that the (pseudo)halide ions form a salt with a counter cation in order to prevent corrosion or other damage to various components such as the chromatograph and column.

[0023] In this specification, the phrase "the (pseudo)halide ions do not form salts" means that the content of cations other than protons and hydronium ions in the mobile phase is 10 mol % or less, preferably 5 mol % or less, more preferably 3 mol % or less, and even more preferably 1 mol % or less of the (pseudo)halide ions.

[0024] A mobile phase in which (pseudo)halide ions are present in the system without forming salts can be prepared by mixing a free acid selected from hydrogen chloride, hydrogen bromide, and thiocyanic acid with other components of the mobile phase. The mixing method is not particularly limited, and may include, for example, a method in which a solution obtained by dissolving each compound in a solvent is mixed with the other components, a method in which hydrogen chloride gas or hydrogen bromide gas is mixed by bubbling, or a method in which liquid thiocyanic acid is directly mixed with the other components. With regard to hydrogen chloride and hydrogen bromide, it is preferable to adopt a method in which a solution of hydrogen chloride or hydrogen bromide is mixed with the other components from the viewpoint of simplicity. For example, to prepare a mobile phase containing 5 mM chloride anions in a 5 / 95 (v / v) water / acetonitrile mixture, a preferred method is to first weigh out a predetermined amount of commercially available hydrochloric acid, adjust the water / acetonitrile ratio to 5 / 95 (v / v) by mixing with acetonitrile as needed, and then dilute with a 5 / 95 (v / v) water / acetonitrile mixture to a chloride ion concentration of 5 mM. However, when using a commercially available high-concentration aqueous solution (e.g., 35% concentrated hydrochloric acid) as a (pseudo)halide ion source, the water content of the high-concentration aqueous solution is low, so the mobile phase can be prepared by directly diluting the high-concentration aqueous solution with the mobile phase solvent without adjusting the water / organic solvent ratio prior to dilution.

[0025] The free acid of thiocyanate ion is unstable and is not commercially available. Therefore, it is desirable to prepare a mobile phase containing thiocyanate ion by the method described below, i.e., by mixing a thiocyanate salt with other components of the mobile phase. In other words, in the mobile phase according to this embodiment, it is desirable for the thiocyanate ion to form a salt with a counter cation.

[0026] The absence of salt formation of (pseudo)halide ions in the mobile phase can be determined by quantitative determination of (pseudo)halide ions, acid-base titration, and evaluation of the presence or absence of volatile residues. The quantitative determination of (pseudo)halide ions can be performed by, but is not limited to, ion chromatography or inductively coupled plasma atomic emission spectroscopy (IPC-AES). Acid-base titration is performed after diluting the mobile phase with approximately 10 times the amount of water to mitigate the effects of solvent composition. If the concentration of (pseudo)halide ions determined by acid-base titration is equivalent to the concentration of (pseudo)halide ions determined by the above quantitative determination, taking into account the measurement accuracy, and if no crystalline residue containing (pseudo)halide ions is produced when the mobile phase is evaporated to dryness (e.g., at 30°C under a pressure of 10 hPa), it can be determined that the (pseudo)halide ions in the mobile phase have not formed salts.

[0027] A mobile phase in which (pseudo)halide ions and counter cations form salts in a system can be prepared by mixing the salts of (pseudo)halide ions and counter cations with other components of the mobile phase. The mixing method is not particularly limited, and may involve, for example, mixing each compound directly with the other components, or dissolving each compound in a solvent and mixing the resulting solution with the other components.

[0028] The counter cation is not particularly limited as long as it is a cation other than a proton or a hydronium ion, but is typically an unsubstituted ammonium ion (NH +) and onium cations such as primary to quaternary ammonium ions; and metal ions such as sodium ions and potassium ions. The counter anion is preferably an onium cation. For example, when amine separation is performed using a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, the onium cation is less likely to interact with the crown ether-like cyclic structure, weakening the retention of the ionized amine. The onium cation is preferably selected from secondary to quaternary ammonium ions, and is preferably selected from secondary ammonium ions and tertiary ammonium ions in view of its applicability to liquid chromatography-mass spectrometry.

[0029] Suitable examples of primary ammonium ions include monoalkylammonium ions such as methylammonium ion, ethylammonium ion, and butylammonium ion. Suitable examples of secondary ammonium ions include dialkylammonium ions such as dimethylammonium ion, diethylammonium ion, and dibutylammonium ion. Suitable examples of tertiary ammonium ions include trialkylammonium ions such as trimethylammonium ion, triethylammonium ion, and tributylammonium ion. Suitable examples of the quaternary ammonium ion include tetraalkylammonium ions such as tetramethylammonium ion, tetraethylammonium ion, tetrabutylammonium ion, hexadecyltrimethylammonium ion, and octadecyltrimethylammonium ion. 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, and even more preferably an alkyl group having 1 to 4 carbon atoms.

[0030] Among the above onium cations, salts of (pseudo)halide ions and unsubstituted ammonium ions (e.g., ammonium chloride) are preferred from the viewpoint of improving separation performance, but because they are not highly soluble in nonaqueous solvents, it is preferable to add water to the solvent to an extent that the salt can be dissolved. Furthermore, when chromatography is performed using a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, the salt may inhibit the separation target from being included in the ligand having a crown ether-like cyclic structure, so it is preferable to select another onium cation as the counter anion.

[0031] In an embodiment in which a (pseudo)halide ion forms a salt with a counter cation in the mobile phase, it is desirable to control the pH of the mobile phase as necessary.

[0032] For example, when separating amines by chromatography, if the amines exist in both the free amine and ammonium ion states in the mobile phase, a slight change in pH can significantly change the ratio of the free amine and ammonium ion, which in turn can affect the chromatographic behavior. Therefore, it is necessary to adjust the pH of the mobile phase to favor either the free amine or the ammonium ion state.

[0033] Furthermore, when separating amines in ion-pair mode, the amines are converted to ammonium ions, and their adsorption behavior is utilized for separation. When the amines are alkylamines, they are sufficiently protonated even in a neutral mobile phase, so the above problem does not occur even without adjusting the pH. However, when the amines are weakly basic aromatic amines, it is recommended to add an acid to the mobile phase to adjust the pH to approximately 4.0 or less.

[0034] Furthermore, when the amine is an amino acid, dissociation of the carboxyl group of the amino acid may weaken the retention of the target substance on the stationary phase. Therefore, it is desirable to suppress dissociation of the carboxyl group and protonate the amino group by adjusting the pH to less than 2.0.

[0035] Therefore, if (pseudo)halide ions form salts with countercations in the mobile phase, the pH can be adjusted by adding an acid, as necessary, to convert almost all amines to ammonium ions and suppress dissociation of the carboxyl groups of amino acids.

[0036] The pKa of the acid used to adjust the pH of the mobile phase in water at 25°C is usually -2.0 or higher, preferably -1.0 or higher, more preferably 0.5 or higher, and usually 4.0 or lower, preferably less than 4.0, more preferably 3.8 or lower, and even more preferably 3.6 or lower. Preferred ranges for the pKa of the acid include, for example, -2.0 or higher but lower than 4.0, -1.0 or higher but 3.8 or lower, and 0.5 or higher but 3.6 or lower. While acids stronger than hydrogen chloride convert chloride ions to hydrogen chloride, increasing the risk of metal corrosion, a pKa of -2.0 or higher can reduce this risk. Furthermore, while acids that are too weak can make it difficult to protonate amino acids, a pKa of 4.0 or lower can protonate almost all amino acids. In this specification, unless otherwise specified, the "pKa" of an acid refers to the pKa in water at 25°C. When referring to the pKa of a polyvalent acid, the pKa refers to pKa1.

[0037] Preferred examples of such acids include phosphoric acid (pKa: 2.15), oxalic acid (pKa: 1.04), and formic acid (pKa: 3.55). Of these, it is particularly preferable to select the acid from formic acid and oxalic acid, as they have high acidity and produce little noise in detection with a mass detector. In addition to the acids mentioned above, organic acids can also be used. When an organic acid is added to the mobile phase, the S / N ratio at short wavelengths in detection by ultraviolet absorption decreases slightly, but this does not impair separation performance.

[0038] When adding an acid to the mobile phase by dissolving the acid in a solvent, if the solvent composition of the mobile phase changes, the baseline of the chromatogram will be disturbed, resulting in inaccurate analysis results. Therefore, it is preferable that the solvent in which the acid is dissolved is the same as the solvent of the mobile phase. The amount of acid added to the mobile phase may be selected appropriately depending on the target to be separated and the solvent of the mobile phase so that the pH of the mobile phase is at a desired value. Typically, an acid is added to the mobile phase in an amount that makes the total concentration in the mobile phase between 1 mM and 10 mM.

[0039] Whether or not the mobile phase contains (pseudo)halide ions in the form of salts with countercations can be evaluated by evaporating the mobile phase to dryness (e.g., drying at 30°C under a pressure of 10 hPa). If the residue contains 0.1 mM or more (pseudo)halide ions relative to the volume of the mobile phase, it can be assumed that the mobile phase contains (pseudo)halide ions in the form of salts. Furthermore, if the countercation is a primary, secondary, or quaternary ammonium ion, and a peak corresponding to an organic group bonded to the nitrogen atom of the ammonium ion is observed in the NMR spectrum of the residue, it can be assumed that the (pseudo)halide ions form salts with the primary, secondary, or quaternary ammonium ions in the mobile phase. Furthermore, if the countercation is a metal ion, and the presence of the metal ion is confirmed by ion chromatography, it can be assumed that the (pseudo)halide ions form salts with the metal ion in the mobile phase.

[0040] The total concentration of (pseudo)halide ions in the mobile phase is usually 1 mM or more, preferably 2 mM or more, more preferably 4 mM or more, and usually 300 mM or less, preferably 200 mM or less, more preferably 100 mM or less, even more preferably 50 mM or less, particularly preferably 20 mM or less, and most preferably 10 mM or less. Preferred ranges for the total concentration of (pseudo)halide ions include, for example, 1 mM to 200 mM, 2 mM to 100 mM, 2 mM to 50 mM, 4 mM to 20 mM, and 4 mM to 10 mM.

[0041] The retention strength of the target substance on the stationary phase depends on the concentration of (pseudo)halide ions; the higher the concentration, the stronger the retention. However, depending on the solvent and other conditions, the retention strength may reach saturation at a certain concentration. Therefore, the concentration of (pseudo)halide ions does not need to be extremely high; as long as it is within the above range, the retention of the target substance on the stationary phase can be sufficiently increased. For example, the examples described below demonstrate that the target substance can be well retained on the stationary phase even at a chloride ion concentration as low as 10 mM or less, resulting in good separation of the target substance. If (pseudo)halide ions, especially chloride ions, are present in the mobile phase without forming a salt, and the mobile phase solvent is a mixture of water and an organic solvent with a low water content or an organic solvent (water content 0% by volume), there is a risk of corrosion of various components such as the chromatograph and column. Therefore, if the various components such as the chromatograph and column are made of a material that is easily corroded, such as steel, it is desirable to keep the concentration of (pseudo)halide ions at 5 mM or less.

[0042] 1-2.Solvent The solvent for the mobile phase according to this embodiment is one or more solvents selected from a mixed solvent of water and an organic solvent having a water content of more than 0% by volume and not more than 50% by volume, an organic solvent (water content: 0% by volume), subcritical carbon dioxide, and supercritical carbon dioxide. In the mixed solvent of water and an organic solvent and the organic solvent (water content: 0% by volume), the organic solvent may be one type alone, or two or more types may be used in any combination and ratio.

[0043] The organic solvent contained in the mixed solvent of water and an organic solvent is not particularly limited, but is preferably an organic solvent capable of dissolving the target of separation. Suitable organic solvents include, for example, acetonitrile, methanol, ethanol, n-propanol, 2-propanol, tetrahydrofuran, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylacetamide (DMAc). Furthermore, as long as it can dissolve the target of separation and various components such as the salts, the organic solvent may contain hydrocarbons such as hexane; ethers such as methyl tert-butyl ether (MTBE); halogenated hydrocarbons such as dichloromethane; and the like. Among these, the organic solvent is preferably selected from acetonitrile, methanol, ethanol, n-propanol, 2-propanol, and tetrahydrofuran, more preferably methanol and acetonitrile, and even more preferably acetonitrile, in terms of high chemical stability, low viscosity, and enabling detection of amines by ultraviolet absorption. Furthermore, the organic solvent may be a mixture of a hydrocarbon having 5 to 8 carbon atoms and an alcohol, which is commonly used in chromatography.

[0044] The water content in the mixed solvent of water and an organic solvent is usually more than 0% by volume, preferably 0.5% by volume or more, more preferably 1.0% by volume or more, and usually 50% by volume or less, preferably 30% by volume or less, more preferably 20% by volume or less, and even more preferably 10% by volume or less. Preferred ranges for the water content in the mixed solvent of water and an organic solvent include more than 0% by volume and 30% by volume or less, 0.5% by volume or more and 20% by volume or less, and 1.0% by volume or more and 10% by volume or less.

[0045] The organic solvent containing 0% by volume of water is explained using the same explanation as for the organic solvent contained in the mixed solvent of water and organic solvent.

[0046] When the mobile phase is used for supercritical fluid chromatography, at least one of subcritical carbon dioxide and supercritical carbon dioxide is selected as the solvent. In this case, since the compatibility between carbon dioxide and hydrogen chloride, hydrogen bromide, thiocyanate, or salts of (pseudo)halide ions is not necessarily good, it is preferable to use water or a water / methanol mixture as the solvent together with carbon dioxide so that the target (pseudo)halide ions are dissolved in the mobile phase at the desired concentration.

[0047] 1-3. Separation target The mobile phase according to the present embodiment is useful for ion-pair chromatography of ionizable organic compounds because it can ionize the ionizable organic compounds by supplying (pseudo)halide ions to the ionizable organic compounds and provide retention to the non-ionic stationary phase. The use of the mobile phase according to the present embodiment makes it possible to separate a mixture of multiple ionizable organic compounds into individual compounds, or to separate an ionizable organic compound from a mixture of an ionizable organic compound and another compound.

[0048] Ionizable organic compounds typically include amines. The amine is not particularly limited and may be any of primary amines, secondary amines, and tertiary amines. Specific examples of the amine include amino acids such as alanine, cysteine, glutamic acid, methionine, leucine, tyrosine, and tryptophan; amino acid derivatives such as esters of the amino acids; amino alcohols such as dimethylaminoethanol, propanolamine, methioninol, and norephedrine; amino group-containing hydrocarbons such as phenylethylamine, aniline, methylaniline, chloroaniline, and aminobenzoic acid; and cyclic amines such as 1-phenyl-1,2,3,4-tetrahydroisoquinoline.

[0049] The use of the mobile phase according to this embodiment can improve the separation performance of chromatography, making it possible to separate mixtures of amines that are difficult to separate due to their similar structures into individual amines. Examples of mixtures of amines with similar structures include mixtures of chain isomers, positional isomers, geometric isomers, and analogs. Furthermore, by performing chromatography using a chiral stationary phase, a mixture of enantiomers can be separated into individual enantiomers.

[0050] 1-4.Stationary phase The stationary phase to which the mobile phase according to the present embodiment is applied is not particularly limited, but as described above, a non-ionic stationary phase is preferable in order to exhibit the properties of the mobile phase. Furthermore, since a size-exclusion stationary phase is used for separation based on differences in the molecular size of the separation target and does not utilize chemical interactions between the stationary phase and the separation target, the mobile phase according to the present embodiment is preferably applied to a stationary phase other than a size-exclusion stationary phase.

[0051] In this specification, a non-ionic stationary phase means a stationary phase that does not have an ionic functional group in the portion that comes into contact with the object to be separated. On the other hand, an ionic stationary phase means a stationary phase that has an ionic functional group in the portion that comes into contact with the object to be separated. However, under the conditions of use, A stationary phase that has a zwitterionic functional group in the portion that binds to the ion and does not have a positive or negative ionic functional group is neutral in charge overall and is therefore included in the category of non-ionic stationary phases.

[0052] The nonionic stationary phase is not particularly limited, and any known stationary phase that is commercially available can be used. Known commercially available stationary phases include: stationary phases in which a ligand having a crown ether-like cyclic structure is supported on a carrier; stationary phases in which polysaccharide derivatives such as cellulose derivatives and amylose derivatives are supported on a carrier; stationary phases in which a carrier is bonded to silica gel via an alkylsilyl group having 1 to 32 carbon atoms, such as octadecylsilyl (ODS) silica gel; and polar phases in which a polar group such as an amide bond is embedded in the alkyl chain of the alkylsilyl group. Examples of such stationary phases include those called embedded stationary phases; stationary phases in which silica gel is bonded with a group containing a benzene ring (e.g., phenylethyl group and phenylhexyl group), a halogen-containing group (e.g., pentafluorophenylpropyl, pentachlorophenylpropyl, and pentabromophenylpropyl), a group containing a cyano group (e.g., phenylpropyl group), a group containing an amide bond (e.g., amidopropyl group), or a group containing an alcoholic hydroxyl group; stationary phases in which amino acids, cyclic oligosaccharides (e.g., cyclodextrin and cyclofructan), cyclic oligosaccharides with modified hydroxyl groups, or macrocyclic amides (e.g., vancomycin) are supported on a carrier; and stationary phases with zwitterionic functional groups.

[0053] Among the above-mentioned stationary phases, the nonionic stationary phase is preferably a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a stationary phase in which a polysaccharide derivative is supported on a carrier. These stationary phases will be described in more detail below.

[0054] 1-4-1.Stationary phase with a ligand having a crown ether-like cyclic structure supported on a carrier In this specification, the term "ligand" refers to a compound that is supported on a carrier and that exhibits physical affinity for the separation target and, if necessary, chiral recognition ability. The ligand having a crown ether-like cyclic structure 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.

[0055] *-O(CH2CH2O) n -* (I) In the formula, n can be appropriately selected from integers of 4 to 6 depending on the amino group of the amine and the hydrocarbon to which the crown ether skeleton is bonded. For example, the ligands represented by formula (II) or (III) described below have n of 5 and have a crown ether-like cyclic structure of a size suitable for encapsulating a primary ammonium group, and are therefore suitable for use in separating primary amines. The hydrogen atom of the ethylene group in the repeating unit may be substituted with various functional groups, but is preferably unsubstituted.

[0056] In this embodiment, when separating enantiomers, a compound having a crown ether-like cyclic structure bonded to a homochiral structure is used as the ligand. Examples of such ligands include the ligand represented by formula (II) described in JP-A-2-69472 and WO 2012 / 050124, and the ligand represented by formula (III) described in JP-A-2014-169259. Furthermore, ligands in which the phenyl groups at the 3- and 3'-positions of the 1,1'-binaphthyl structure in formula (II) are replaced with 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 used, but are not limited to these. Furthermore, ligands effective for separating enantiomers are often also effective for separating molecules that are similar to each other.

[0057] [ka]

[0058] The ligand is used as a stationary phase in a state supported on a carrier. Known methods can be used for the support, and preferred methods include those in which the ligand is supported on a carrier via a chemical bond such as a covalent bond. Specifically, a method can be used in which a reactive group is introduced into the ligand, a ligand raw material, or a ligand intermediate, and the substituent is reacted with the reactive group present on the carrier surface. The reactive group present on the carrier surface may be a group present on the surface of an untreated carrier, or a group introduced onto the carrier surface by surface treating the carrier with a surface treatment agent, such as a silane coupling agent such as 3-aminopropyltriethoxysilane or 3-glycidyloxypropyltrimethoxysilane. In addition to methods that form bonds between the ligand and the carrier, an insolubilized layer can also be formed on the carrier surface by forming so-called cross-linking bonds between atomic groups containing the ligand. Furthermore, the ligand may be supported on a carrier by other known support methods, such as physical adsorption (coating) of the ligand onto the carrier.

[0059] The carrier is not particularly limited as long as it can immobilize the ligand by a chemical bond such as a covalent bond. Such a carrier may be an inorganic carrier or an organic carrier, but an inorganic carrier is preferred. Examples of inorganic carriers include silica gel, alumina, magnesia, glass, kaolin, titanium oxide, silicates, and hydroxyapatite. Examples of organic carriers include cross-linked polystyrene, cross-linked polyacrylamide, cross-linked polyacrylate, and polysaccharides. These organic carriers are preferably insolubilized by cross-linking with a cross-linking agent.

[0060] The shape of the carrier is not particularly limited, and examples thereof include particles, porous cylinders (monoliths) that are liquid-tightly housed in a column tube, etc. The inner wall of a capillary may also be used as a carrier.

[0061] In this embodiment, the carrier is preferably silica gel. This is because silica gel has the above-mentioned properties, i.e., excellent separation performance, and is hard and durable. As the silica gel, in addition to fully porous silica, so-called core-shell type silica gel may be used, or silica gel with a chemically modified surface may be used.

[0062] 1-4-1.Stationary phase with polysaccharide derivatives supported on a carrier As the polysaccharide derivative of the stationary phase in which the polysaccharide derivative is supported on a carrier, any polysaccharide derivative known to be usable for separating optical isomers can be used. Known polysaccharide derivatives include, for example, cellulose, amylose, and β-1,4-xylan. , β-1,4-chitosan, chitin, β-1,4-mannan, inulin, or curdlan, in which the hydroxyl groups of the polysaccharide are converted to benzoate, phenyl carbamate, 3,5-dimethylphenyl carbamate, 3-chloro-5-methylphenyl carbamate, 3,5-dichlorophenyl carbamate, 2,4-dichlorophenyl carbamate, 3,4-dichlorophenyl carbamate, 2,5-dichlorophenyl carbamate, 4-fluorophenyl carbamate, 4-chlorophenyl carbamate, 4-bromophenyl carbamate, or 4-iodophenyl carbamate. More specifically, examples include amylose (3-chloro-5-methylphenylcarbamate) described in JP 2018-054608 A; cellulose tris(3,5-dichlorophenylcarbamate) described in WO 2008 / 102920 A; cellulose trisbenzoate, cellulose tris(phenylcarbamate), and cellulose tris(3,5-dimethylphenylcarbamate) described in WO 2005 / 075974 A; and cellulose tris(4-chlorophenylcarbamate) described in WO 2002 / 030903 A.

[0063] The carrier may be the same as the carrier of the stationary phase in which a ligand having a crown ether-like cyclic structure is supported on the carrier.

[0064] Furthermore, a method for supporting a polysaccharide derivative on a carrier can be a known method, such as those described in JP 2018-054608 A, JP 2018-030965 A, WO 2014 / 087937 A, or JP 7-138301 A, but is not limited thereto.

[0065] 2. Separation method of ionizable organic compounds A second embodiment of the present disclosure is a method for separating ionizable organic compounds, including a separation step of separating ionizable organic compounds by liquid chromatography or supercritical fluid chromatography. The mobile phase used in the separation step is the mobile phase according to the first embodiment of the present disclosure. The stationary phase used in the separation step is a non-ionic stationary phase (excluding size-exclusion stationary phases). The explanations of the ionizable organic compounds and the stationary phase are given in "1-3. Object to be separated" and "1-4. Stationary phase" above, respectively.

[0066] The separation method according to this embodiment may include other steps, such as a step of qualitatively characterizing the separated sample and a step of quantitatively characterizing the separated sample, in addition to the separation step. Liquid chromatography and supercritical fluid chromatography can be performed using commercially available liquid chromatographs and supercritical fluid chromatographs, respectively. Column equilibration conditions and flow rate and other conditions can be appropriately selected depending on the column size, sample volume, type of mobile phase, and the like.

[0067] Furthermore, the separation method according to this embodiment may be applied to liquid chromatography-mass spectrometry (LC-MS) to perform various analyses of the separated sample, such as quantitative analysis and qualitative analysis. The analysis method using LC-MS includes a separation step of separating ionizable organic compounds by the separation method according to this embodiment, and a mass analysis step of analyzing the sample separated in the separation step by mass spectrometry.

[0068] As the mass analysis in the mass analysis step, a known mass analysis method used in LC-MS can be adopted. For example, the ionization method in mass analysis can be appropriately selected from atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), electrospray (ESI), fast atom bombardment (FAB), thermospray (TSP), etc. depending on the type of sample, the purpose of analysis, etc. Furthermore, as the mass spectrometer, a quadrupole mass spectrometer (Q-MS), an ion trap mass spectrometer (IT-MS), and a time-of-flight mass spectrometer (TSP) can be used. A suitable analyzer such as a TOF-MS can be selected and used depending on the required sensitivity, resolution, etc.

[0069] 3. Use of (pseudo)halide ion-containing solutions as mobile phases A third embodiment of the present disclosure relates to the use of a solution containing one or more anions selected from chloride ions, bromide ions, and thiocyanate ions at a concentration of 1 mM to 300 mM, and the solvent being one or more solvents selected from a mixed solvent of water and an organic solvent, with a water content of more than 0% by volume and 50% by volume or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide, as a mobile phase. The solution in this embodiment is the mobile phase according to the first embodiment of the present disclosure. That is, the anions, solvent, target to be separated using the mobile phase, and stationary phase to which the mobile phase is applied in this embodiment are as described above in "1-1. Anion species," "1-2. Solvent," "1-3. Target to be separated," and "1-4. Stationary phase," respectively. [Example]

[0070] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited to the following examples as long as it does not deviate from the gist of the disclosure.

[0071] Example 1: Chiral separation of dl-tryptophan (Preparation of mobile phase) 5.00 mL of commercially available 1.00 N hydrochloric acid was weighed out with a volumetric pipette and added to a 1.00 L volumetric flask. 74.4 g of acetonitrile was added to the hydrochloric acid, and then the mixture was diluted with a 5 / 95 (v / v) water / acetonitrile mixed solvent to prepare mobile phase A, which contained 5 mM hydrogen chloride and a 5 / 95 (v / v) water / acetonitrile mixed solvent.

[0072] (Amine separation) Amines were separated using a column packed with a chiral stationary phase consisting of a silica gel-supported ligand with a crown ether-like ring structure represented by formula (IV) (Daicel Corporation, "CROWNPAK CR-I(-)" model, 3 mm internal diameter, 150 mm length) and a high-performance liquid chromatograph (Shimadzu Corporation, "LC-20AD" model). The target dl-tryptophan was dissolved in a water / acetonitrile (1:1 (v / v)) mixture containing approximately 5 mM hydrogen chloride to a concentration of approximately 0.1% w / v. 2 μL of the resulting solution was injected onto the column using an autosampler. Mobile phase 1 was pumped through the column at 0.43 mL / min, preheated at 30 °C. A photodiode array detector (Shimadzu Corporation, "SPD-M20A," detection wavelength 220 nm) was used as the detector, and the data acquired by the detector was analyzed using data analysis software (Shimadzu Corporation, "LCsolution"). The resulting chromatogram is shown in Figure 1.

[0073] [ka]

[0074] Example 2: Chiral separation of dl-tryptophan (Preparation of mobile phase) According to the method described in Example 1, a mobile phase B containing trimethylammonium chloride at a concentration of 5 mM, oxalic acid at a concentration of 2.9 mM, and a mixed solvent of water / acetonitrile (5 / 95 (v / v)) was prepared.

[0075] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase B was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0076] Example 3: Chiral separation of dl-tryptophan (Preparation of mobile phase) According to the method described in Example 1, a mobile phase C containing trimethylammonium chloride at a concentration of 5 mM, oxalic acid at a concentration of 10 mM, and a mixed solvent of water / acetonitrile (5 / 95 (v / v)) was prepared.

[0077] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase C was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0078] Comparative Example 1: Chiral Separation of dl-Tryptophan (Preparation of mobile phase) According to the method described in Example 1, a mobile phase a containing perchloric acid at a concentration of 5 mM and a mixed solvent of water / acetonitrile (5 / 95 (v / v)) was prepared.

[0079] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase a was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0080] Comparative Example 2: Chiral Separation of dl-Tryptophan (Preparation of mobile phase) According to the method described in Example 1, mobile phase b was prepared containing trifluoroacetic acid at a concentration of 5 mM, oxalic acid at a concentration of 10 mM, and a mixed solvent of water / acetonitrile (5 / 95 (v / v)) as the solvent.

[0081] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase b was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0082] Comparative Example 3: Chiral Separation of dl-Tryptophan (Preparation of mobile phase) According to the method described in Example 1, a mobile phase c containing oxalic acid at a concentration of 10 mM and a mixed solvent of water / acetonitrile (5 / 95 (v / v)) was prepared.

[0083] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase c was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0084] Example 4: Chiral separation of dl-tyrosine (Preparation of mobile phase) According to the method described in Example 1, a mobile phase D containing hydrogen chloride at a concentration of 5 mM and a mixed solvent of water / acetonitrile (10 / 90 (v / v)) was prepared.

[0085] (Amine separation) Chiral separation of dl-tyrosine was carried out in the same manner as in Example 1, except that mobile phase D was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0086] Comparative Example 4: Chiral Separation of dl-Tyrosine (Preparation of mobile phase) According to the method described in Example 1, a mobile phase d containing perchloric acid at a concentration of 5 mM and a mixed solvent of water / acetonitrile (10 / 90 (v / v)) as the solvent was prepared.

[0087] (Amine separation) Chiral separation of dl-tyrosine was carried out in the same manner as in Example 1, except that mobile phase d was used instead of mobile phase A. The obtained chromatogram is shown in Figure 8.

[0088] Example 5: Chiral separation of dl-tryptophan (Preparation of mobile phase) According to the method described in Example 1, a mobile phase E containing triethylammonium chloride at a concentration of 5 mM, oxalic acid at a concentration of 10 mM, and a mixed solvent of hexane / ethanol / water (100 / 100 / 4 (v / v / v)) was prepared.

[0089] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase E was used instead of mobile phase A. The obtained chromatogram is shown in FIG.

[0090] Comparative Example 5: Chiral Separation of dl-Tryptophan (Preparation of mobile phase) According to the method described in Example 1, a mobile phase e containing trifluoroacetic acid at a concentration of about 67 mM and a mixed solvent of hexane / ethanol / water (100 / 100 / 4 (v / v / v)) was prepared.

[0091] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase e was used instead of mobile phase A and the flow rate of the mobile phase was changed to 0.25 mL / min. The graph is shown in FIG.

[0092] Example 6: Chiral separation of racemic 1-phenyl-1,2,3,4-tetrahydroisoquinoline (Preparation of mobile phase) According to the method described in Example 1, a mobile phase F containing triethylammonium chloride at a concentration of 5 mM, oxalic acid at a concentration of 2.5 mM, and a mixed solvent of water / acetonitrile (2.5 / 97.5 (v / v)) was prepared.

[0093] (Amine separation) Chiral separation of dl-tryptophan was carried out in the same manner as in Example 1, except that mobile phase F was used instead of mobile phase A, a chiral stationary phase (CHIRALPAK IB N-5, manufactured by Daicel Corporation, inner diameter 4.6 mm, length 250 mm) in which cellulose tris(3,5-dimethylphenylcarbamate) was immobilized on a silica gel carrier via a chemical bond was used as the stationary phase, and the flow rate of the mobile phase was changed to 1.0 mL / min. The resulting chromatogram is shown in Figure 11.

[0094] Examples 1 to 5 and Comparative Examples 1 to 5 are experimental examples using a chiral stationary phase having a ligand with a crown ether-like cyclic structure. Such a stationary phase is effective for chiral separation of primary amines, and the primary ammonium ion is included in the crown ether-like cyclic structure by hydrogen bonding, and the chirality of the sample is recognized by interaction with the chiral binaphthyl structure.

[0095] In Examples 1 to 4, the peaks of each enantiomer were farther apart than in Comparative Examples 1 to 4, indicating that dl-tryptophan or dl-tyrosine was well separated. In particular, in the chiral separation of dl-tryptophan in Examples 1 to 3, it was confirmed that the retention of the target substance on the stationary phase was stronger and the peak separation was greater than in Comparative Example 1, which used a mobile phase containing perchloric acid, and Comparative Example 2, which used a mobile phase containing trifluoroacetic acid. Furthermore, in Examples 1 to 3, no peak splitting was observed, as was observed in the second peak in Comparative Example 1.

[0096] It has been known that using a mobile phase containing trifluoroacetic acid and an organic solvent such as acetonitrile or a hexane / ethanol mixed solvent as the main solvent can result in weak retention of amines on the stationary phase, resulting in poor separation of amines. In contrast, the mobile phases of Examples 1 to 5, which are primarily organic solvents with low water content, contain chloride ions, enabling stronger retention of amines on the stationary phase than the comparative examples. It was also found that high retention performance was achieved even with a low chloride ion concentration of 5 mM in the mobile phase. Comparative Example 5, on the other hand, exhibited separation performance comparable to that of Example 5, but the trifluoroacetic acid concentration (approximately 67 mM) required to achieve such separation performance was found to be more than 13 times the chloride ion concentration (5 mM) in Example 5.

[0097] Example 6 is an experimental example using a chiral stationary phase in which cellulose tris(3,5-dimethylphenylcarbamate) was immobilized on a silica gel carrier. When using such a stationary phase, problems are known, such as inability to adequately retain ionic substances or perform chiral recognition, when using a mobile phase that is primarily composed of an organic solvent, such as acetonitrile, and has a low water content. In contrast, the results of Example 6 confirmed that, even when using a mobile phase that is primarily composed of an organic solvent and has a low water content, adding triethylammonium chloride can achieve good retention of amines and chiral separation. [Industrial Applicability]

[0098] By using a mobile phase according to at least some embodiments of the present disclosure, high amine separation performance can be achieved by performing liquid chromatography. Furthermore, by selecting highly volatile (pseudo)halide ions and acids, adverse effects on mass spectrometric detection can be suppressed, which may enable application to LC-MS. Therefore, such mobile phases can be widely applied to analysis and purification by various types of liquid chromatography or supercritical fluid chromatography, and are expected to be used in fields such as organic chemistry, medicine, and pharmacology.

Claims

1. containing one or more anions selected from chloride ions, bromide ions, and thiocyanate ions at a concentration of 1 mM or more and 300 mM or less; A mobile phase, wherein the solvent is one or more solvents selected from a mixed solvent of water and an organic solvent having a water content of more than 0 vol.% and not more than 50 vol.%, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide.

2. The mobile phase of claim 1 , wherein the anion does not form a salt.

3. The mobile phase of claim 1 , wherein the anion forms a salt with a counter cation.

4. The mobile phase according to claim 3 , wherein the counter cation is at least one selected from the group consisting of a secondary ammonium ion, a tertiary ammonium ion, and a quaternary ammonium ion.

5. The mobile phase according to claim 3, further comprising an acid having a pKa in water at 25° C. of −2.0 or more and 4.0 or less.

6. 2. The mobile phase of claim 1, wherein the anion is a chloride ion.

7. The mobile phase according to any one of claims 1 to 6, which is used in liquid chromatography or supercritical fluid chromatography using a non-ionic stationary phase (excluding size exclusion type stationary phase).

8. 8. The mobile phase according to claim 7, wherein the non-ionic stationary phase is a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a stationary phase in which a polysaccharide derivative is supported on a carrier.

9. a separation step of separating the ionizable organic compounds by liquid chromatography or supercritical fluid chromatography; The mobile phase used in the separation step contains chloride ions, bromide ions, and thiocyanate ions. ions at a concentration of 1 mM or more and 300 mM or less, and the solvent is one or more solvents selected from a mixed solvent of water and an organic solvent having a water content of more than 0 vol% and 50 vol% or less, an organic solvent, subcritical carbon dioxide, and supercritical carbon dioxide; A method for separating an ionizable organic compound, wherein the stationary phase used in the separation step is a non-ionic stationary phase (excluding a size-exclusion type stationary phase).

10. The method of claim 9 , wherein the anion does not form a salt.

11. The method according to claim 9 , wherein the anion forms a salt with a counter cation.

12. The separation method according to claim 11, wherein the counter cation is at least one selected from the group consisting of a secondary ammonium ion, a tertiary ammonium ion, and a quaternary ammonium ion.

13. The separation method according to claim 11, wherein the mobile phase further comprises an acid having a pKa in water at 25° C. of −2.0 or more and 4.0 or less.

14. The separation method according to any one of claims 9 to 13, wherein the non-ionic stationary phase is a stationary phase in which a ligand having a crown ether-like cyclic structure is supported on a carrier, or a stationary phase in which a polysaccharide derivative is supported on a carrier.

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